10:02:27 So welcome back everyone to the metabolism forum. Great to have you all back. This is the second last session for this, this forum that we have.
10:02:40 And before introducing Shelley copy. I just want to sort of announced for the next for the last session which I agreed to do.
10:02:53 And Terry and I were talking about what would be a good format for that so the original plan was to talk about metabolic processes in my program that's and I see want to do that.
10:03:04 But I rather than doing a research talk, I want to review and capture on some of the fundamental principles that we have been talking about, and put them together in a complex ecosystem like a microbial Matt, and we heard about gut microbiome, and then
10:03:22 you will see the various similar patterns but important differences that I it was in Craddick for each environment. So, rather than having a research talk I wanted to do this more as it sort of tutorial but also invited, or to invite questions that you
10:03:37 have, and things that you would like to have reviewed after having these 10 sessions. Now, in this form that you would like reviewed by us.
10:03:49 And I would encourage you to send me emails before time so I can prepare and organize this a little bit.
10:03:56 So feel free to send me an email, spormann@stanford.edu.
10:04:03 And then I will work with that and integrate questions and ideas that you have of concepts and then flow this put this into the last session.
10:04:17 So, it is our great great great great pleasure to have Shelley company here today.
10:04:24 Shelly is well known for KITP and, and also in the summer course that we have been teaching at the Hopkins marine station. I think every year we had invited.
10:04:36 Shirley for 12 years to be part of the summer course because of her
10:04:45 stunning expertise how to think about microbial evolution in terms of pathways evolution of neural pathways and taking the view of a biochemist and surely has done some extraordinary work and really has the reputation in the field to help us how we think
10:05:05 metabolic pathways involves, what is the plasticity of pathways, and also to put this into an ecological context. So I'm very delighted that Shelley is here and virtually here with us.
10:05:21 And thanks for taking the time Shelley and, the floor is yours.
10:05:27 Thank you, that was very nice introduction. Thank you very much.
10:05:51 Um. Alright so I'm going to be talking about the evolution and metabolic pathways but this will be a very wide ranging talk, it's going to go everywhere from prebiotic chemistry to bacterial genetics to evolutionary biology.
10:06:08 And I'm going to try to avoid using jargon, or at least explain my jargon, but feel free if I start talking about using terms that you're not familiar with, or concepts that are, that you're not familiar with please feel free to ask questions, and I've
10:06:29 built in a number of pauses in this talk to specifically stop and say, Okay Are there questions so there will be multiple opportunities to ask questions.
10:06:43 Okay, so you've seen this picture, several times now I think, and maybe the shock value has worn off or maybe not.
10:06:59 But the question I'm going to be asking today is, where did all this come from.
10:07:01 And we're going to start at the beginning, like relate the beginning with the formation of the earth, which the earth accreted about 4.5 billion years ago.
10:07:15 and obviously this is an artist consumption here on the left.
10:07:19 But the earlier was extremely hot as a result of a very heavy bombardment from other objects in the solar system, as the solar system was for me.
10:07:32 So it was extremely inhospitable place and of course there was no life at that time.
10:07:40 But within about point 3 billion years things calm down a bit to the point where the surface of the earth cooled enough to allow all of the water vapor that was in the atmosphere to rain down, and to fill the oceans.
10:07:58 Okay and so this was the first time that life might have had a chance to begin. And if the best estimates right now are that life had emerged by perhaps 3.8 billion years ago which is actually not that long after the surface of the earth cooled enough
10:08:23 to allow liquid water. Can you see my cursor.
10:08:28 Good. Ok.
10:08:30 Ok. So, The.
10:08:35 Since that time, the first life on Earth has given rise to an astonishing diversity of organisms. This is a phylogenetic tree that attempts to depict the relationships between organisms that belong to three domains, or three, three domains of life.
10:09:00 So up here in the blue are the bacteria. Down here are the archaea, and then these are the you carry ons, and the root of this tree is somewhere in here.
10:09:16 The last universal common ancestor, or the Luca, And since the time of the Luca approximately 3.8 billion years ago. Some organisms have gone off in this direction and diverge from the bacteria, others went off in this direction and diverge to form the
10:09:35 archaea, and the eukaryotes.
10:09:40 So we, of course, do not have much direct information about the Luca, but we can infer some things about the Luca based upon the characteristics of excellent life.
10:09:57 So for example, if there is a particular characteristic that is found in all three domains of life, then it likely had emerged. By the time of the Luca.
10:10:10 So here are some things that we can say about the Luca, okay it was certainly microbial no fish or plants, of course, that early.
10:10:21 We know that it had ribosomes because all life that we know about on Earth has writing songs and writing songs make proteins so we know it had proteins.
10:10:30 We also know that it had some type of compartmentalization because that is critical for the, what we call the vertical inheritance of genetic information that is passing on genes to offspring.
10:10:48 We also can estimate what kinds of proteins were present in the Luca.
10:10:55 This is a kind of squishy thing to do, but the Luca had approximately 670 genes, including enzymes for synthesis of amino acids nucleotides sugars fatty acids, co factors like in add an ATP.
10:11:14 And we know these things because these are characteristics that are conserved in all three domains of extent life.
10:11:25 But surely just and here's a one question that might be worth just doesn't know.
10:11:30 Is it known that is exactly one worker or could life have evolved independently multiple times.
10:11:38 life could certainly have evolved independently, multiple times.
10:11:44 It is even possible that life evolved and then got from eradicated by a giant Meteor multiple times.
10:11:53 So it, it definitely could have evolved multiple times, I think you're asking could it could there have been parallel origins.
10:12:03 If there were parallel origins, either some of them died out, or they all happen to converge upon the same types of metabolism and nucleic acids, presumably bits and pieces can have come from different organisms like bits and pieces of the carrier coming
10:12:23 from okay and and bacteria so but this is sort of a minimal set rather than some description of. Right. This is a minimum set to everything now has in common.
10:12:34 That's right. Right, so whether one thinks there exists things such as a looker, then this is still a set of the as far as the enzymes. That's right. Which one expect.
10:12:44 Yeah.
10:12:44 So, one of the things that comes out of thinking about the Luke in this way though is that it was already a pretty sophisticated organism.
10:12:55 So it already had red zones and proteins and metabolism and a lot of metabolic pathways. So this is sort of like getting to a movie 10 minutes before the end.
10:13:08 So you get to see the end. And then you can try to figure out what happened beforehand. and you may or may not be very correct.
10:13:20 And so a lot of the most interesting evolution of metabolism actually happened before the look up.
10:13:29 Okay, so I'm going to be focusing on enzymes in this talk because almost every reaction in this network is catalyzed by an enzyme, there are a very small number of non enzymatic reactions in extent metabolism.
10:13:51 And it is very obvious that Catholicism was critical for the emergence of life.
10:13:57 This is a list of a number of chemical reactions that occur in living organisms. And these are the T one half values that were estimated by Dick wolf Clinton's group.
10:14:11 20 years ago.
10:14:13 And you can see that, well here's one.
10:14:17 I summarization which is a reaction in glycol says has p one half of only two days, but dicker box elation of ONP which is involved in biosynthesis of nucleotides has a half react, half life of 1.1 billion years.
10:14:33 Okay so clearly catalysts were critical for accelerating the kinds of reactions that that contribute to metabolism.
10:14:44 But there's a second reason that fatality is important. And that is the catalyst structure metabolism, but accelerating specific reactions. So, consider this slightly messy cartoon of a reaction network, in which you've got multiple inputs and multiple
10:15:07 outputs.
10:15:11 If you can discover catalyst say for these two steps.
10:15:16 What will happen is that this catalyst will accelerate the reaction of be with C. So, C will no longer effectively be transformed to E.
10:15:31 And then if you can accelerate this reaction of DNID will be central channel toward K and won't have the opportunity to react with H, or with I.
10:15:46 So just adding a couple of catalysts to this reaction network.
10:15:52 Soon proves the network and results in the production of primarily k and this kind of thing is important because instead of making lots of different products in small amounts.
10:16:06 It makes it possible to make fewer products but in larger amounts.
10:16:11 Okay and so playing this game.
10:16:15 Again, if we have, say, This catalyst again but instead, we have this catalyst.
10:16:21 Then we can prove the network to form, primarily and Jay.
10:16:29 And if we have just this one catalyst up here that converts see to eat.
10:16:37 We would pro the network again so that the outputs are primarily in in be.
10:16:43 So the point of this is that catalysts are extremely important for structuring networks pruning off certain branches and allowing production of a smaller number of products that in larger quantities.
10:17:04 Okay, so what were the earliest catalysts.
10:17:08 So the earliest catalyst will probably mineral surfaces metal ions in small molecules. And generally, these were probably not very effective catalyst, but any amount of fatalis us is helpful in terms of both accelerating reactions and pruning networks.
10:17:28 And so those initial inefficient catalysts could have helped to generate somewhat larger quantities of, say medium sized molecules which might include peptides, which are small chains of amino acids, or short RNAs likely complex with amino acids co factors
10:17:53 or metal lines because RNAs are not actually that great that fatalis us. And I have RNAs in quotation marks here because this is what RNA looks like in extent life in the earliest, quote unquote, RNA might have had a different backbone so they might have
10:18:16 had different kinds of sugar they might have had different linkages. They might have had different basis, which is what we call these companies are nuclear bases attached to the sugars.
10:18:28 So the earliest RNAs will probably probably almost definitely not this. Okay.
10:18:34 And on the right hand side of this slide I'm showing you that these are structures of RNA so we typically depict RNA, in a line like this but actually our news fold up into structures, and you can easily imagine a metal ion, or even an amino acid or a
10:18:56 factor, being sort of tucked into a cavity in an RNA like this, and providing catalytic on silver ring that would make the RNA a better catalyst.
10:19:10 Okay. And so, as better catalysts emerged in in the in these medium sized molecules, they again could have accelerated reactions PR networks allowed production of new compounds and compounds in higher quantities, eventually leading to you, bonafide RNA
10:19:33 or at least some, you know, close progenitor that again could have catalyzed reactions but likely with catalytic distilleries like amino acids or co factors or melons.
10:19:50 And then, Ultimately, the, the fabulous protein enzymes that are characteristic of extend life emerged. And we know that RNA came before protein because proteins are made by rhizomes and ribosomes are made of RNA, so proteins had to be the sort of the
10:20:11 pinnacle of this evolutionary evolutionary process.
10:20:18 Okay, so what sort of stage of here, RNA replication so we don't do is it thought that those already would involve amino acids and various other other factors.
10:20:39 RNA by by itself just doesn't have the functional groups in molecules to be a very good catalyst. So, you know, my prejudice.
10:20:51 Coming from a protein perspective, obviously, is that RNA probably needed some help to do all of those kinds of reactions, whether it was metabolic reactions or replication reactions.
10:21:07 So, yeah, I don't believe in a pure in a world. And I guess since the amino acids are much easier to create by natural processes in the RNAs in the first place.
10:21:17 Um, that presumably as far as thinking about the early very early biochemistry that's it's a cheap and on in some ways. Yeah, yeah.
10:21:44 So, a lot of those cool factors in AD and ATP
10:21:52 actually look like pieces of RNA.
10:21:57 And so, they, they very well may have evolved sort of somewhere in this range, and have been retained by metabolism over billions of years, because they're useful.
10:22:13 They do good chemistry.
10:22:15 Yeah, so they, they are kind of considered to be probably relics of a world that involved RNA.
10:22:24 So they were probably very early.
10:22:29 Okay, so next question is What did the first catalyst have to work with.
10:22:34 So what kinds of chemicals were available on the earlier. Okay, so there was a lot of carbon, including organic carbon delivered to the early Earth from the heavens.
10:22:51 So from comets meteorites enter interplanetary dust particles and electrical discharges.
10:23:02 And I mean a lot so 4.4. billion years ago it's estimated that interplanetary dust particles delivered tend to the ninth kilograms of carbon per year.
10:23:14 Comets about 10 to the six kilograms per year and meteorites about 10 to the fourth.
10:23:23 kilograms per year.
10:23:25 So that's a lot of material that accumulates over millions of years.
10:23:32 And some of the most interesting carbon that was delivered was found is found in some types of meteorites that are called carbonaceous contracts.
10:23:42 The key there is carbonaceous which means that they contain a lot of carbon.
10:23:47 So on the left I'm showing a, an analysis of three different meteorites gra CR to and La PTCR to these were meteorites that fell in Antarctica, and the merchants and meteorite which you may have heard of which fell in Australia, and the y axis is the
10:24:06 abundance in ppm. And so there are lots of amino acids in these carbonaceous contracts, there's ammonia means elder hides and ketones hydrocarbons and carb oxalic acids and the relative quantities of these things and very among meteorites
10:24:29 the merchants and me right has recently been found to contain sugars. So die have dark the acetone is related to a compound and glycol says, A ravenous ribosome silos and Lexus are all sugars that are found in living organisms.
10:24:49 One of the things I want to point out though, is that that meteorites typically contains thousands of organic compounds. So there are lots of things in them but generally relatively small amounts of each thing.
10:25:06 But some of these compounds that are found in meteorites are really quite interesting. So, this is a table of classes of compounds, and then specific examples.
10:25:18 So acidic acid Eleni lactic acid perfect acids accent gas or plus Eric acid acid outside are all found in living organisms.
10:25:30 So mean is not.
10:25:34 And this one is particularly interesting, Nick authentic acid is part of in AD, which you've heard a lot about as being a critical co factor for reacts reactions and added me is the, is part of ATP.
10:25:53 And it's also part of nucleic acids, RNA and DNA. Okay.
10:25:58 Okay. But meteorites also contain a lot of things that are not found in life, like appropriate propane Napoli and ice equivalent. And they also contain about 70% of the carbon is just this insoluble tar like material that is polarized aromatic rings.
10:26:21 So the supply of organics to the earth was also augmented by electrical discharges in the atmosphere. And you may have heard of the famous Miller URI experiment where.
10:26:40 Exactly.
10:26:45 Okay, that's better.
10:26:47 So they passed electrical discharges through various mixtures of gases in this flask and then collected the chemicals that formed and analyze them.
10:26:59 And in this type of experiment.
10:27:02 You can make a number of protein a gigantic amino acids. So those are the amino acids that show up in proteins, as well as a lot of non energetic amino acids.
10:27:16 So again, many, many different things, but relatively small amounts of each one.
10:27:41 One question is, how did these organic compounds form in the meteorites what is sort of the backstory, how they arose and where they came from.
10:27:37 So, that is a subject of much investigation. They seem to have formed in the interplanetary dust particles and as the, as larger bodies accreted UV radiation may have played a role, also maybe some heat that's generated by collisions.
10:28:02 So there are there are people who are working on trying to figure out how, how these compounds formed.
10:28:11 So it's a very interesting question how they formed.
10:28:14 It is indisputable that they did for.
10:28:19 So sorry I can't give a better answer than that. Thanks.
10:28:23 Okay, so, um, all of this carbon bringing down from the heavens could have led to an origin of life in a warm little pond on the surface of the earth and the warm little pond was actually first articulated by Darwin.
10:28:40 Long before anybody really knew very much about the molecular basis of life.
10:28:48 So for a long time this the the idea that like originated in the warm Mill Pond was the only game in town.
10:28:57 But there are some things that are a little problematic about this idea, and that is that all of these sources of carbon produce have very complex mixtures of compounds that only partially overlap with molecules are used in life.
10:29:17 And so it's a difficult problem to grapple with, why were some chemicals chosen and others not chosen. And if you have a complex mixture with small amounts of lots of things, how do you ever build up enough to actually make something useful.
10:29:39 Okay, so an alternative to this idea is that life might have originated in hydrothermal vents and so hydrothermal vents were first discovered in 1979.
10:29:52 And what was shocking was that these hydrothermal vents host lush ecosystems. And these ecosystems are far from the sun, they're very deep there's no white.
10:30:04 They are completely fueled by Redux reactions that are catalyzed by microbes that live in the walls of events that around events, and the carbon from those microbes actually feeds, these ecosystems that contain huge tube worms and Trump in clams.
10:30:25 So, clearly life conform in this kind of environment.
10:30:33 This is a particularly interesting picture of a cross section of a sulfide chimney from one of these events. So this is the inner conduit where the hydrothermal fluid is flowing.
10:30:48 And this hydrothermal fluid which is hf is that 302 degrees centigrade.
10:30:55 And on the outside of the event we have seawater at two degrees centigrade. And the interesting thing about these structures, is that they are porous, so that the hydrothermal fluid can percolate into the wall of the vent and the sea water can percolate
10:31:11 in from the outside, and strikingly, there are microbes that are found in every one of these four zones, through this porous wall. So zones one through three which are a little cooler have between 10 to the six and 10 to the eighth microbes per gram so
10:31:33 phi.
10:31:34 There are fewer in the wall that's right next to the portion of the wall that's right next to the hydrothermal fluid, because it's so hot.
10:31:45 But these microbes are continually fed by small molecules that are present in the hydrothermal fluid that diffuse into the walls.
10:31:59 So, these porous walls, provide things that are useful for microbes. Today, and could also have provided a start for the origin of life in these systems.
10:32:14 So they provide. Whoops, sorry.
10:32:17 They provide compartmentalization, which, as I said before is critical for passing on genetic information but it's also critical for keeping all the good stuff inside and not letting key metabolites leak out.
10:32:46 because they are lined with minerals and minerals can catalyze chemical reactions. And the there is a continuous supply of reactive small molecules that concern is
10:32:55 that can provide the basis for Redux reactions for harvesting energy, and also for carbon fixation.
10:33:03 So this is an example of a black smoke or field.
10:33:10 At the bottom of the ocean, and these black smokers have very acidic meant fluids that contain lots of hydrogen and methane, co2 hydrogen sulfide in ammonia.
10:33:21 So these are important ingredients for for sustaining the microbes that live in these porous walls of events, but could also have fostered the emergence of life in these environments.
10:33:38 There's also a second time scale on that picture.
10:33:45 What's going back.
10:33:48 Going back. This is the submersible elven.
10:33:53 To give you an idea.
10:33:56 I can't actually even see the scale over here, but albums not all that big.
10:34:05 But these, these towers can be many meters high.
10:34:10 And did you might have missed but did you mention phosphates.
10:34:15 No, I did not mention phosphate because that is one of the difficulties is where to find phosphate, in, in any of these scenarios where to get phosphate, so I can't actually tell you what the most up to date theories are fun origin of the phosphate.
10:34:40 Okay so this. The second type of hydrothermal. That was discovered in 2000 and it's very different.
10:34:48 The, the fluids are alkaline.
10:34:51 So, these are like 60 meter tall towers of various types of carbonates, but again there are the hydrothermal fluid that's coming through here is enriched in things like methane and hydrogen, and other hydrocarbons, the temperatures are much milder in
10:35:10 these.
10:35:11 So,
10:35:15 you know, getting back to the question of how did we even get to the Luca.
10:35:22 We really can't say because there's there's really a Philosopher's debate going on between people who believe that life originated hydrothermal vents.
10:35:36 In which case, the origin of metabolism required built up of a very sparse set of small molecules into larger molecules.
10:35:46 And then the other side of the fence is people who believe that life originated warm little ponds at the surface of the earth, in which case the metabolic networks we see an extent life required massive printing of the complex set of metabolites and choosing
10:36:07 some metabolites rather than others to become part of life.
10:36:12 Okay, so it's really not very satisfying. And I told you, you know, a lot of the interesting stuff happened by the time the Lucas emerge.
10:36:24 But now I'm going to get back to the question for did all this come from in the, the, what I'm going to talk about next our series that really apply, mostly to the stage at which there were at least macro molecular catalysts have some type.
10:36:44 So there are two dominant theories that have been proposed for how metabolic pathways evolved.
10:36:54 And the first was proposed in 1945 by Nathan Horowitz at Stanford right about the time the world war two was ending in Europe, and her wits proposed what's called the retrograde hypothesis.
10:37:13 So, at the time, the warm little pond was, as I said, the only game in town. And the idea was that life originated in the context of a rich primordial soup that have lots of useful compounds available in the environment.
10:37:32 And so, what would have happened is, over time, as life became more abundant. And if compound, he was critical for life.
10:37:43 compound, he could have been depleted from the environment. And then,
10:37:53 if an organism could find an enzyme that would generate, he from some other compound in the environment D, then that organism would have a selective advantage.
10:38:06 And here I'm calling this enzyme one.
10:38:10 And then over time, it would have happened again D would have been depleted from the environment, and it would have been necessary to find an enzyme to convert some other compounds, C, and D.
10:38:20 and so on.
10:38:22 Adding be adding a so haurwitz his idea was the pathways would evolve in a retrograde direction from the input in product of the pathway working back as things got depleted from the environment.
10:38:42 So actually 20 years later haurwitz added another idea to this hypothesis.
10:38:50 And his new idea was that enzymes in a pathway should be related. So if an organism is found in enzyme one that can convert TT, and now it needs to convert C to D.
10:39:04 Then a logical place to find that enzyme might be enzyme one because enzyme one can already bind D and C is presumably structurally somewhat similar to D.
10:39:20 And so maybe enzyme one could be recruited to catalyze this new reaction.
10:39:30 And what would then have happened would be that the gene encoding, one would duplicate and diverge to give you two related enzymes, one into.
10:39:43 Okay, so let me show you a picture of how that might happen.
10:39:46 Okay, so let's suppose that we have a gene that encodes inside one, and it has an inefficient secondary activity to catalyze reaction to based on its ability to bind compound D.
10:40:04 So, when reaction to becomes important.
10:40:09 There may be selective pressure to knit for the gene to duplicate, or even amplifier to provide more of that inefficient secondary activity that has now become important.
10:40:24 And over time, these multiple copies kids to caustically acquired mutations, some of which will improve the ability of the enzyme to catalyze that newly knee jerk reaction, and some of which will
10:40:42 regenerate the enzyme that was initially responsible for catalyzing. The first reaction. Okay. And so, when sufficiently good capitalists are present for both reactions.
10:40:58 These extraneous copies, can be lost, and you will end up with one gene encoding enzyme one in one gene encoding enzyme too.
10:41:10 Okay, so that was more which is idea for how you would in a stepwise manner, evolve, new enzymes, each of which would be related to the enzyme catalyze the previous step in the pathway.
10:41:31 Okay, so, in 1974 1976, years, and Jensen came up with a new hypothesis, which is in some ways similar, but in a really key way different from what more which had proposed.
10:41:51 So, the, the key idea that you could and Jensen hypothesize was that early enzymes were promiscuous ok so the promiscuity in terms of enzymes means the ability to catalyze secondary reactions.
10:42:12 So this cartoon is showing a blue enzyme, which has the ability to catalyze the blue reaction, but it may have an inefficient ability to catalyze the red reaction.
10:42:29 And if that's the case, then this this enzyme could be recruited to perform this red reaction in another pathway. Okay so Maura which would have said it would be recruited to provide the another step in the same pathway, your cousin Jensen are saying
10:42:53 be recruited to perform a reaction in another pathway. And so this is the general idea that promiscuous activity promiscuous enzymes could be recruited from various places in the metabolic network and patched together to form a new metabolic pathway.
10:43:09 Okay, so these enzymes would not necessarily be related to each other, they would have been recruited, simply because they had the ability to catalyze a newly knee jerk reaction.
10:43:25 Okay. And again, once you've recruited an enzyme to do a new function.
10:43:32 Most of the time you're going to need to go through this process of duplication and divergence to end up with a specialist enzyme that can catalyze a newly needed reaction.
10:43:47 Okay, So, let me pause for questions.
10:43:58 If there are no questions.
10:44:00 I will keep going there will be more opportunities.
10:44:08 Okay, So now let's, let's talk about these two hypotheses. Okay, so I see three problems with the retrograde hypothesis. The first is that it requires a b c d and e to be present in the environment.
10:44:24 And, you know, if you're thinking about this in terms of the warm little pond and maybe there are all of these things present in the environment. If you're thinking about this happening at hydrothermal vents, where the inputs are very sparse.
10:44:39 Then, this is not so likely to happen.
10:44:44 but a more serious problem is that this hypothesis requires ABC and D. to be stable.
10:44:56 And that's really a problem.
10:44:57 So we know that there are many intermediates in metabolic pathways that are not steady stable. So what I'm showing you here is the pathway for synthesis of the amino acid prolly, which is one of the amino acids that's incorporated in two proteins in life.
10:45:21 And let me take a little, just a little side diversion here on, so I'm showing you all of these compounds, but I want to explain the way that chemists draw these compounds.
10:45:35 Okay so here is the detailed view of the amino acid glutamate which is the precursor for this pathway, and it shows all of the atoms
10:45:48 chemist, have developed a shorthand, because basically we get tired of writing carbon and hydrogen all day long.
10:45:56 And so this is the shorthand version of glutamate, and each vertex here. That's not labeled as a carbon.
10:46:06 So we typically use. We don't write the carbons, we do right the other atoms like oxygen and nitrogen.
10:46:15 And we don't write all of the hydrogens. So, carbon conform for bonds. So, what you can see here is this carbon has two bonds. One, two, you can infer that the other two bonds are two hydrogens.
10:46:27 Okay, so this is the shorthand for, for all of the structures that I'm going to be showing you for the rest of the talk.
10:46:42 Okay, so the point of this slide is that here is the path way for biosynthesis of probably in
10:46:51 essentially all forms of life, this intermediate here is extremely unstable.
10:46:56 It has a half life of probably on the order of milliseconds, in its cycle wises to five Oxo probably, which is a dead end metabolite okay so this is a pathway that certainly could not have evolved in a retrograde manner because this compound would never
10:47:12 have have been present in the environment.
10:47:17 Okay. And then the third problem which really kind of puts the nail in the coffin for this hypothesis is that bioinformatics evidence that we have now of course more words in 1945 certain again.
10:47:30 But we now know that enzymes in a pathway are rarely related to each other. They're like one or two pathways where there are there are some examples of sequential reactions catalyzed by related enzymes, but generally enzymes in a pathway are not related.
10:47:50 OK, so the retrograde hypothesis, really hasn't stood up to the test really good stuff about the related. So you mean if you look at the sort of crucial catalytic domain, those are those don't seem to be related.
10:48:04 So the way we judge this is by looking at the sequence of the inside the amino acid sequence of the enzyme.
10:48:13 And if you do a pairwise sequence alignment of two proteins, you can determine whether there is any similarity between those sequences that suggests that they are related.
10:48:28 So if they are more than about 25% identical.
10:48:33 Then they are likely related, but I guess I'm, I'm where the difference between the third catalytic domains and the rest of the protein since the domains get shuffled around and redraw the other thing anyway and then it.
10:48:49 related but the overall relatedness is is low, because of things getting shuffled around.
10:48:59 So there.
10:49:02 So there. Okay, this is more complicated question. There are examples of enzymes that catalyze the same chemical reactions that are clearly not related.
10:49:14 Their structures are different their sequences are different.
10:49:17 Even the catalytic strategies can be different.
10:49:21 There are also cases where the sequence identity between two enzymes is less than 25%, but they have the same overall structural fold.
10:49:35 And in those cases, we think that they probably are related that the the sequence identity has just been lost over long periods of time.
10:49:44 Okay, so, so we can tell by looking at the sequences and the structures, whether they are likely to be related or not.
10:49:52 Hmm.
10:49:55 Okay, so now let's, let's look at the feasibility of this patchwork hypothesis. So the patchwork hypothesis.
10:50:05 Depends on this promiscuity of enzymes.
10:50:09 And so one of the things that I want to tell you about promiscuous enzyme activities, is that even very inefficient promiscuous activities can accelerate reactions by orders of magnitude.
10:50:22 So what I'm showing you here is our reactions that are catalyzed by an equilibrium zone alkaline FOSS potatoes, which catalyze is the hydraulic cleavage of this po bond in a variety of what are called phosphate esters which is basically phosphate attached
10:50:42 to something else. Through this oxygen bond.
10:50:47 And the K cat over km for this kind of reaction is about three times 10 to the seventh per dollar per second. So you've seen kicked out over Kim mentioned before, if you don't remember it is the second order rate constant for the reaction of the enzyme
10:51:06 and the substrate, when the enzyme is it saturated.
10:51:11 Okay so Auckland phosphate haze can also catalyze cleavage of.
10:51:17 So bonds.
10:51:23 Oops.
10:51:22 But cat over km is much, much lower.
10:51:26 And it can also catalyze the cleavage appeal bond in a more complex molecule. Again the cake however can is much lower.
10:51:37 But even though the these promiscuous reactions are very inefficient, they accelerate reaction rates by many orders of magnitude so the proficiency column over here is the ratio of the K cat over km for the enzymatic reaction to the rate of the uncatalogued
10:51:59 reaction.
10:52:01 So, the rate of the catalyst reaction is 17 orders of magnitude higher than the rate of the young catalyzed reaction.
10:52:11 And that's pretty impressive, but they just so you know, the most efficient enzyme most efficient enzyme that's known catalyzed as a reaction by 26 orders of magnitude over the undercapitalized reaction.
10:52:27 But notice that these inefficient promiscuous reactions still catalyzed rates by nine and 11 orders of magnitude relative to the uncompromised reaction.
10:52:38 So what that suggests is that promiscuous activities, even if they're not very efficient can be very much better than nothing. And so, they may provide a selective advantage when an organism needs a new enzyme decided to take the high temperatures and
10:52:58 thing and the other conditions that the vents, make this a bigger effect of smaller factors What are these blue a lot of the most existing enzymes actually studying don't don't work in the in some of the extreme conditions as against inside the cells
10:53:13 but.
10:53:27 actually some, there are some affiliate organisms that can live it, temperatures above the temperature of boiling water, as long as there's enough pressure to keep the water. Liquid so there are partying enzymes that can function that you know 110 hundred
10:53:34 20 degrees centigrade.
10:53:36 You're still going to get many orders of magnitude rate acceleration, relative to catalyzed reactions, even at the high temperatures.
10:53:50 Okay, catalyze ones presumably are typically faster but you still you still pick up the menu.
10:53:55 Yes.
10:53:58 Okay, another important thing about promiscuity that relates to the patchwork hypothesis is that promiscuity is extremely common.
10:54:07 So, you know, I think most of us grew up being taught that enzymes catalyzed very specific reactions.
10:54:18 But we're becoming aware that probably all enzymes have promiscuous activities, and maybe a surprising number of them. So this is a very interesting paper that was published a few years ago, it was a high throughput substrate profiling experiment where
10:54:39 large group of people, essayed over 200 enzymes, with 167 different substrates, and everywhere you see read in this plot is means that the enzyme could catalyze, a reaction with that substrate.
10:54:58 So there's a lot of blue and the blue means that the enzyme did not turn over the substrate, but there's a lot of red too. And there's this cluster up here of enzymes that can catalyze, in some cases, over, 150, different reactions, with the among these
10:55:18 hundred and 67 substrates. And then there are some that are much more specific but even the ones that are fairly specific you see, you know, a fair number of read blogs.
10:55:28 So, we're coming to understand that enzyme promiscuity is very common, and it is just a feature of the fact that active sites of enzymes are highly reactive environments, they've evolved to have lots of acids and bases and metal ions and co factors in
10:55:51 nuclear files and electro files and oxiana and holes and they know you want to understand some of those words but the point is that they are very reactive environments.
10:56:00 And if a non canonical substrate can wander into that environment sometimes chemistry can happen. So, promiscuity is very common.
10:56:11 We will never know the total number of risks activities because obviously nobody's ever going to say every possible molecule in the universe.
10:56:23 For a given enzyme.
10:56:25 But
10:56:28 let's just make a guess that maybe, on average, enzymes have 10 promiscuous activities.
10:56:36 This is a table showing the number of enzymes in several different microorganisms. So equal I has about 1600 enzymes, and if each of these enzymes has on average 10 promiscuous activities.
10:56:53 That means that he call I could have a repertoire of about 16,000 promiscuous activities that could be recruited to do new things. And the common editorial possibilities.
10:57:07 Obviously, are huge when you have 16,000 things to play with.
10:57:12 Shelly.
10:57:32 After we have a versus the enzyme, the kind of the strange molecule that had some kind of activities, you know, on the way.
10:57:51 It'll go well what Where are you, so I am, I'm in the era of macro molecular panels. So, either RNA or proteins.
10:57:53 Okay.
10:57:55 Some of these ideas could be sort of extrapolated back. But the these ideas specifically applied to macro molecular catalyst, which were present probably before the Luca, okay, but I'm, I'm, I'm not necessarily talking about the age of small molecule
10:58:19 Patel Asus.
10:58:23 Okay. But then, so you already have. Proteins are you talking about small peptides, which is just to be a bit more specific.
10:58:30 So, this would have been either a macro molecular RNA or proteins because I'm, I'm talking about the possibility of gene duplication and divergence, to make new enzymes.
10:58:45 So, we have to be at least at the stage where there are genes.
10:58:51 Okay, so you're really okay so you think of ribosome and all of that could happen before, but before the enzyme that effectively catalyzed metabolism.
10:59:07 No, I don't think the writing songs evolved before there were enzymes for fatalis. I think that there were not protein enzymes before the right Islam evolved.
10:59:14 They could have been.
10:59:17 As I said, RNA or some precursor to RNA, that had catalytic auxiliaries.
10:59:27 But you had RNAs there, there would have been a stage where there were RNA genes that were being transcribed to make more RNA.
10:59:42 Okay, so I specifically talking about the stage where you would have had some kind of genetic material so that you could have had genes duplicate and diverge to form new enzymes.
10:59:55 And these genes could be RNA jeans with the yeah they could have been RNA genes. At the beginning of ultimately they can DNA jeans, but where would protein come from this one.
11:00:08 Well the protein could not have emerged until after you had RNA that could have made ribosomes.
11:00:15 Okay, and then they okay so the whole topic of how translation emerged is huge and speculative and more than I can actually talk about No no, but I'm still asking my question.
11:00:32 Because you're thinking about proteins capitalizing on these reactions. Right. And you'll also talk about these reactions happening before I was on before right the zones but there could have been catalytic RNA before writing songs.
11:00:51 Making proteins, somebody catalytic RNA so RNA can actually catalyze chemical reactions if you have macro molecular or it could have catalyze Yes, yes, yes.
11:01:03 But you don't but then you also mentioned you needed a protein so did I hear you know you don't need, you don't need.
11:01:13 Okay, you might have amino acids, or metal ions are co factors, participating in Qatar.
11:01:22 Yes, so this, you could push these ideas back to sell that had an RNA genome.
11:01:34 You need to do all the metabolic analysis, but can you sort of intermediate intermediate one which is still what's been explored with RNA is plus. Your amino acids or possibly, you know, very small peptides are sort of pre proteins, and what they can
11:01:52 catalyze is the head of investigation has been done, of not saying okay can catalyze some particular reaction that's important now, but asking whether particular RNA RNA amino acids can catalyze anything of interest I mean all sort of the range of things
11:02:05 that they can, yeah, there were a few examples of RNA catalyzing reactions of small molecules RNA catalyzing ligation of to RNA or cleavage of an RNA.
11:02:23 So there has been actually a fair amount of work looking at what kinds of catalytic reactions RNAs can catalyzed.
11:02:34 They're not great by themselves, but even RNA, plus amino acids are plus little, little peptides and.
11:02:42 So, I've seen studies of RNA, like with an amino acid.
11:02:49 Yeah, so, so people have done a little bit of work with RNAs and catalytic auxiliaries
11:02:58 wondering was whether some of the catalytic remains in the proteins that the amino acid part is small enough that if you had a sort of structure to stabilize it and other things that was RNA, but had the little, little bit of the few amino acids in both
11:03:14 of in the key way
11:03:18 there and whether that sort of people have built up or looked at scenarios like that. Yeah, a little bit, and I think that that that's really the key that don't there never was a pure in a world, but RNA could have provided sort of the structural scaffold
11:03:38 to bind.
11:03:40 So just like protein enzymes provide the scrub structural scaffold to bind co factors now RNA could have provided a structural scaffold to bind co factors and amino acids could have been co factors back then.
11:03:55 Yeah, yeah.
11:03:58 Sorry, Shelley, I mean, maybe because I started one of these questions I can sort of contact I'm wondering, the main thing I'm wondering about this, so you're imagining that there are already auto catalytic systems that self reproduce in some way in that
11:04:13 have some sort of genetic information maybe not DNA maybe RNA, and they have some sort of rudimentary enzymes, maybe not produced by results.
11:04:26 Is that is that is that correct Is this the sort of phase that you're sort of imagine, oh yeah so that would be the earliest part of the phase that I'm talking about and then this is what I'm talking about now could have started at that phase, but it's
11:04:39 continuing all the way through the present.
11:04:43 Okay.
11:04:46 So, eventually, he had you.
11:04:49 Yeah, but that I mean, they said this is where difficulty comes in because, I mean, once you have arrived zones and everything and Gene duplication, the way that we know it works today, I can, I can sort of assess the plausibility of these various scenarios.
11:05:04 When we go back into some more rudimentary states where it's really unclear how the replication really sort of works at all.
11:05:14 It becomes much more shaky to me to discuss.
11:05:18 It certainly is. So all of these ideas, apply very easily in the context of the Luca who that that had protein enzymes.
11:05:32 All I'm saying is that some of these concepts might actually, you could push back a little bit to an error of where maybe proteins work providing calluses but macro molecular RNA was all was providing to tell us more wit and Nickerson Jensen.
11:05:56 We're only thinking about protein enzymes.
11:06:02 And really that's mostly what I'm talking about. I'm just suggesting that some of this might have been applicable like push back before you know when you came in by the end of the movie.
11:06:13 Right. Yeah.
11:06:15 But yes, definitely speculative, and mushy, but, you know, at least a little bit of an attempt to generalize some of these ideas to maybe how we got to the local okay but I'm pushing it back to a ribosomal is definitely pushing the concepts in a direction
11:06:44 that where things get much harder to even talk about.
11:06:59 So I say too much about that part of it.
11:06:56 Because I'm waving my hands but I'm just saying some of this might have been applicable if you had. If you had jeans.
11:07:06 And you had a macro molecule encoded by those genes, whether it was RNA or protein.
11:07:13 And those macro molecules could catalyze reactions, then you could have started patchy together things in new ways.
11:07:24 Okay, thanks.
11:07:26 I think part of the argument might also be that it is easier to make all ego peptides, and only go nucleotides.
11:07:38 And it's easier to synthesize amino acids then nucleotides.
11:07:46 And there's more kind of potential in the amino acid side chains, then in the nuclear nucleotides.
11:07:55 Right.
11:07:57 That is true.
11:07:58 Yeah, and those are all reasons why I don't think there was ever a pure RNA world.
11:08:05 You know, there would have been a, A world that had peptides in short nucleic acids and metal lions and minerals and all of these would have been contributing to the generation of a proto metabolic network.
11:08:26 Okay, so we've got a lot of misc is activities.
11:08:31 There is.
11:08:34 There are some nice examples of pathways that I think we're clearly patched together by this patch patchwork mechanism from promiscuous activities. So here are a couple of examples of metabolic pathways in two different micro organisms that were isolated
11:09:02 from contaminated soils that were contaminated with natural targeting, which is not a naturally occurring compound. And in the last century, microbes that have been faced with a new compound in the environment that can provide a new source of carbon and
11:09:14 nitrogen have patched together metabolic pathways that will allow them to access that carbon and nitrogen.
11:09:25 And what what you see here are these two different microbes have passed together completely different things. Okay, so in the top. The Pseudomonas has starts by oxidizing this ch three group to see OH minus which is a carb oxalic in the bottom Mycobacterium
11:09:48 starts by reducing this natural group, you know to to an amine in each two.
11:09:56 OK, so the reason that these two pathways look different is that these two different organisms have different collections of promiscuous ability activities at their disposal to patch together to make a new pathway.
11:10:15 And another example that I want to show you that's going to pop up in the second part of the talk is the pathway for synthesis of paradoxical five prime phosphate, which we call PLP.
11:10:28 This is vitamin B six, and it is an essential co factor in all forms of life. It capitalizes a range of different chemical reactions. One of the most important of which is the production of amino acids for protein synthesis.
11:10:47 So, most organisms that make PLP do up with a gorgeous to enzyme complex.
11:10:56 That takes glutamine glycerol to high three phosphate and rivals five phosphate and spits out PLP.
11:11:05 But the ancestor of some proteome bacteria, apparently, lost the genes for these enzymes probably during a time in which there was some compound in the environment that could be taken up and converted to PLP by a salvage pathway.
11:11:25 And then, ultimately, those organisms, may have found themselves in an environment where that precursor was no longer present, and they had to either come up with a new way to make PLP or die.
11:11:41 And so they came up with the even more complicated looking pathway. And this is what the pathway looks like.
11:11:51 In here I just want to define for you.
11:11:55 This is another example of shorthand.
11:11:59 We get tired of writing out all the oxygens and bonds and phosphates so P with a circle around it means phosphate.
11:12:08 Okay, so this is the pathway that was patched together to reconstitute PLP synthesis when it became important. And we know that this was patched together because we can see the origins of many of these genes.
11:12:26 So this first enzyme epd is related to an enzyme in glycol Asus PDXB is related to an enzyme in searing biosynthesis CRC is actually the same enzyme that is used in certain sentences it's been recruited to serve a second function.
11:12:47 DXS performs exactly the same chemistry in the pathway for synthesis of vitamin b1.
11:12:55 And SPDXH is part of the ancient salvage pathway that is present in most organisms. Okay, we don't know where PDXANPDXJ came from. But, we can see that this was patched together by recruiting promiscuous enzymes that did other reactions in other parts
11:13:16 of the metabolic network, network, or in this case, the same reaction in other parts of the metabolic network.
11:13:26 Okay, so we have some nice examples of pathways that look like they've been patched together.
11:13:32 So are there any additional questions.
11:13:40 Oh, I just I certainly don't know anything about this album chemistry but to chain eyes of a how surprised, are you when you see a pathway like that evolve, I'm asking whether you can predict that but is there some sense of oh this is difficult to do
11:14:02 and no surprise me to go this is easy to do.
11:14:08 So, the whole second part of the talk is going to be addressing that kind of question. Okay. Okay, so hang on to the thought and then come back and ask me again.
11:14:21 If you're not clear.
11:14:26 I have a question.
11:14:28 I get very inspirational, but I'm really new to this topic.
11:14:31 So, I wonder when there is the possibility possibility to have pathways pitch together like you just showed.
11:14:42 Then, I wonder if like stress reaction in such a system is always to just express whatever they have and see if that fits.
11:14:55 Concerning the enzymes. Is that the case, or if you know what I mean.
11:14:59 Oh, well okay so if you're going to recruit a promiscuous enzyme to do something new. It has to already be expressed.
11:15:12 So, or microbes can only work with the collection of enzymes that are actually expressed at a particular time, which is actually an interesting
11:15:25 sort of puts an interesting twist on this, because what it suggests is that so microbes don't express all of their enzymes all of the time because that would be a waste.
11:15:35 They only express the things that are needed.
11:15:39 So what that suggests is that the the suite of mysterious activities that might be available might vary depending on what the environment is because different enzymes expressed depending on the environment.
11:15:56 And that's actually something that we're working on in my lab right now is looking at how how things can evolve depending on what the environment is because that will affect what enzymes are available to be recruited.
11:16:16 So it's a great question.
11:16:20 I think actually just to end and I don't know if this was Pinterest idea but sort of this this observation, especially if you take e coli on a subset limited conditions, all kinds of pathways I do started though, the substrates on our president, and maybe
11:16:37 the question wasn't the direction is this may be a biological strategy to take over with coma diabolical promiscuous activity to have a metabolic escape from a stressful situation I don't know if this was sort of the deeper thought of this question.
11:17:01 Okay, I'm not quite sure I understand completely what you said. But I would say promiscuous activities are not there because they can provide an escape or, or something that might be useful in the future because evolution can't anticipate what might be
11:17:26 useful.
11:17:28 So, people often ask me why, why are these promiscuous activities there and they are there, purely because enzymes are such reactive environments.
11:17:40 And they are maintained, not because they will be useful in the future. But because there's no selective pressure to diminish them below a level where they don't cause harm.
11:17:56 So, there will never be selective pressure to make an insight any better than it has to be right so first thing is biological perfection.
11:18:08 So, if, if a secondary activity actually interferes with something natural selection will not get down to the point where it doesn't matter anymore.
11:18:20 So that's right. The reason that these promiscuous activities exist, not because they might be useful in the future. But because natural selection can no longer see them after they've been knocked down to a very low level.
11:18:36 I think that there's another part of that is that evolution by its by its nature will always look as if there were things that were selected for for their future benefit because everything is conditioned on on success.
11:18:48 And so it's there's a lot of things where, obviously, if they can't be as you say I mean they can't be too bad along the way, but they could come in for some completely accidental reason there's nothing to do with these particular reactions they end up
11:19:00 for and so on. But it's always going to look as if they were doing something would select for the, for the future.
11:19:10 I suppose it depends on your perspective so I don't look to me like this.
11:19:16 were because they would be useful in the future.
11:19:20 When they did the flip side of that would be okay since, you know, natural selection will never and should never get rid of promiscuity right.
11:19:29 If biology actually can take advantage of this promiscuity. And if under some, you know, stress conditions and stresses not stress so it depends on what.
11:19:41 But let's say low subset concentrations and depending on the ecology of the organism to express more metabolic pathways which has a cost, which also has then as you worked out more promiscuous activity for an organism to get hold of any hydroxyl group
11:19:59 that can be oxidized to an energy age, which can go through the respiratory chain is success, even though there is no great pathway that we can draw on.
11:20:12 So the question was that.
11:20:15 Is that part of the biology of, you know, effects of stress response, actually a way how the organisms play with the intrinsic biology of promiscuity.
11:20:29 Hey, don't know so you know there are situations for okay so if you starve.
11:20:47 Eco lie, it will turn down a bunch of pathways and it will turn up a bunch of other pathways and turning up the bunch of other pathways is basically this desperate attempt to find something that will that will work and provide carbon but those are those
11:20:55 are like bonafide well evolved pathways. And the response is just to turn on the genes that encode those enzymes. The.
11:21:07 The promiscuous activities I mean I think it's just being this very large reservoir of catalytic potential that can be used when the environment changes, either to evolve a single new enzyme, or to evolve, new pathway.
11:21:29 Thanks Thanks Jerry, let me ask our questioning another way.
11:21:35 The so Alberto to us and the first tutorial that the all of the biochemical reactions in the cell that basically will how many kinds of 12 papaya, or something like that, how many well even take Yeah.
11:21:51 So, in principle, an organism can collect a setup at 12 enzyme.
11:21:58 Each, let's say, can do a little bit of those each type of reaction but not care about the side groups and things like that.
11:22:06 Is that does that happen. Is there such and time that we can identify it so generally just, you have an enzyme that will oxidize hydroxyl group that inside will often have promiscuous ability to catalyze oxidation of hydroxyl group in different substrates,
11:22:28 as long as they can fit into the active site.
11:22:31 So, promiscuous activities take advantage of the catalytic machinery that is there, they usually don't work as well because you can't position the substrate as nicely relative to the catalytic groups as the canonical substrate can be positioned.
11:22:54 but they will.
11:22:56 So all these promiscuous activities involve using the sort of generic catalytic machinery to catalyze either a very similar chemical transformation, or occasionally a transformation that looks quite different, but that can still use that same catalytic
11:23:15 machinery.
11:23:16 Right. So, what I'm asking is, are there, enzymes that, that sort of, let's say, go out of its way, not just select for side groups of pockets and things like that so so anything.
11:23:29 Are there. Okay, yes, there, there are there are some broad substrates specificity enzymes that have actually evolved to have brought substrate specificity.
11:23:42 So an example would be enzymes that are called Bluetooth on this transfer cases, which are detoxification enzymes in your liver.
11:23:51 And they have evolved to detoxify many many different things because your body can never anticipate what you might ingest.
11:24:01 So there are actually enzymes that have very capacious active sites that can handle many different kinds of substrates, and that's a very different thing though because in those circumstances, the enzymes actually evolved to have multiple physiological
11:24:20 roles.
11:24:21 Okay, and these promiscuous activities are physiologically irrelevant they're just accidents. Yeah. And, but they are very important in an evolutionary sense, but for organisms that the the a bacterium that we study equalizer the monarchs whatever I do
11:24:42 not know of example but could that be, why couldn't they collect a set of such and such a hard time. Okay, so there are, there are some okay so there are some fairly non specific images Amadeus and is that will release a means from chemicals.
11:25:07 But generally, so most metabolic enzymes are fairly specific and they've evolved to be that way because
11:25:20 it's easier to regulate metabolic pathways. So if you have specific enzyme for this function and this function.
11:25:30 You can turn, you can turn this one off when it's not needed and you can save the resources so most metabolic enzymes have evolved to be pretty specific for reasons of regulation, and efficiency.
11:25:48 Also, if you have an active site that is sort of broad specificity that means you can't bind the substrate in the perfect position relative to the catalytic groups and so you will compromise catalytic efficiency.
11:26:10 So as I've said that evolved from sort of multi specific to, I understand that the specificity rate and all that, the better you are working for towards a particular substrate.
11:26:29 Imagine the better the rate will be in everything right, and especially facility.
11:26:23 But just. But, but it, I wouldn't want to know it doesn't cost that much to carry a set of 1020 anthems around that, that's broad, broad head balls specificity and low rates
11:26:39 in concert.
11:26:42 Just one enzyme won't necessarily give you an advantage. So if you have a non specific enzymes that will help catalyze the transformation and a bunch of different things that may not get you to the point where you can either harvest energy, or carbon
11:26:59 or nitrogen out of it.
11:27:01 But so we have a, one for each of the trial reactions.
11:27:07 One.
11:27:10 That's an interesting idea.
11:27:13 So, I don't, it doesn't happen as far as I know, but it's an intriguing idea.
11:27:23 I would argue that it actually is important for directing metabolic flexes into certain process for example take an organism that grows on acetate whether acetate is useful for energy conservation or to produce membrane is a huge difference.
11:27:41 And, and this this the substrate specificity of the enzyme helps to fellow metabolic flexes then ultimately relevant for for for biosynthesis and assimilation of carbon into new biomass which is the ecological success for specificity right just that when
11:28:01 you run out of things, you have a new compound, you have some new compound but your normal things are not working then releasing a set of these kind of things might be useful.
11:28:12 And it comes at a huge cost. It comes at a huge cost if you don't want to express them no during normal time, but but in a strange way you're not replicating anyway why, why not, why not release them to see whether they can get something to happen, and
11:28:36 and.
11:28:29 Look, if you have a set of things like that. Maybe, maybe that would be kind of the know look for metabolism and that that that, something, something can start with you ultimately you need to find some pathways that sustained pathways.
11:28:43 Among the set of all compounds to to get you some way.
11:28:48 Yeah, I mean, the idea was that the, The Luca did have a small number of enzymes that had multiple functions. So I think, but what I think you can say from the, the effect of Super 8 billion years of evolution is that organisms have tended to evolve more
11:29:08 specific enzymes that are organized into nice metabolic pathways or networks that are regulated. So, even though it sounds like a cool idea biology doesn't seem to have gone that direction.
11:29:27 So there's actually apparently a discussion in the chatroom about, you know, expressing of Jesus, let's say terrorists universal genes of, you know, the 12 thousands and just throw everything in in in something we work that comes enough times of stress
11:29:44 at a huge ecological cost because protein synthesis is costly. And if it's unclear which proteins. I useful especially you know the level of proteins which is related to the flex.
11:30:20 I mean, It's different at the individual level the population level if different individuals in the population, express different ones, then it's not as not as costly, but I actually want wonder how if there were enzymes where one can make a case that
11:30:36 their origin might have been the wizard, new compound that was somewhat poisonous and to catalyze something which stops the bad effects of that first step which doesn't have to be useful for producing for producing anything.
11:30:49 And then you know later on that video becomes something part of the
11:30:56 catabolic pathway to get absolutely i mean antibiotic resistance is generally generally evolves by recruitment of a promiscuous enzyme that can detoxify in a single step.
11:31:12 The next nasty toxic compound. So the selective pressure for recruitment. A promiscuous enzyme and improvement of its of its enzymatic activity can be detoxification.
11:31:30 It can be harvesting energy can be accessing the new source of carbon and nitrogen, phosphorus, anything that makes the organism more fit.
11:31:42 And the detoxification can be one was a huge selective pressure as against doing some getting a little bit from some planning.
11:31:50 Yeah.
11:31:52 I wonder if you could comment on the fact that in anabolic reactions, right, you have enzymes that are extremely non specific like RNA polymerase is that do transcription and rhizomes that make proteins and DNA replication.
11:32:11 They work on anything. Right. So is there is there some meaning to this that when you putting copies together of yourself from the basic constituents, you do use very general purpose enzymes, but not when you're.
11:32:28 Yeah. So those are so DNA polymerase is an RNA polymerase is, and the ribosomes are things that have evolved to have sort of multiple specificities in the sense that, yeah DNA polymerase can recognize a CG or tea nucleotides.
11:32:54 And their active sites are essentially designed so that in the case of DNA polymerase, the specificity is provided by the strand of DNA that's been copied.
11:33:10 So it's not provided by the active site, it's actually provided by the, the nucleotides that are the basis that are present in the strand that's being copied.
11:33:21 And so that's also true for RNA polymerase is copy DNA to make RNA, and also for the right assume, so the specificity is provided by whatever the template is that is in the active site, rather than the active site itself.
11:33:42 Okay, but it doesn't happen in capitalism as far as I know, a template.
11:33:50 Kind of directed you could imagine Terry's 12, you know, general purpose enzymes that are matched with templates to decide when they're when they're supposed to act on what substrate, ya know the templates are all involved in DNA, RNA and protein synthesis.
11:34:10 Okay, so I think that it's probably time to take a break but let me just leave you with the, the thought that's going to launch the next part of the top.
11:34:21 Okay.
11:34:23 And that is.
11:34:26 So the patchwork hypothesis is supported by the evidence, but how does it actually happen. I showed you a couple of examples of pathways that seemed to have been patched together.
11:34:40 In this way, but what we're missing is information about the process. So, we might or might not have some information about what type of organism.
11:34:53 A new pathway originator Dan. So, if a pathways only found it man Rob's we can say yeah, that must have been involved in an anarchic environment but we often don't really know what kind of organism, and what kind of environment.
11:35:11 We're present when a new pathway involved. We don't know anything about what promiscuous activities were available in the proteome of that organism. And we don't know but mutations were involved in
11:35:29 allowing promiscuous activities to be recruited and then, increasing the flux of a pathway. And that's because we are sitting at the end of the process, looking back.
11:35:43 And so the way we go about this is to use laboratory evolution.
11:35:47 So, in the laboratory, we can specify the organism we know exactly what it is, we know what its genome is, we can specify the environment exactly because that's what we make up our growth medium today.
11:36:04 And we can evolve, something new, and then we can sequence the genome again at the end of the experiment and identify the mutations that were responsible.
11:36:17 And so that's the second part of the talk is going to be about the laboratory evolution of a new metabolic pathway, which then allows us to really get at the process rather than just the end result.
11:36:34 Well thanks so much for this this exciting first part of the tutorial jelly. And let's take a 10 minute break and get back to a quarter to the hour.
11:47:20 All right. I don't know whether I'll get through all of it but we'll see.
11:47:25 Okay, so what I'm going to tell you now is a story about the evolution of anonymous novel metabolic pathway. So this.
11:47:38 Most of what I'm going to tell you about, was published in this paper in 2019.
11:47:46 And the goal of this paper was to understand how a new metabolic pathway might evolve. But I have to take a step back first and lay the foundation with some experiments that we started.
11:48:02 Gosh close to 15, years ago. And at that time, we were actually asking a different question.
11:48:09 So at that time we were interested in trying to discover unexpected promiscuous activities.
11:48:17 And so we did a high throughput genome wide unbiased search, otherwise known as a fishing expedition to look for promiscuous activities that were unknown.
11:48:34 And we were doing this with a technique that's called multi copy suppression and I'll explain what both of those words me.
11:48:41 But the idea is that we started with. Strange, Nikolai in which genes had been deleted that were required for growth on glucose so this x means that a gene was deleted in the genome which is depicted by the orange circle.
11:49:00 So these cells could no longer on glucose, and we introduced into those cells, a plasma library in that had a collection of every metabolic enzyme that is normally encoded by Nicola except for the missing gene which would not have been very interesting.
11:49:20 And so we introduce that library into these deletion starting so this library is called the ESCA library.
11:49:29 So it's a complete set of eco like a 12 open reading frames cloned gene by gene into a plasma backbone and the plasmid one plasmid will be introduced into a cell but it will rapidly replicate up to maybe 250 copies so that's where the multi copy comes
11:49:52 in.
11:49:53 Okay, so we would introduce this library into these deletions strings, and then show you this each so just get one member of the library, each, each store gets one plasma.
11:50:05 Okay, and then that plasma will replicate to 250 copies, but they will all be expressing the same GB other cells will get a different plasmid. Right. Yeah.
11:50:19 So your population, you will end up with every possible plasma, that's in the library.
11:50:28 Okay, so then you you play these cells on selective plates. So in our case would be glucose as a sole carbon source and the parental strain can't grow, and you look for colonies the girl on the plate.
11:50:44 And then you isolate the plasmids and you sequence, this insert to discover what gene which what when overexpressed can restore growth of this deletion strip.
11:50:58 And the idea is that if a gene on the plasmid encodes an enzyme with an inefficient promiscuous activity that corresponds to the missing enzyme.
11:51:11 Then, if you express a huge amount of it. On this multi copy plasmid maybe there will be enough activity to restore growth.
11:51:20 And so this suppresses the mutant phenotype so that's why it's called suppression and multi copy comes from the fact that there are 250 to 300 copies of the plasma didn't sell.
11:51:35 Okay, so we did this experiment, so everybody okay with the idea of this experiment. So we're basically searching for inefficient promiscuous activities, this way.
11:51:47 So we did this experiment with a number of different deletion strengths. But the most interesting turned out to be a gene called or a protein called PDXB, which is Earth marinate for phosphate dehydrogenase.
11:52:05 And this enzyme oxidizes this alcohol to a carbon Neil. And that ends up transferring a hydrate to NAD to form NADH.
11:52:18 And I want to make a little note about jargon now.
11:52:21 So bacterial genes are designated by metallics, and they're in lowercase, the proteins that are encoded by the genes are capitalized, and not italicized.
11:52:34 So, the gene PDFs be lowercase italics encodes the enzyme PDF speak, which has the long name of originate for phosphate dehydrogenase.
11:52:46 So we're throwing like four or five baby hydrogenated is is much more descriptive. But this is another one of those shortcuts that bio interest in biochemistry use.
11:52:58 It gets tedious to say overthrown it for phosphate dehydrogenase for many times. So we typically just call the enzyme PDFs be.
11:53:09 Okay, so, PDFs be involved in the biosynthesis of PLP, which I introduced before.
11:53:18 It's vitamin D six.
11:53:21 And this is the pathway in E coli for synthesis of PLP.
11:53:28 And I only want to make two points on the slide. One is that PDFs be capitalizes the second reaction in this left branch of the pathway.
11:53:39 And the other is that equal I can take up paradoxes from the medium and convert it to pop by a salvage pathway. And this is important because what that means is that we can delete a gene that's involved in pop biosynthesis, and we can keep the cells alive
11:54:04 by giving them paradoxes so that they can make PLP by this in excuse me, the salvage pathway.
11:54:08 Okay so this allows us to do some genetic experiments.
11:54:15 Okay, so when we did this multi copy suppression experiment we were surprised to find that overexpression of any one of seven genes would restore growth of this delta PDFs be straight and equalize so delta PDFs babies that PDXB has been deleted.
11:54:35 So what I'm showing you here are pictures of auger plates on which we have strict bacteria.
11:54:45 The Delta PDFs be string.
11:54:49 Where, where we have overexpressed the genes in colors on a multi copy plasmid. So up here on the left is the positive control, if we express PDFs be on a plasmid after two days, we get robust growth on the plate.
11:55:08 So the plus two means we took the picture after two days, down here in the bottom right is the negative control. This is a vector a plasma, that doesn't have an insert.
11:55:20 And in that case, we don't see any growth on the plate. After seven days.
11:55:26 And then for each of the seven genes we over express these individual genes on a plasmid, we get robust growth, after as few as two days, or up to five days.
11:55:41 So this was a pretty astonishing result because a lot of people were doing this kind of experiment at the time. And typically people would find one or two or at most three genes that would replace or that would restore growth of a strain that lacked an
11:56:01 important gene.
11:56:03 And we'd found seven.
11:56:06 So, that was, that was pretty exciting, but it was also puzzling when we looked at what enzymes are actually encoded by these seven jeans.
11:56:17 So PDXV is a dehydrogenase, and you might expect that some other dehydrogenase the call I would have a promiscuous ability to catalyze the PDFs be reaction.
11:56:32 And among these seven enzymes one is actually dehydrogenase it's PDXA which is also involved in a different step in pop biosynthesis, but the rest are really odd collection of essays.
11:56:47 A kinase a dehydrator taste of predicted hydro hydro delays. And worst of all, a concert protein of unknown function.
11:56:57 And this just doesn't make sense.
11:57:01 I said, you might expected the hydrogen is to have a promiscuous activity to replace a dehydrogenase, but there's really no way that a dehydrator taste, which causes elimination of water, or a hydro lays which breaks bonds, is going to be able to catalyze
11:57:20 it the hydrogen nation reaction.
11:57:23 And in fact, none of these enzymes actually catalyze is the PXB reaction, even the dehydrogenase PDSA.
11:57:35 So in a sense the this experiment failed we thought we were looking for promiscuous activities that could replace PDFs be.
11:57:44 And we didn't find it easy but what we found was actually, I think, much more interesting.
11:57:50 And that is that by expressing any one of these seven enzymes, we were essentially rerouting metabolism and pulling material out of someplace else in the metabolic network and patching it through a new pathway, that's patched together from promiscuous
11:58:10 enzymes and patching it in downstream of the break in the broken pathway, and thereby reconstituting synthesis of PLP.
11:58:22 So we call these kinds of pathways serendipitous pathways to denote the sort of fortuitous patching together of promiscuous enzymes that normally serve other functions in the cell.
11:58:37 And this is actually metabolic innovation.
11:58:42 So this is evolution of a new metabolic pathway that we're talking about.
11:58:49 So we've.
11:58:53 A number of years ago we figured out the sequence of reactions involved in one of these so we call it serendipitous pathway, or SP one, and it pulls material out of the Syrian virus with this this pathway at the stage of three festival hydroxyl debate
11:59:10 and patches it through a series of four reactions, tying in to the PLP synthesis pathway downstream of the block caused by deletion of PDFs be.
11:59:24 So the first step in this pathway. As I said pause material out of searing biosynthesis.
11:59:33 And this is catalyzed by a protein called nude L, which was one of the enzymes that we found in our screen.
11:59:43 Nude ln codes of predicted co a power phosphor lies.
11:59:48 And this enzyme capitalizes the cleavage of a PO bond between two phosphate groups.
11:59:56 And in this serendipitous pathway it capitalizes cleavage of a PO bond between a phosphate remember that a circle around a p means phosphate, so cleavage between a phosphate and an organic molecule to form 300 oxy parfait.
12:00:17 The next step in the pathway was to our surprise actually non enzymatic. It is a dicker box relation reaction of three Hydrox you parfait to form glycolaldehyde.
12:00:33 And we looked long and hard for an enzyme that could do this, we found some enzymes that could do it but they obviously we're involved in vivo because we could delete the genes and coding those enzymes and the cells could still make PLP when one of these
12:00:56 enzymes was over expressed. The third step in the pathway is at what's called an alcohol condensation between glycolaldehyde and KYC to form for hijack see three me.
12:01:08 So, this part of the molecule here, came from glycolaldehyde, and this part came from glassy. Sorry.
12:01:18 And they've been condensed form for hijacks the three na.
12:01:22 This is catalyzed by an enzyme that is annotated as low specificity three any now delays or LTE for short, the physiological function of this enzyme is not known, but it is unlikely to be this reaction because for hijack see three and he is not a non
12:01:46 Nikolai and actually it's quite toxic. And the last step is catalyzed by three B, which is homocysteine kinase the name three becomes because it is involved in threatening biosynthesis kinases are enzymes that phosphor late substrates.
12:02:05 So, three be in the serendipitous pathway is false correlating this hydroxyl group.
12:02:11 So ATP is donating phosphate to this for hydroxyl group to form for fossil hydroxide 3d. And now we're back in the regular PLP synthesis pathway.
12:02:25 So, this serendipitous pathway requires three promiscuous activities, and one non enzymatic reactions.
12:02:33 Okay, and this obviously doesn't look like any standard metabolic pathway, these enzymes are involved in other functions and other pathways, they have no particular relationship with each other, but they happen to provide the promiscuous activities that
12:02:51 were required to catalyze the steps.
12:02:57 So now I want to explain why this pathway works if you over Express either nude L, or three be.
12:03:07 So if you offer Express New down, it's like opening the faucet and allowing material to flow into this pathway.
12:03:15 If you over Express three Be it pulls material through the pathway.
12:03:20 And that's because this alcohol condensation of glycolaldehyde and glycine is thermo dynamically a pill. So it favors the reverse direction.
12:03:31 glycolaldehyde is small and uncharged and can diffuse out of the cell.
12:03:37 But if you couple this uphill reaction with an irreversible downhill reaction. That is forced by the this extra chronic reaction of phosphorylation reaction from ATP, you essentially pulled material through the pathway.
12:04:00 So
12:04:03 are there any questions about this piece.
12:04:12 I'm just curious, what what did so you deleted you started in your equalize train with the PDFs a minute penalty.
12:04:22 So yeah, yeah.
12:04:25 So yeah, yeah. Can you explain that and what would have happened if you would have made a mutation or deletion in the catabolic gene of glucose utilization, as opposed to, co factor biosynthesis.
12:04:38 So,
12:04:41 that is a very interesting question. And you could imagine doing this kind of laboratory evolution. Experiment with after deleting anything.
12:04:54 I only know of two laboratory evolution experiments that have
12:05:01 evolved new metabolic pathways, one is the one I'm about to tell you about, and the other is one where a gene in branch chain amino acid biosynthesis was deleted.
12:05:15 But in, in practice, you could play this game with energy, you might not get anything.
12:05:25 But you might.
12:05:28 The differences sort of the flexes that you need to generate right. So, for co-factor biosynthesis and and deviation of flexes some amino acid to, let's say, co-factor biosynthesis is relatively modest rerouting as opposed to catabolic rerouting.
12:05:47 Yeah, that's right. So, I think that the fact that, so I'm going to show you that we managed to involve a new pathway and. In, only about 100 generations and I think the reason is that it was, it's a relatively low flux flux pathway.
12:06:06 So, the bar is much higher.
12:06:09 If you're making an amino acid or you're doing sugar metabolism.
12:06:15 So I think, you know, we were lucky in the fact that we ended up working with a low flex pathway.
12:06:23 But, you know, to tell you the truth, we didn't start out at all with the intention of. Let's evolve a new metabolic pathway and let's pick a low flux pathway.
12:06:35 We sort of stumbled into it, based on this early experiment where we were just looking for promiscuous activities. So we didn't start off by sort of intelligently saying the PLP synthesis pathway would be a good way to do this.
12:06:55 It also shows I think nicely that in this case, it was easier to to suppress the mutation by rerouting metabolism, rather than by sort of duplicating and the end the last enzyme activity which I think is really interesting lesson on the plasticity of
12:07:14 flexes and pathways.
12:07:17 Yeah, and it's also a testament to the fact that you can actually recruit multiple promiscuous activities and patch them together. Yeah, yeah,
12:07:31 yeah, I have a question about the experiments so the you play them on glucose, play, but before glucose, where they enrich medium.
12:07:52 So we grew them in glucose with paradox in present in the day, they could they could grow within the wash them very carefully to remove all the paradoxes so that if something early all prepared of everything except for production.
12:08:05 Yeah.
12:08:07 Okay, so I've been telling you about a serendipitous pathway that was facilitated by either over expressing three B or nude L, that there are five more genes in this picture, and by genetic means we know that there are at least two other serendipitous
12:08:26 pathways that are facilitated by overexpression have any one of these five.
12:08:35 But we decided not to go after those other two pathways, because at that point our question and changed. So remember our initial question which is can we find promiscuous activities that replace the missing enzyme.
12:08:50 But our question at this point had changed to, can we get equalized to evolve a novel metabolic pathway.
12:09:02 And this multi copy suppression technique is really not suited very well for addressing that question because I mentioned that these plasmids will replicate to a level of 250 or 300 in the cell.
12:09:21 And so using that kind of the system. You can express much higher quantities of an enzyme than you could ever get by a more physiological mutation, like a promoter mutation that just increases gene expression or gene duplication or amplification, to be
12:09:42 quantitative um if you took the strongest promoter in the cell and put it in front of the gene, how big a factor would you get, compared to this plasma plasma factor.
12:09:52 But that is a good question, um, you know there's sort of an upper limit to the amount of any given protein that you can make even with a strong promoter.
12:10:20 So, you know, I'd say maybe you can increase expression by thousand, so you can show it similar similar to what you're doing by putting in the know.
12:10:20 Motor over as well. Yeah, if you have both of those. You can basically make bucket loads of protein, but your plasma has a strong promoter on it so the amount.
12:10:30 Yeah, so you're just trying to max out the amount of protein that you can make. Yeah, and that's going to be just much more than you could ever achieve by, say a mutation in the genome.
12:10:43 But I guess I don't want a single mutation the genome. I mean, these big populations, you can move around the whole promoter and he's not that rare. Other thing to have a promoter moved.
12:10:52 Oh yeah I just said, I just want to do it by moving, moving a very strong promoter, is that, I mean how what fraction of what you're getting here with that could that do in principle, I'm just with a promoter mutation.
12:11:10 So, a promoter mutation cricket, I don't want them to promote a rotation I want to move a promoter motor. Okay, yeah. If you make a promoter very strong.
12:11:17 Yeah, you can.
12:11:20 You could in theory, elevate the level of an enzyme by, you know, 1000 fold if it wasn't being expressed to a very high level. Anyway, but you're never going to be able to make as much as you can, off of a multi copy plasma, with a strong promoter.
12:11:39 If you're so even if you had this the same strong promoter on the plasmid, and in the genome, you'd be 300 fold different
12:11:50 make sense.
12:11:52 Just because you've tried cricket plasmids already. They have a very strong promoter on them and they have a very strong promoter on the plasmid, and you've got 300 copies of the gene.
12:12:02 Okay.
12:12:03 I just looked at the abundance of note, l and three be equal right growing on glucose in our stream. So, to be there's already 1000, copies. And so, so you cannot push that the entire cycle on a billion proteins, but not l that only like 1020 little very
12:12:25 very low number of companies, and so predominant cosmic can give it a big boost.
12:12:32 Okay, but bottom line is that this is not a very physiological thing to do. So we decided that we would like to try laboratory evolution.
12:12:42 So, you know, typical laboratory evolution experiment would involve putting your strain into a culture, in our case with glucose as a sole carbon source growing it up, and then doing serial dilutions over and over and over until you evolved to the point
12:13:01 where you're happy, or your postboxes, I'm not going to do this anymore.
12:13:07 But this is sort of a problematic experiment in this case because I told you that PDFs be catalyzed is this reaction in the synthesis of PLP, which is an essential co factor.
12:13:26 So if you delete PXB you wouldn't expect the cells to grow. And it is a fact that dead cells don't evolve.
12:13:31 So this experiment should never have worked but fortunately, my postdoc decided to give it a try anyway.
12:13:42 And, interestingly, when he put the cells into glucose, as a sole carbon source. After about 10 or 11 days. He actually saw a little bit of timidity, which meant that something was growing.
12:13:56 Now we don't know whether they were growing because there were some very very low efficiency the key serendipitous pathway that allowed them to make just a little bit of PLP, or maybe there was already in the population, a mutant that had taken a step
12:14:14 toward evolving a new population. But either way, once you have cells that grow, then you can evolve them.
12:14:22 And so, my postdoc carried out this serial dilution experiment for between 110 and 150 generations. In, 11 replicant lineages.
12:14:36 And at the end of that time, he had evolved.
12:14:42 Several strains that grew almost as well as well take the call I. So what I'm showing you here, our growth curves on the y axis is the od 600, which is a measure of the ability of the culture, which is proportional to the number of cells in the culture.
12:14:59 And this is as a function of time.
12:15:23 The wild type equal ly which means the standard non mutant straight is shown in black. So, it, it grows very well.
12:15:17 And it kind of plateaus after about a day.
12:15:23 And in colors are 11 different clones from the end of the experiment. And you can see that the lag phase.
12:15:32 Before the cell stop growing varies among these lineages, but once they get growing, they grow.
12:15:40 Almost as well as well tied up to 60% of the growth rate of the wild type streams.
12:15:48 And so these strains, I should mention, we call them JK one two JK 10 and the JK comes from 200 Kim did these experiments.
12:16:00 So nothing mysterious about the name, they're just do one strings.
12:16:04 Okay, so this was pretty astonishing because the you know the initial culture took 10 days to reach a little bit of timidity. And at the end of 110 250 generations.
12:16:18 We have strays that have obviously reconstitute PLP synthesis pretty well. So the two questions that were interested in asking are, how are they making PLP, and then how did mutations improve their ability to make PLP.
12:16:36 Okay, so most of the work I'm going to talk about now was with this one strain JK one which is the red curve so it was sort of a. average performing strain
12:16:51 and JK one has completely reconstituted its ability to make PLP so the level of PLP in JK one is comparable to the level in the wild type stream.
12:17:07 So we first asked as JK one use this previously identified serendipitous pathway one.
12:17:14 And so we went about this by deleting genes involved in SP one to see whether this, the cells could still grow.
12:17:24 So we deleted LTE in the cells could still grow.
12:17:29 So that means LTA is not required. So, the cells are not using SP one.
12:17:37 But we found that three B is required. So if we delete three be the cells won't grow even if we supply threatening in the medium to compensate for loss of three B's normal function in three any biosynthesis.
12:17:56 So that suggests the possibility that there is some other way of making this for hijack c three any intermediate.
12:18:06 Okay, so I covered reams of papers with possible ways to make for hijacks the 3d using the 12 canonical types of enzymatic reactions that you typically see in sales.
12:18:23 And eventually, an ad, and I would bring them into the lab and I showed him Did you hon and he taking heat.
12:18:29 And so finally I came up with one that sounded plausible enough that he was interested in testing it.
12:18:37 Okay. And so this is going to be SP four.
12:18:40 So SP two and three remember where the two that we decided not to pursue.
12:18:46 So this is sp four.
12:18:59 Okay, so I'm going to blow up this part of the pathway.
12:18:57 And the first reaction.
12:19:01 In this proposed pathway is the cleavage of the phosphate off of this hydroxyl group on for phosphor thousand eight to give a resonate.
12:19:12 And this would be catalyzed by an enzyme called a fast pace so enzyme names, always end in a race, so fast fatigue is an enzyme that takes off a phosphate.
12:19:27 The next step would be a dehydrogenase, which would catalyze the oxidation of this alcohol to a carbon Neil.
12:19:38 And then the third step would be a trans emanate, which would convert this alpha keto acid to an alpha amino acid. OK, so the acid group is this carb oxalic here in the alpha position is a keto ketone, so this is an alpha keto acid.
12:19:59 And this is an alpha amino acid. And then at that point we would be back in familiar territory. We already know that three be can catalyze this reaction which generates for fossil hijacks the three any downstream of the break in the PLP sentences pathway.
12:20:17 Okay, so this is a, this is an appealing pathway because it involves only four steps, one of which we'd already shown works. And all of these are very common enzymatic reactions in sucks.
12:20:34 So we set out to identify which enzymes were responsible for each of these steps, and we hit a brick wall right away.
12:20:43 So there are two enzymes in the literature that had been reported to catalyze this reaction.
12:20:51 But they are not involved. Well, if we deleted both of those genes, either individually or together.
12:21:02 The cells could still grow.
12:21:04 So that suggests that either those enzymes are not involved in vivo, or maybe they are involved but there are multiple phosphate cases that can do this reaction.
12:21:23 And it turns out that he cola has about 40 small molecule foster cases that might have this promiscuous ability to do this. And there was just no way we're going to knock out 40 G's to test them, especially since we might have had to double knockouts
12:21:35 and triple knockouts.
12:21:37 And so that was crazy. So we're just going to say that these, this is either an unknown FOSS potatoes or a collection of hospitalizes that will do this reaction.
12:21:52 So we had better luck with the next step because we had a good candidate for this reaction and this is an enzyme called sere a.
12:22:01 So sirree is involved in Syrian biosynthesis it capitalizes The first step in the biosynthesis of Siri, and it pulls three phosphor Glister eight out of glycol assist as the progenitor for this pathway.
12:22:18 The long name for series three hospital grocery dehydrogenase.
12:22:24 And we were interested in sirree because we had seen mutations in Syria in multiple of these involved strengths.
12:22:32 So this is a list of the genes in which mutations were found in each of our evolved strains.
12:22:41 And we'd seen mutations in sirree in four of them.
12:22:45 So there was a whiff of something going on with Sarah.
12:22:49 Sarah also catalyst is the right kind of chemistry. So, in the pathway for Syrian biosynthesis it oxidizes an alcohol, that's alpha to occur box relate to a carbon Neil.
12:23:05 And that is exactly the same kind of chemistry that's involved in this postulated SP for oxidation of this alcohol to a carbon Neil offer to a box like okay so it was a good candidate.
12:23:19 And in fact sirree can catalyze this reaction has a promiscuous ability to catalyze this proposed reaction. So, k cat over km for this reaction in Syria by synthesis is 1.6 times 10 to the third promoter per second, which is a pretty average kick out
12:23:39 over km for metabolic enzymes
12:23:44 and sirree will catalyze this reaction kick out over km is only point 07 promoter per second, which is kind of pathetic, compared to this but remember that I told you that even inefficient promiscuous reactions or enzymes can catalyze reactions by orders
12:24:03 of magnitude, relative to a non capitalized reaction.
12:24:08 Okay, so we knew that Siri could do this reaction in vitro, but the critical question is does it do it in vivo, because not everything you can measure in a test tube actually happens inside cells.
12:24:21 So to address that question, we deleted Cirie ANRJK one stone. And we asked him to grow.
12:24:31 So this is another one of these growth curves, as a function of time.
12:24:37 And these are her bubbles.
12:24:41 Sorry about that, but bubbles happen.
12:24:45 Essentially, this strange doesn't grow.
12:24:48 But of course it doesn't grow because it can't make Sarah.
12:24:52 So if we add Siri.
12:24:55 It's still can't grow. And notice that this is a log scale so this isn't very very low D so essentially the cells, can't grow.
12:25:08 But if we add both Syrian and pure Doxey so remember that paradox he can be converted to PLP by salvage pathway.
12:25:17 Then the cells can grow. So what this is telling us is that sirree is required for synthesis of both Siri, and paradox.
12:25:27 Okay so that is good evidence that Cirie a is actually serving a function in PLP synthesis in the cells.
12:25:41 Okay, so the next question was, well what enzyme is catalyzing this trans emanation reaction. And we had a very good candidate in CRC, which is also involved in Syria biosynthesis so seriously is actually a by functional enzyme, it's involved in serious
12:26:00 synthesis, but also PLP synthesis. So in PLP synthesis. This is on catalyze is a trans emanation reaction, in which an alpha keto acid is converted into an alpha amino acid.
12:26:16 And that's exactly the chemistry, we propose an sp for trans emanation of an alpha keto acid to an alpha amino acid.
12:26:29 And, interestingly, the only difference between the what the native substrate which is the normal substrate and the substrate and SP four is a phosphate group on this hydroxyl group, so it's missing over here.
12:26:44 So it capitalizes the right kind of chemistry on a structured way related molecule.
12:26:52 And in fact CRC can catalyze this chemistry.
12:26:55 So we have to actually measure k cat over km in this case in the reverse direction because the substrates for the forward reaction or not commercially available.
12:27:09 But if an enzyme capitalizes a reaction in the forward, or in the reverse direction, it will certainly catalyze it in the forward direction.
12:27:19 Okay so k cat over km for the native reaction 727 per dollar per second. Whoops.
12:27:31 And for the permissiveness reaction is point 007 promoter per second. Again, pathetic, but probably orders of magnitude better than nothing.
12:27:40 Okay and then at that point we're back in familiar territory we know 3d can do this reaction.
12:27:50 Okay, so there are any questions on that piece.
12:28:00 Okay.
12:28:03 Okay, so then, now that we've figured out how they're making PLP, we can ask how do mutations improve the synthesis of pop so now we're really getting at the process.
12:28:17 Okay, so I showed you this before, these are the genes in which mutations were found in the evolved strains remember that genes are denoted by lowercase metallics.
12:28:30 And this is kind of a scary prospect, because there are a lot of jeans and a lot of mutations. And so how are we going to zero in on what really matters.
12:28:43 Okay, so the way we zero in is that we look for genes that were affected by mutations in multiple evolved strains.
12:28:52 There are about 4200 genes and E. coli, and there.
12:28:57 If each one is randomly hit you wouldn't expect to see a mutation crop up in more than one strain out of 11.
12:29:07 But we have mutations in the same gene cropping up in multiple evolved lineages, which is a strong sign that these mutations are actually beneficial.
12:29:20 Okay, so I'm going to talk about the mutations in YBHAPTL Cafe and Syrah.
12:29:29 So first the mutation why BHA.
12:29:34 So, the mutations in YBHA.
12:29:41 Obviously cause loss of function. They were either deletions of all of the gene, or a premature stop code on that would truncate synthesis of the protein, or a frame shift mutation that would disrupt the reading frame and make garbage out of the rest
12:29:58 of the protein. So, clearly all of these mutations cause loss of function in it's easy to see why.
12:30:05 Why BHA is a sort of broad substrate specificity foster taste but one of its favorite substrates is PRP.
12:30:16 So make sense that if the cells are having trouble making PLP that deleting an enzyme that destroys PLP would be a good thing.
12:30:27 But that doesn't tell us how mutations are helping the cells, make PLP, which is the more interesting question.
12:30:36 Ok so now I'm going to talk about the gabay in PG on mutations together because they contribute to the same thing.
12:30:44 Okay, so the mutations in gappy decrease the catalytic efficiency of the enzyme. So, the activity of the enzyme in cell extracts is depleted by 80% in JK one relative to wild type cells.
12:31:05 Okay, so the mutations in YBHJ, obviously cause loss of function because they're deletions of all are part of the gene.
12:31:16 Okay, so now if you are confused about why mutations in PGL and gap a might help the cells make PLP, you should be.
12:31:26 Because neither PGL nor gabay is involved in this novel metabolic pathway.
12:31:34 So I'm going to give you the punch line first and then I'm explain how we got there.
12:31:41 So, turns out that together these two mutations, increase the ability of sirree.
12:31:48 To perform this newly needed reaction.
12:31:52 Okay, so how did they do that.
12:31:55 Alright, Cafe is an enzyme in glycol assist by zoomed in on the sort of upper part of glycol assist in this figure, Cafe capitalizes this reaction oxidation of an hour to hide to what's called an ACL phosphate that generates NADH.
12:32:18 So, offers talk some about how glycol Genesis is used to generate in a dh.
12:32:26 Okay, so that's what cafe does a mutation that knocks gabay activity down by 80% is going to cause a bottleneck in the pathway, so that the flux through the lower part of the pathway is going to be diminished.
12:32:46 Okay, so that's what the gap a mutation does.
12:32:49 Now let's look at PGL. Okay, the long name for PGL is six boss boo boo Kono lactamase it capitalizes this reaction over here, opening up this six member drain.
12:33:05 And PGL is the second enzyme in the upper part of the penthouse phosphate pathway.
12:33:10 Okay, which is shown in the blue cloud. And this is kind of a messy network of reactions.
12:33:19 The whole purpose of the pencils phosphate pathway is to make two things. One is right both five phosphate, which is required for synthesis of nucleotides, including ATP and the nucleotides that make RNA and DNA.
12:33:38 The other is Earth rose for phosphate, which actually is a Tetris, not a Pinto's soda for carbon sugar. Earth rose for phosphate is the precursor of all the aromatic amino acids, and also the precursor of PLP.
12:33:55 So, I'm going to simplify this messy pathway.
12:34:01 Like this.
12:34:02 Okay, so the penthouse phosphate pathway.
12:34:06 The key is that it's making these two critical intermediates.
12:34:12 Okay, so if you normal under normal circumstances, about 24% of the glucose that comes into the cell is diverted into the Pintos phosphate pathway to make rebels five phosphate and northwest for phosphate.
12:34:27 But if PGL is lost this upper branch of the pathway is no longer functional but all is not lost because these intermediate can be synthesized by pushing up material from the bottom.
12:34:47 And this requires glycerol to high three phosphate intermediate in in the glycolic pathway.
12:34:54 So, in the absence of PGL closer alto high three phosphate has to be diverted from college, to make these critical intermediates.
12:35:08 Ok so now if we combine the effects of those two mutations.
12:35:14 What we're doing is, we, I told you before that, the 80% reduction in gap activity, cause it decreases the flux through the lower part of glycol assists.
12:35:27 And then, by diverting glycerol to high three phosphate into the Pintos phosphate pathway. It's going to diminish flux, even further. So now we have lower levels of glycerol the high three phosphate because it's being diverted, and we have a very poor
12:35:44 enzyme. So material is just going to trickle through the bottom part of glycol Genesis
12:35:51 and evidence of that is that we can't even detect this downstream intermediate three possible glycerine in strange JK one.
12:36:03 So this is a result of metabolism x, we can easily detect three fastball cluster rate in wild type cells but we can't detect it in JK one. So, this indicates that there is much diminished flux in the lower part of like Hollis.
12:36:22 Okay now I'm zeroing in on three festival glycerine for a specific reason. And that is the three festival glossary is the native substrate for CRA, which is serving a new function and SP four.
12:36:37 So, if a single enzyme is catalyzing two different reactions. Each substrate is competitive inhibitor of the other reaction because they're competing for access to the active site, and three fossil glycerin, the native substrate from the enzyme will be
12:36:54 a very, very powerful inhibitor of this newly needed reaction. So by depleting three festival glass rate in the cells, it provides better access for Earth donate for the active side of sirree and will increase flux in this new SP for pathway.
12:37:20 But there's actually more to the story than that. So I mentioned that three phosphor Glister rate is the precursor for the series about a synthesis pathway and you might expect that if three phosphor glycerin is in low supply to sales might be making
12:37:37 less serious than normal.
12:37:39 Okay. And in fact, that's true.
12:37:45 In JK one series is undetectable, even though it is easily detectable and wild type cells.
12:37:49 So why might that matter.
12:37:53 Well, remember that the first step in the pathway for Syrian biosynthesis is catalyzed by Siri, and Siri is subject to feedback inhibition by Siri and so this is a common feature in bio synthetic pathways that the end product of the pathway will feed
12:38:12 back and inhibit the first enzyme because if there's enough of the end product.
12:38:18 The cells want to shut off flux through the pathway to avoid expanding resources on something that's no longer needed.
12:38:27 Okay. So why does this, how does this connect to PLP synthesis.
12:38:33 Okay well remember that sirree is involved in, as before.
12:38:41 So, the combined effect of the PGL mutation in the gap a mutation is to diminish the concentration three possible cluster right and the concentration of Siri.
12:39:02 And what that does is it increases the ability of Siri to perform its new function in s before by decreasing inhibition by the native substrate.
12:39:07 And by decreasing feedback. In addition, by Siri.
12:39:13 Okay now I just want to quickly mention the mutations in sirree, which we observed in four different evolved strains, although not in JK one.
12:39:25 And it turns out that the mutations in sirree do one of two things. They either decrease the feedback, the bit inhibition by Siri, which will mix your a more available for this new function.
12:39:40 For they actually slightly improve the ability of Siri to do this new reaction.
12:39:47 Okay, so I've been talking about Siri a lot, everything seems to be pointing to Siri. And so what it looks like. is that improving the ability of sirree to catalyze this reaction, seems to be the key to making this whole thing work.
12:40:07 And in fact, we've come up with seven different ways that the function of Siri in the new pathway could be improved. So I'm showing this schematic of series normal function.
12:40:24 The new function and the feedback inhibition by Siri.
12:40:31 So, we could improve the ability of Siri to catalyze this reaction by decreasing the concentration of the native substrate for the enzyme.
12:40:40 And we saw that in strange AK one, we could improve the accessibility of the ability of sirree to catalyze the new reaction by decreasing the concentration of Siri, and therefore diminishing the feedback inhibition, which will affect both activities.
12:41:00 And we saw that also NGK one.
12:41:05 We could in theory, increase the concentration of Earth reunite, which would help push material through the pathway.
12:41:12 We did, didn't see that but we only did metabolize comics on one string because it's really expensive.
12:41:25 And actually, it's our Will you heard from was our collaborative, on, on the table. Next, We could diminish the ability of searing to inhibit sirree.
12:41:32 We saw that in three evolved strengths.
12:41:36 We could in theory decrease the affinity of sirree for its native substrate which would decrease its ability to competitively inhibit sirree.
12:41:49 We didn't see that.
12:41:53 We could increase the efficiency of this newly needed reaction, and we did see that in starting JK sex, and in theory we could also increase the amount of the inside of itself, and we didn't see that but again we only in j k one.
12:42:11 Okay, so I'm going to wrap this up with the really key points. One is that multiple novel pathways can be patched together using promiscuous activities in the podium.
12:42:23 So, this is evidence for this patchwork model of evolution of new metabolic pathways.
12:42:34 One of the most important findings, was that mutations that elevate flux to a novel pathway, need not occur in genes encoding enzymes in the pathway. So this is the list of genes in which we found mutations.
12:42:50 For only for strange acquired mutations in a gene encoding an enzyme in SP four and that was sirree, none of the other enzymes acquired mutations.
12:43:06 And I think this was, you know, perhaps the most unexpected finding, at least, in retrospect I think it makes sense but at the time we were pretty surprised.
12:43:20 A third important lesson is that even very inefficient promiscuous activities may be sufficient to launch a new pathway. And even if the flux through this pathway is very low to begin with.
12:43:34 This can provide the starting point for evolution of a higher flux really bonafide standard metabolic pathway.
12:43:44 And the same phenotypic result that is improving the ability of sirree to function in this new capacity, can be achieved in multiple ways, which means it's more likely to happen.
12:44:01 Okay so finally just circling back to the whole idea of the patchwork hypothesis.
12:44:06 I think what this work has shown is that yes it can be fairly easy to recruit promiscuous enzymes from various places in the metabolic network and patch them together to form a multi step metabolic pathway, and that the promiscuous or that the flux through
12:44:27 this pathway can be improved in a number of ways.
12:44:33 Some of the most important seem to be just improving the ability of each enzyme to catalyze the new immediate reaction indirectly by decreasing inhibition by other metabolites in the network.
12:44:50 Ultimately, you would expect.
12:45:02 Gene duplication and divergence to improve the efficiencies of these enzymes. All right, and that is the story.
12:45:07 This was started by 200 Kim, who is forever memorialized in the names of the strange.
12:45:14 When you have left the lab, it was taken over by two graduate students, Jake, and Andrew who have since graduated with help from serious and Michael who were undergrads.
12:45:24 And we had helped with proteomics from the lab of will old at CU Boulder, and the tableau mix with the sour from eth, and with high throughput sequencing from Vancouver at University of Pittsburgh, in this work was funded by the NIH and by NASA.
12:45:45 Okay, that's, that's the story.
12:45:49 Awesome.
12:45:52 Thank you so much. This was awesome. This was an amazing good amazingly beautiful story, and I really liked how you DD convoluted sort of the complex metabolism and evolution of metabolic pathways.
12:46:07 Clearly within the principles of the enzyme ology and the enzymes and the chemistry that involved, so it's it's a beautiful ways sort of to demystify evolution of pathways and clearly sort of grounded in basic you know chemistry and biochemistry, so thank
12:46:25 thanks so much. This was awesome. So why don't we open the talk now for for general questions.
12:46:38 Because I have one which maybe is more of a comment but there was a few slides back that was a seven at the top of it.
12:46:53 And as far as I understood the number of different ways that you'd found that something happened. There is no there was no, it was it seven earlier, maybe.
12:47:04 So, I went through seven different possible ways we can think of this happening. We found four of them, we didn't find the other three okay so but we only really looked hard in one strength.
12:47:21 So, yeah, I think that the other three could easily occur. Yeah. So I think this is just, I think one of the things which somehow isn't emphasized enough in discussions of evolution is not that there are very many ways to do better, but even to get better
12:47:39 in some particular way which is what you're looking for here, there were very many different ways of doing it. Each one of them may be improbable, but there's so many possibilities that some of them happen.
12:47:51 Yes, and the populations are huge, and the population is huge, but if you start comparing what can happen in. In, you know populations outside the lab.
12:48:01 Right, so then the process by which you would have some mutation occurring and moving a strong promoter in the same in the same cell division is not that improbable like it's quite it'll happen all the time.
12:48:14 And so things which really sort of bootstrap the, um, what can happen in the, in the lab by very big factors to be able to get two things to happen at once, even if some deleterious.
12:48:25 They'll serious intermediaries. So I think, oh you know I doubt if both of those things would really happen in the same cell division, because I mean if you just take all the cola in the Bay Area, something like that, in people's guts you know that's
12:48:40 a sort of thing that's plausible than the same to get in the same.
12:48:45 Although it's just very very big numbers, and with the pressure with the bacteria so some of the sort of, you know, combining processes that one can see either directly in the lab like these ones or ones that you forced in the lab like during the very
12:48:57 high expression that you know in nature is going to manage to combine those, and that's and that's to do a particular task but of course the nature may be you know he's going to do some different task and do better by doing that so I think in some ways
12:49:11 this the sort of thinking how rare things be all the, all these points awards Geez, you know, Rob, seemingly amazing things, not, not all that rare by standards of the awareness that evolution can can manage.
12:49:25 Yeah, so we've had 3.8 billion years to experiment with vast, vast populations of microbes. Yeah. Yeah.
12:49:35 And I'll be interesting happens.
12:49:40 why I find remarkable that all these distribute types he gets all by loss of function mutations. Yes, there is another game of a strong promoter, or any new activity it's all about loss of function.
12:49:53 We did get one. So we got one mutation that increase the efficiency of this promiscuous reaction that that you can consider a gain of function mutation, but most of them are loss of function mutations.
12:50:08 And this makes sense because loss of function is so much easier than gain a function. There are so many more ways to destroy or diminish an activity, than there are to improve in it to me.
12:50:23 But this brings up a very interesting problem for evolution, which is, if the easiest thing to do is to destroy a function. So we call these expedient mutations, because they're solving the immediate problem better at a cost to a presumably well evolved
12:50:47 function. And this does not seem to be how evolution should happen in the long term. So we actually have a new grant from NASA to address this question of Okay, if this is the way, you know, evolution.
12:51:04 Initially happens, how good life and actually ever gotten anywhere.
12:51:08 So we're going to be addressing this question of how do you either avoid like mutations or how do you repair them.
12:51:20 After a new function is sufficiently well evolved.
12:51:25 Other way is sort of how do we get this you know doesn't 12 or 15 prototype reactions, for which you know everything is evolutionary progress from that could be all by a loss of function.
12:51:41 in a sense in terms of metabolic sense right.
12:51:43 Yeah, but then you know eventually you have to go back and fix things and we've seen, I know. Another system we saw the winner in an evolutionary experiment had deleted.
12:51:56 About 100 genes.
12:51:58 Okay, that's really a bad idea because now if those cells, end up in a different environment, they're just gonna die. Yeah.
12:52:10 It means is one of them to get some of the genes transferred back from from someone else and again the big numbers one.
12:52:19 A lot
12:52:19 of the hope for monster version of evolutionary stories, I'm rich small, is that something to skate scraped by in the conditions which you're in, you do something which is otherwise bad.
12:52:35 and then with all the sort of recovery from that from the pollution which may will be by sort of small, small steps, but it's it's some it's very hard to disentangle that after the fact is the order things, things
12:52:47 that I told you about the, The, the most abundant strain had thrown out 100 genes, but there was another lineage that hadn't thrown out 100 genes and it wasn't quite as prevalent.
12:53:02 But if the environment changed. Yeah, the guy that throughout the hundred and 10 would have lost out, and the guy that was a little less fit would have become more fit so you know that's another beauty of these laboratory evolution experiments is that
12:53:19 you can see the dynamics of the competing clones in the population and then think about, you know, why you see the dynamics that you do and what would be the implications of the mutations.
12:53:39 If the environment, changed and those functions really became critical again.
12:53:46 So I'm wondering actually that's what quitting for Terry. I was intrigued by the gap a phenotype, which was sort of reduced flex through blood I guess I'll have to force it would you expect that this mutant still has an acetate overflow metabolism.
12:54:05 Because you restrict the flow through like calluses within energy conservation and therefore the whole idea about flux of ATP, so that you don't predict that the mutant would be more fit to have a complete oxidation of whatever acetate makes it through
12:54:24 this pathway, so I'm wondering, do you have any thoughts on that guy. You're, you're basically saying this Muta makes a good costume.
12:54:36 Right. Yes, yes. Less advantage just to lean back colleges vs DC.
12:54:41 So, the, actually I was curious especially. So in your midterm, was this one. Well, one fits gap play was like one fifth of the activity.
12:54:55 How fast compared to water
12:54:59 it actually did reasonably well so to go back to the slide the growth curves
12:55:09 in mind that red curve in the middle right. Well, yeah, it's not similar. it's hard to read up what what what
12:55:19 record my.
12:55:21 Yeah, here we go.
12:55:25 Okay, so it's the red curve. Right.
12:55:29 So, it grew, maybe half the rate of the wild type and interesting thing about the loss of function mutations, is that, yeah we saw, mostly loss of function mutations but you can't completely lose cafe function because then you would have no glycol Genesis.
12:55:48 So, the mutation and gabay had to be a diminution of function, not a complete loss of function.
12:55:59 And that makes it harder to find just the right point mutation right right pocket down enough much.
12:56:21 So, alpha, just by slowing down growth that much me that that stay ready to be on the, on the other side of the acetate. Okay. Yeah, yeah, yeah. I'm Shelly.
12:56:25 I have a question and I think there's a question in the chat room is similar nature so the question in the chat room is, how do you know this this new metabolic pathway on, you know, the patch of pathway is not a older version of some, some relatives.
12:56:47 and and the way I will ask this was the clearly there's a striking parallel between the way, so the searing is a made from a trigger so fastly, and the way PLP is made, and that in fact Searcy is already used by Nicola footballs purposes.
12:57:09 Right, so, so maybe in some other organism asteroid also have been used for both, or no so so what do we know about the, the evolutionary relatedness of of sure a and, and, and actually, the bands that that's performing the, the job.
12:57:37 The, yeah. Yeah, so the question of whether this is a relic. So there's actually two parts to that question is, if this is a relic of some previous pathway.
12:57:48 You know, I don't think so because three B's involved in three any biosynthesis and these are involved in Syria biosynthesis, and this is probably two or three enzymes, do other things.
12:58:03 So it really does seem to be patched together from enzymes from different places in the metabolic network. It happens the two of them come from Syrian biosynthesis, but I think this is definitely an example of patching together promiscuous activities
12:58:19 that happen to generate this pathway.
12:58:24 The question of whether this kind of pathway occurs in nature.
12:58:30 The, there were only two pathways that are known in all organisms that have been studied so far to the ancestor one that I told you with last in the ancestor of some proteome bacteria, and then the pathway that I showed you is, that's presently co lie.
12:58:52 That doesn't mean that there aren't other pathways.
12:58:58 And we're actually, we're trying some experiments in other bacteria where we delete PDFs be to see what we can evolve in other bacteria that have might have different collections of promiscuous activities, and at least one case, we're running up against
12:59:19 the enzymes that we expect to be involved in PLP synthesis, don't seem to be. And so, we haven't figured out what's going on in those cells.
12:59:32 Maybe it is a variant of one of these pathways that isn't known yet So the short answer is, there are two pathways that are known, but we don't know everything right so there may actually be there could be something out there in nature that looks more
12:59:51 like this.
12:59:53 Yeah, I guess will be difficult to tell but in principle, there could be a number of organism were not what he called us associate football so the second reaction.
13:00:06 But the cookies organism out there they use the same story for both searing sentences and for PRP production, right there.
13:00:23 Yeah, and that that's one thing versus yeah acting might be true at all. it's actually a by functional enzyme that does
13:00:29 both a reaction in Syria biosynthesis, and this reaction, and since you don't need much flux through this. maybe that organism has needed to duplicate diverge and specialized two different enzymes.
13:00:46 And I think, you know, a sort of a bigger part of that kind of question really is, Why do we see the pathways that we see.
13:00:57 Right, so if these pathways had been patched together from promiscuous enzymes that are available in the proteome.
13:01:13 together. So there may have been a lot of historical contingency in generating the metabolic pathways that you know we see sort of canonized in extent organisms, just based on what were the resources available, back when pathways were evolving.
13:01:35 And again, a lot of them. A lot of the most interesting stuff had happened by the Luca, so you know we can only speculate.
13:01:44 Can I can ask to be, you know, given the parameters of this pathway basically different only by the phosphate group on the side here, man how this is very common occurring themes that in a multiple pathways that basically variation or theme of the same
13:02:06 enzyme marching same series as a marching band. The Wizard different site group, or their pathways like that Yeah.
13:02:17 Are there pathways like that Yeah. Is that a common thing right because if it is, then, well, maybe the answer originally came from that and then that makes the kind of a patchwork easier, you just take these concepts.
13:02:28 Yeah, so there are there are examples. So, clearly the PLP enzymes,
13:02:37 were related to the series synthesis enzymes. I wouldn't say that there are a lot of examples of that.
13:02:46 And maybe all hedge on that.
13:02:49 So for pathways for degradation of anthropogenic pollutants.
13:02:55 A lot of those, I suspect have evolved from pathways there were already present and sort of basically the entire sequence of enzymatic reactions condemned just be ported over to a new substrate.
13:03:13 And, you know, each enzyme may not be very good at, at an alternative substrate but there. There are cases where I think that you could essentially recruit an entire pathway to metabolize a substrate that is structurally related to the normal substrate
13:03:35 for the pathway.
13:03:39 Did that answer your question.
13:03:53 Rhea has a question maybe not sure what he's after the Searcy need PLP Floyd's reaction.
13:03:55 Yes. And that is a really strange thing.
13:04:01 Just an enzyme is needs a co facto that is make making this might explain the longer lag time to juice. Yes, exactly.
13:04:12 So I'm looking for my PLP synthesis pathway.
13:04:20 Yeah, so a very strange thing in the normal PRP synthesis pathway is that CRC requires PLP which is the end product of the pathway.
13:04:31 Okay, this does not seem to be a very smart way to run a pathway.
13:04:37 This is not the only example of this in metabolism though because in a different time environment parts of the system requires Simon, and the ATP synthesis pathway requires ATP.
13:04:54 So, this is a strange way to do things for sure, but this is one of three examples that I know of.
13:05:05 And somebody said that could explain the different phases, and that's exactly my hypothesis to that, you know you you sort of have to exceed a threshold, before you have enough PLP to populate the enzyme in the pathway that needs PLP before the growth
13:05:30 can really take off.
13:05:33 So it's sort of a positive feedback, until you make a certain amount, you can't get going.
13:05:42 I think the 12 synthesis also has a bit 12 dependent step. Is there a.
13:05:48 I think so yeah okay that would make for examples.
13:05:57 Wow, what a morning.
13:06:00 I think we'll call it could see a Terry What do you think, well thanks again surely this was amazing. This actually brought a lot to the discussion and an insights on how to think about pathways pathway I, I could take to Fluxus and evolution and you
13:06:21 did a marvelous looked up to really complicated complex problem and rooted in very simple physical, chemical protein chemistry principle so I really really appreciate this.
13:06:35 As always, so thanks again.
13:06:37 Shelley, well thank you for listening. I'm sorry I just went so long, but it's a KITP talk they always go until every loss it's a good thing. It's a good thing.
13:06:48 Yeah.
13:06:49 All right. Thanks. Thanks again.
13:06:53 Bye everyone. See you next next week.
13:06:56 I thank you Shelley.