Chemistry and biology: alkaloid biosynthesis in plants

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Chemistry and biology: alkaloid biosynthesis in plants
Elucidating and Engineering Natural Product Pathways in Plants
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Many enzymatic transformations are utilized in the biosynthesis of the monoterpene indole alkaloids, a group of structurally diverse natural products. Here we describe the discovery, functional characterization and mechanistic study of several enzymes involved in the biosynthesis of the monoterpene indole alkaloids in Catharanthus roseus. Also discussed are the implications of this work in the metabolic engineering of natural products.
Keywords natural products biology

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by the year.
i would be giving need election organic chemistry i i don't really consider myself like a card carrying organic and this but i do love organic chemistry and i hope that will come across in the seminar and i i have to warn you i do have a couple of heat maps and in this seminar but they're in the search.
this of the loo sitting organic chemistry so i i i hope you know you'll find them somewhat somewhat interesting because of that ok so first of all the four i go any further i just want to.
i acknowledge on my group of all the former members and then i also want to acknowledge the current members and i'm primarily going to be talking about nats and offer nando's work and a little bit of west glance work and i really want to think john fernando west and nat for helping me move by i've moved to the uk.
from the states just about a year and a half ago and it was those of people especially john chang who really did a bull the bulk of the organization who i have to thank off for me being able to actually stand up here today and tell you about some science so i'm very very grateful to that and then.
i'm also grateful to these agencies for for funding ok so i'm sure as many of you know the centuries of successful medicine has had some of its origins in plants and i just show here some of natural products so called natural products also called secondary metabolites or spa.
specialized metabolites that are derived from plants and each one of these molecules on is used in the clinic today so of morphine isolated from opium poppy of beautiful alkaloid used for thousands of years as as an opiate tax all very potent anti-cancer agent have been. aston again another potent anti-cancer age an isolated from capture at this was a as the jocks an isolated from fox love i used to treat a variety of a cardiac cardiac ailments i even today so this is just a small sampling of some of the compounds that have been isolated from plants that that are that are used in in modern medicine. medicine and i want to highlight that plant derived medicinal or on advanced intermediates of those missiles of content that can then be used in sammy synthetic procedures high are still today in many cases extracted from the plant so total synthesis on has made incredible strides on in the last fifty years or so but today we.
we are still nevertheless are reduced isolating these compounds on from from plants today the because plants are very very effective very efficient in terms of making them and and i'm going to him talk about ogden blast and i as an example throughout the course of this talk on and the blast in an and other compounds by a synthetic lee related to them.
aston and i just want to remind you of them glasses a is a highly potent anti-cancer drug it's been it was discovered in the nineteen fifty's and and has been used since then it's isolated from catherine this was a sleeve in in very very low yield get his is still katherine's rose a sleeve still remains the primer. the source of this compound on and i would argue that this is one of the most complex natural products no not not that they're there are certainly many many complex natural products but i would put this one up there with with with the best of them. and so just to sort of asked the question why study even blast in another related natural products from katherine's this rose a us arm and first of all i hope as will come across in the talk today that by elucidated discovering the enzymes that make up the bio synthetic pathways on the metabolic pathways that construct this small.
fuel that's going to enable us to undertake engineering strategies to really think of ways to improve the production levels of this compound from of from of bioengineering perspective and i think a whole host of very interesting issues in bioengineering and biology are going to be raised his throughout this process of thinking about issues. like transport how various bias and that intermediates move throughout the plant how different and signs are regulated about what levels to the enzymes have to be relative to one another and so forth those are all going are going to be raised and then finally i this is really arm of of my favorite reason and i think it's a reason that everybody in this audience will agree with. is that really i think the complexity of this molecule means that it's a given that we're going to discover interesting an unprecedented and novel biochemistry within this pathway so i think they're going to be some very interesting and symes involved in constructing this molecule in and i hope i'll tell you about a couple of interesting examples problem today.
so just a little bit more background on been blast in is a member of the monitor peeing in all alkali class of natural products and i guess it's its. a particularly a i i i do feel a little bit of confidence in terms of giving an organic chemistry seminar because i do think that the monitor been in the local is really had an amazing history in organic chemistry and some of the most beautiful since the seas biomimetic studies and so forth were done with this. last of of natural products and it's a family of about three thousand different compounds i've just shown here couple of examples their found within a wide variety of different types of plants and you can just see that the chemical structures of the compounds are very very different but despite the chemical diversity of these up. this class of compounds each one is derived from the same starting materials namely trip to mean which is just the car box slated trip a fan and then this funny looking up monitor pina compound called seek a logan and so just on how do we if if we want to elucidate been blasted by a synthesis or any natural product bias and. this is how do we go about thinking how to attack the problem and the story with the monitor been in allowed lloyds has been is that historically there were some very careful labeling studies done in plants and plant extracts from their high was a great deal.
for put into isolation of various intermediates from plant extracts and i fell off my life that some of those intermediates here on the slide those intermediates after they were isolated within structurally characterize and sort of form the core of framework for a bio synthetic pathway and then biomimetic chemistry so model chemical react.
fans could be used to probe the realm of what what was chemically reasonable and i just i really want to emphasize that in thinking about of bio synthetic chemistry and i'll be talking about a lot of biological tools sequencing tools but we absolutely need chemical intuition and chemical knowledge on to real. we think in india in a serious way about how these these pathways have put together so this this is kind of the the that the chemical foundation of thinking about how the blast in and and alkaloids a natural products more generally are are made and we can extrapolate a little bit more about the by. the synthesis of been blasted and this is a a kind of a simplified schematic of how them blast in his mates starting on this very simple monitor been idle talk a little bit more about in just a few minutes geranium all i'm going all the way from geranium all to than blasting here and i've shown on summer.
read dashed arrows and those steps are all cryptic on we have absolutely no idea what type of enzyme war or what the sequence of that and sime catalyze would would catalyze this step and in fact some of the art of bias that intermediates have shown in brackets there's a putative by a synthetic in.
it's they haven't been isolated they're just proposed based on all the the the realm of of of chemical possibility so there is a tremendous amount that we need to that yet for mean remains to be discovered offer this vile synthetic pathway and i think it's important to point out that of them to have been blast in is not some obscure little.
compound that was discovered a couple of years ago and hasn't really been on paid much attention to the blast in is an incredibly important compound that has been subjected to decades of study both in terms of biological activity and in terms of understanding on its by a synthesis and this really highlights on a point that that i want to make.
in that many plants natural product pathways of remain unsolved so in fact with the exception of morphine on our every compound on the slide has an incomplete by a synthetic pathway we don't know all of the enzymes for example that are involved in the synthesis of taxol. the blast and jocks and and honestly the majority of important well known of plant derived natural products fall into this category we just don't know how most of them are made and the reason for this is simple it's that are working with plants is hard it's a pain in the neck.
so i'm just a kind of of this gig give you a sense of of this problem in just one slide in microbial bio synthetic pathways and amusing here are the example of act in a row which was actually one of the very first bacterial natural products are that was have studied. using modern genetic technology on it is nearly always the case that the gene setting code the enzymes of the boss and that pathway are actually organized in a little group a little gene cluster on the chromosome so if you can sequence of bacterial genome in.
you know a couple of days which is now possible you can actually go through using a technique which is now commonly called genome mining and just look for these little groups of genes that looked like they might in code and signs that would potentially be involved in the blast and this is of a pollock he tides and days and while i don't want to imply that.
elucidated the bias that pathway of bacterial are derived products is easy it's certainly not and there's absolutely seven explicit chemistry and biochemistry that has come out of the studies and is continuing to come out of the studies that this this field of microbial natural products is just in a in a very very wonderful state right now.
on this fact that the genes are clustered together in one place in the genome really streamlines the process and has just a really enabled this this field to to to take off so in contrast of plants plants are different so first of all plants have very large genomes many plant gene.
films are larger than the human genome some of the much larger some plants even have more than one genome their facts of a week for example is hacks a ploy to aso they're very very complicated genetically and there's lots and lots of genetic material that you have to sift through if you're going to find these genes and many time.
the ends the genes that cause the enzymes have a plan by synthetic path we are not clustered on the genome or chromosome on so they're scattered throughout the chromosome not that's not always the case but that's very frequently the case and so this sort of leads to the statement that i've heard a lot you're never going to get anywhere of studying plan pathways and i think that. that was kind of odd that that had it been been the view forum of a very very long time on and i would argue that that's changing. not just in my lab but in many many labs on of across the world and and i all hope i'll give you some insight into some of the techniques that are are enabling that change ok so decline of them exemplify that what i thought i would do is talk about some of the unknown steps of monitor been in the lock away by a synthesis namely that leads to the bio said the swiss have been black.
aston and i thought that i would start with a relatively simple example just the kind of illustrate how are thinking works and namely to look at a offer a missing step in the conversion of this bias and that intermediate to burst mean that is responsible for converting to person mean on into been doing.
eventually he is ultimately converted into been blasted and in fact if you look closely you can see that been to lean is really just one half of the been blasted molecule and specifically what we can do is we can go back to the chemistry side of the problem and really look at the chemical framework that's been mapped out for us. buy organic chemists and and biological chemist and it's a very nice section in the pathway in that all of these files and that intermediates a stable and each one of these biases that intermediates had actually been isolated and structurally characterized and over the last i would say twenty years or so. each one of these steps has been assigned a particular enzyme and the enzyme has actually been discovered and cloned so we know exactly what it looks like we know it's who we know it's amino acid sequence and so on there is still to be missing steps this step hear this funny hydroxyl nation step here and then this particular step here which. his of methylation and so we thought obviously there's going to be an enzyme that looks like an n.f.l. transfer raised that's going to be required for been blasted by a synthesis ok so now here is sort of where the biology and the bioinformatics comes in and again i used to think that this stuff was incredibly bore. during until i sort of realize how it could be applied to understanding chemistry so well well well we'll see if i can convince you to feel safe like so i'm the first of all we have obtained a catheter and this not a genome but a transcriptome so we've gotten a sequence of all of the transcribe gene so we haven't seen.
his d.n.a. we have sequenced messenger r.n.a. essentially and this is actually this is part of a very large and i each funded consortium effort and all of this data is are located at this website also the data for all the whole number of other bisnow plants on.
including on the producer camp with the sun for example on and somehow we managed to get hot cannabis in there and how we manage to get that through the united states congress i'm not really sure but anyway there's a transcript on the cannabis of being a downloadable net website if you want to take a look at it ok so. in addition to having the sequence of all of the trend what i'm going to call transcripts namely these are the the genes that are actually expressed by the plant so the messenger or a day we also have what we're calling expression profile data and this is just a snapshot of that expression profile data this is an excel spreadsheet so this is the data in its rauf.
form and what we were looking at is here on the y. axis each one of these lines called the sea or a locus this is a catheter at this was a as transcript so this is a gene this is some gene that is being expressed by catherine this was a is and there's about thirty seven thousand of them in cabaret of those rules a us and then on the x. axis each. one of these is a different to issue so we've taken seventeen what i'm calling tissues or conditions on so we're taking leaf were taking flowers were taking stands for taking roots were taking little seedlings were taking seedlings that we've subjected to treatment with a hormone that we know up regulates natural product production is as it's essentially us.
stress response so we know that we have some ceiling set only produce a little bit of alkaloid and we have some ceilings that produce lots of lots of alkaloids and so on so so seventeen whole variations on catherine throws a as and what we've done is using on something called a luminous sequencing next generation sequencing we measure the essentially.
the the expression that level of each one of these genes in each single tissue and that's all that this excel spreadsheet is showing. ok so now how can we use this data to try to find for example this and metal transfer race which we would like to know because it's involved in been blasted by us at this as and the traditional approach is on we say ok let's guess what type of the enzyme is going to be involved so for example for and oxidation we would say ok maybe a p four.
fifty's involved if we're going to be methyl aiding and mean group will probably looking for an enzyme that looks like a metal transfer race and then we look at these are transcripts or contacts sometimes these are called context on and there are plenty of automated annotation a software packages so you can just feed. that data into an automated software machine is going to automatically annotate each one of those transcripts that each one of those thirty seven thousand transcripts are going to say ok let's pick out all the ones that are automatically annotated as methyl transferees as and them are going to say ok now on even though the genes have been blasted by a synthesis might not be.
located next to one another on the chromosome maybe they're related in terms of expression profile so we're going to say ok we know actually in the case of the transformation from to person individually we actually know five by a synthetic genes so we're going to look at the expression profiles of those five known boston that genes were going to pick we can pick those out of those thirty seven thousand different candidate.
and then we're going to say ok which one of the two hundred or so methyl transferees as look like they have a pattern of expression across the seventeen different tissues that are most similar to those known of bias and set of genes involved in the that's other people have already identified that convert tubers and he didn't have been doing it now there's some. problems associated with this traditional approach them to talk about the sort of the next example but we thought that this would be a pretty good approach forearm of looking at looking for this and the whole transference this kind of how we look at the city heat map so this is just software you download off the web it's free and all i'm showing here is here are different tissues.
i don't think we used all seventeen different issues in this example and then here is for example one known bile synthetic genes so i've just highlighted here and then here's the whole list of of of the genes of our desire protein family and then it turns out this i'm just tell you the answer right now this is our desired unknown by a synthetic gene. that we've had and what and we've been able to prove this experimentalism to show you in the next couple size but this is actually what the data looks like so what we would do as we would actually go in and we would actually on fish out all of these different transcripts or contacts were genes whatever you want to call them from our katherine's this rose a us on the genomic material that with that we've gotten.
the lab put it in an expression vector go and had are all basically express an ecologist sort of the standard of bought biochemistry technique and then asked say it for function.
ok so that's essentially just sit there and. and so this is what we did and so we wound up isolating on and i characterizing about eight of possible muscle transfer races and one of them actually did the reaction that we on that that we wanted that we were looking for now i just want to point out on we this is believed to be the natural substrate of this.
the metal transfer a shown here this is a little bit of a pain in the neck to synthesize and to isolate of from catherine this was as we can only i say that isolated in very tiny quantities but what we can do is we can very easily semi synthetically generate this model substrate here which is just a little bit. simplified compared to this and then we can do a biochemical last say where we've got authentic standards of our model substrate and our methyl a good product and this isn't l.c.s. trace and then we can incubate are enzyme with and without an acid in a sealed or sand co-factor the same co-factor is actually. fighting methyl groups of four this reaction and we can see in the presence of both enzyme and the required sam co-factor are seeing conversion to the product in the kinetic states taken at a constant seem to be perfectly reasonable so this very nice evidence that we can in a period of about two months or so that's about how long it took davis come to do this to go.
go through this family of thirty seven thousand different transcripts and find the one that actually cares about this reaction and we're by no means the the only people to do this we certainly did not invent a coke special analysis many many other people working out on this i just one and the size this is an example of of of how far.
for the field of of plant metabolism it has come and there are examples from many other labs that are that are our are very very beautiful using using this this this type of approach now i mentioned that we used a model substrate and that's not great we you know you can always imagine that now you we we we've we like to.
we see the activity with with the real substrate and also on and signs sometimes do funny things in vitro they might not exhibit the same specificity in vitro are that they might in vivo i mean it's it's very often the case that you can take an enzyme out of its natural environment for speed at some natural substrate and it will do some conversion.
but it might not actually do that function in vivo so really the way to nail down the function of of an enzyme in a metabolic pathways is to actually silence it knock it down knock down its expression in the plants and then asked me to see if you've actually stop production of the natural product and so.
it turns out that on de lis come before a post-op in the lab worked out a way to do this and catherine force rose a us that's very very convenient this is a technique called virus induce gene silencing or big sur and it's a way to silence on a gene very rapidly implants essentially what we do is we take arm.
of the appropriate plasmon on in in its its of its of of vector of from of from a virus and we take put in that sector a little piece of our and muscle transferees and then we transfer that virus are plasmid into a young ceiling.
and we essentially just do this by sort of taking a pair of tweezers and dipping it into the plasma solution and then just pinching the stem of the plan it's very not technologically a difficult to do at all and then we wait i put down a week here we often way to two to three weeks on and on that's how long it take. for the silencing effect to happen and essentially what is happening is the plant is recognizing this viral vector so it thinks that it's being infected by a virus and its ramping up its immune mechanism and it's saying ok we need to shut this virus down and in the process of trying to shut the virus down it's also shot. going down that little piece of d.n.a. that's in coating that and metal transfer is that we've sort of slipped into that virus plasmon without the without catherine this was a is noticing so the process of she trying to shut down the virus catherine this was a says also shutting down the and methyl transfer is because we put that and nothing transferees into the virus plasmon.
and so what we do is after this one to two to three weeks we caught off of a few of the leaves we grind them up in a liquid nitrogen and extract them out from ethanol and the shoot them on the l.c.s. on and we analyze for metabolic production so essentially we're looking for the our loss of into lean production. and then we can also analyzed the messenger r.n.a. so look at the message or an a and hopefully we will see a decrease in the levels of messenger r.n.a. that correspond to our and metal transferees and i just shown here a kind of visual example we can use this as just sort of control we can silence a gene that's involved in core filled by a synthesis so. for full stops being produced in leeds turned yellow so this just shows you that this method works pretty nicely in enough for to serve a visual point of view and so we did this for this and muscle transfer race and these are again i'll c.m.'s traces and what we can see his arm in arm are negative controls.
see very nice production of these lean as on and we know this is the only because of the massive because we have an authentic standard and then as we are in fact are as ceilings with these viral constructs the container and nothing transfers gene we see a big decrease in the levels of injury that are being produced and we see this build up. of this new compound the mass of which corresponds to what is predicted to be the native substrate of this and nothing transfer so that's very nice evidence that be because we're saying the buildup of this compound this is very likely the substrate of and methyl transfer this and muscle transfers and its accumulating because there's no enzyme that for four to act upon.
so this data told us that not only can the enzyme do this reaction in vitro it also appears to be doing it on it in the vote and that this is very likely the physiological role of of this enzyme and i also just want to highlight that a we think a little bit about. i'm not going to give you any great answers but we do think a little bit about how these enzymes might have evolved i think is a very interesting question you look at the complexity of been blasted and you say to yourself. how did the plant managed to come up with a pathway that manage to construct this unbelievably complicated molecule and the current dogma is and i think this is probably true at least the first approximation is the plants managed to pull in various and signs for that were already existing possibly enzymes from. the primary metabolism and i think that this is the case with this and metal transfer rates so if you look at the sequence of this and metal transfers and you compare it to lots and lots of other different muscle transfer he says this is a phylogenetic tree that dave made you can see that it's actually scary similar to a type of vessel transferees called the gamut to. offer all seem a little transferees so this is actually many transfers it doesn't do it and metal and methylation it is a scene methylation on this on aromatic position here and this is a step that's involved in to cough or all by a synthesis which is essential for plants survival so our thinking is that nature manage to do duplicate. a copy of of gallic awful muscle transfer race this extra copy was sitting around some random mutations occurred and those mutations have changed the activity of the enzyme such that it could instead of it doing a c. methyl reaction on this subject.
but here could do at and methyl reaction on that to diversity means of bias of that intermediate and that in such a way was actually recruited into alkaloid by us and says oh and there's currently a student in my lab whose of richard pain who's looking more closely at the sequences of the n.f.l. transferees and the gamut a car from nothing transferees and is trying. trying to rigorously establish exactly what residues are responsible for differentiating between these two catalytic activities and maybe from that we can infer problem maybe the evolutionary history of other at least one stepped in last of hours services so that was really our first example in my lab of using.
this transcript on the data and we did at first because it was easy i mean an n.f.l. transfer his reaction it's a time up from a chemical perspective it's simple and we know it muscle transfer is look like it's very easy to go into that that transcript database and just look at that automated assignment of what the transcript look like and just see muscle transfers muscle transfers. that'll transfers its it's a it's a relatively i would say straightforward problem but there are some steps in been blasted by us that this is i think where the chemistry is much more cryptic where we might not have any idea of what what the enzyme looks like and so i wanted to talk a little bit more about some of the steps involved in the bias. this is of this very funny looking monitor penis is called an era droid class of her pain and us specifically this to a peanut is your twitter feed is called seek a logan and just a little bit of background to seek a logan and the year droids on are not just found or not just involved in monitoring and all alkaloids. and there also are incredibly large class of compounds in their own right so your boys are found in many many plants and many have a very potent form of the logical and agricultural type applications in an of themselves and in fact one of. this this this is just great. this very simple. early compound here never to lack town is the active ingredient of napa to kotecha terry of which is catnip so this is actually the compound that gets cats high and so i just love the fact that we work on this compound on its its it's fun to actually work on compounds that do something.
the new entertaining. and then also on steroids are found in insects and the bios and whether the insects actually generate these erodes to know or whether they're somehow sequestered from plans he is really not clear but nevertheless on many many your voice on or found from of four found that a whole variety of different and speed as and and so on and these are. while almost certainly on is exhibiting some sort of pheromones arm and signalling type type roles enabling the the insects to communicate with one another. ok so as i mentioned for the presenter we did this on traditional approach but you know there are a lot of problems on namely that it you know this annotation are really doesn't it's not foolproof on and sometimes genes or transcripts their mis annotated we get it wrong on and we just missed them on and all. so we have to know what type of enzyme is going to be involved in the inn in the transformation aftermath of transfers that's that's work we're probably not going to get it wrong that's pretty easy example but for some of the more unusual biochemistry and these pathways week we really we might not guess right and so a better approach is required.
really to not just select a subset of genes are based on their annotation but to really look at the whole database on and to think of the clever ways arm to to to shrink down that list of thirty seven thousand transcript while maintaining a more open minded view about what.
that type of of of enzymes might might be involved in the pathway so is what a a post-op in the lab fernando florist really pioneered in the last year or so and what he did was he so i'm taking back to this expression profile list of this list of thirty seven thousand different transcripts and he said.
ok i'm going to come up with non biased ways so new filtering methods to eliminate thirty thousand or so of these transcript so we're going to be left with a subset of of seven thousand different genes and instead of saying ok we're we're going to do that by taking all the muscle transfer all the contacts that look like muscle transfer he says. when he came up with sort of more biological our criteria and so namely of fernando said ok we know that second logan is expressed in leave so every single one of these contacts that is not expressed in leaf or just going to cut out we're just going to delete them and on and we also know that second logan along with other alkaloid.
are subject to induce a bill expressions so i mention we had the seedlings we can treat these seedlings with a plan for moan and that's going to raise the levels of second logan in production we made it a criteria that have forgiven transcript it had to have a higher level in the us ceilings of that had been in contact with this hormone.
and if the if that was not the case then we also eliminated that transcript so using criteria it like this on and sort of tweaking around with the parameters a little bit fernando got this list down to from thirty seven thousand to about seven thousand and that's a little bit more manageable to to to look at and so with a group of seven thousand.
the transcripts we can start feeding this these expression profiles into commercially available software and looking for expression profiles will and so what fernando said was ok but how we got what are we going to do on and the most interesting step i would argue of second located by a synthesis is the cyclists a. patients and this step is absolutely it not not known and this is the step that converts this linear substrate into the cyclists substrate and and we do know one of the steps in early second located by a synthesis and this is a p for fifty enzyme called geranium all ten hydroxyl research. the ten age which is involved in putting the hydroxyl group ear on and this has been cloned it was closed by traditional biochemical methods i think about fifteen years ago and we said ok probably the cyclades is going to have a similar expression profile to this g ten age i have no idea what the cycle he says i was going to look like but let's look and see. we all have the contacts or transcripts or genes that have expression profiles similar to g ten age and this is just to get a snapshot of of our expression profile data so this is g ten age at the top and then this is the list of genes in you decreasing order with similar expression profiles to g ten th and i. i want to thank professor you walk we at michigan state whose statistician who kind of helped us really set the south in her lap. ok so what we could have done as we could have taken the first you know i don't know hundred or two hundred genes in this list and just ask the them biochemically but again this is i think really were chemical intuition can really of have combined with biology in violent for maddux can really crack this problem.
and i hope this was illustrate that we thought ourselves ok can we think a little bit more carefully about what the cyclades cattle a catalyst might look like it and so this is what we know about the chemistry of your toy bios as we know this is a the upstream pathway is the not were normal level onik. massive blast that pathway that makes these precursors involved an altarpiece bias of this is the map an i.p.p. and then there's an end same an enzyme geranium power of phosphates in phase which condenses the map an i.p.p. to make sure a deal by phosphate and then on and this was largely work done by my heart sank. i should add geraniums some things will convert to drain field i phosphate into this compound called draining all at which point this p. for fifty that i mention geranium ten hydroxyl a skin hydroxyl eight this position. and then it is presumed it's not known what we now know but who wasn't if it wasn't really known at the time that we started this there is a some sort of oxidase probably in an alcohol dehydrogenase second oxidize this dial into the dial to hide and then it was my heart sank the proposed that this dialled hide was in fact the substrate. the offer the cyclades hands on. and so i just want to point out this is completely different from the canonical monitor people cyclades mechanisms of were typically work for people like these mechanisms were typically for example in modern turkey by a synthesis we start from terranea all die phosphate the phosphate his eye nuys generates a car book.
that i'm intermediate and that cargo cat island coupled with the presence of this out keen here can undergo any variety and hundreds of different are rearrangements to form any number of all monitor people structures but clearly this us like was a should reaction is is going to be very very different and i think tanks key insight. was that he and took crude plan extracts that were known to make second loaded another other ear towards and he partially purified these extract so he purified away all of the small molecules in co-factor is but kept the proteins and threw in the style to hide substrate alone. along with some other substrates but this was the substrate potential substrate that worked into this mixture of crew plan extract and found that it was only when he added any the p. h. co-factor did he really restore the activity and we were able to or he was able to convert this dial to hide substrate called ten after draining out into the cyclists. last year to a skeleton here so based on this work out by zheng we could say to ourselves ok we need to have an enzyme that's going to utilize in any the p. h. co-factor and that provides us with a big clue that's going to enable at that enabled us to really narrow down our search.
much much more easily so we on went back to our list were looking at these are genes that i have expression profiles most similar to our geranium ten hydroxyl ace and what we did was on we look for our genes that were allocated as and signs that would be likely to use of. but an energy ph co-factor and its mechanism and the very first such it was an enzyme were transcript in code annotated as a protest drone beta ruddock tastes and for reasons i actually don't really know why but it turned out that another posts i could actually clone this and signed on in our lab on a. that a year ago are or are year previously for reasons that he couldn't really explain but we actually we had this of plasmid in our freezer so often and so just went to the freezer says he can insult was a post-op who closed it in very kindly gave it to fernando and us we're.
but as the of that very quickly and that's so i i normally tell people that we asked say this protest on better doctors have long because it was highest up on our list.
but in all honesty it was because it was already in the freezer so anyway. but if you think about this also logically from a chemical perspective if you look at the reaction that this purchase room baiter a duck taste catalyze is what it does is a hive drive from the any the p. h. is added into this double bottom tier enabling the formation of an eagle species and then. it's believed that this in all species will spontaneously to tom rise back to the key to see the effect of lee reduce this this double blog here and if you look at the structure of ten oxo geraniums you can kind of year you can you can see the similarities in this the structure of of of the substrate so we we we thought maybe we. when we might actually be on to something and i just want to highlight that the project a vibrator doctors are found widely throughout the plant kingdom and the and dodges functions of these enzymes these projects stronger dr bases in plants are really not clear very few plants actually make for just rome. and so it's really not clear why these why these plants are or are actually making a lot of these and simon and i suspect that many of these enzymes other annotated as progesterone beta ruddock faces implants their mis annotated they're actually doing something else like perhaps this a second logan your to it's like was a shoe. the auction so at any rate we could take our clone of this enzyme on we could have over express it in the collide this isn't s.t.s. page gel and we could just to a preliminary asked say by the modern information by t.l.c. on so we could synthesize the substrate we consider the size and authentic standard of the product and lonely. the incubated all the components of the enzyme co-factor said all we could see formation of a product with the retention time matching that of the synthetic like coal standard and that so that was very nice preliminary evidence we could go and week to characterize the kinetics based on consumption of any the p. h. by u.v. this spectroscopy and get very real. reasonable studies take kinetic values again and then finally really and this is the key thing is we could actually do a large scale and the magic reaction purify the major and the magic product buy property of t.l.c. and then really rigorously characterize the product by and a more and compare it to a synthetic standard that that made see that it was identical so. this this really was and is in fact the and cinematic product as evidence by and more. and again we wanted to do you know this is a we wanted to make sure that we weren't just seeing some sort of spurious activity and that this as a medic activity was really arm of this was the un's on catalyzing this reaction in the plant so we could do our big star silencing in katherine's this was a us of our cyclades and what we did. it was we look for decrease levels of alcohol is derived from second logan in and i just show here two examples kept the ranting indians lean and we could see in our silence to lines we could see is statistically significant decrease in the levels of these compounds again says suggested that this for just over a decade homologous actually catalyzing the cyclists ation reaction. in vivo now we look for the accumulation of this dialled to hide substrate so like with our in bethel transfer as we saw this nice accumulation of that of that by a synthetic precursor that the and nothing transfers would normally react with and we did not see any accumulation of this dialled to hide and this style to hide you might imagine is. in the reactive probably pretty toxic to the plant and the plan is probably doing is absolute best to metabolize it to get rid of it and so it just kind of check can see that we mean you know the sort of confirm that maybe we were seeing some evidence of accumulation of a diet the metabolites derive to derive from art. while to hide what we did was we looked at new compounds that were being formed in our silencing experiment i just show here three examples that i have no idea what these compounds are except that they have masses of five thirty one five thirty three and forty nine on and then we on tried to replicate the bigs phenotype by taking a catheter at this was a. relief and feeding it on a whole bunch of this dialled to hide over the course of a day and then doing else of mass spectrometry of this leap that had been fed the substrate and we sought many of the compounds that we saw in the silence leave also being formed in this league that had been that the substrate so that suggest of that sort of the. new compounds that we're seeing in our biggs our silence lines are actually derived from this from the style to hide substrates i think that's reasonable evidence that that the dial to hide it's not accumulating but to reduce of the metabolites of the dialled accumulating ok and then we could also just is another sort of check of functional validation in vivo.
you can actually look at the local ization of this and i'm so plants are in and i won't have time to get into this book plants are incredibly complicated in that i mean these are not unique cellular organisms these are these are our are very complicated structures and these biases and katich pathways just don't take place in in the side us all have one cell. these different enzymes are localized to different cell types and different and times a localized different apartments within the cell and with the help of some collaborators and friends professor of in support of all and professor vincent burlap we were able to check the localisation of the cyclists and what we found of vincent are found.
that are cyclists was actually localize to the site us all which was very nice piece of information because we know that the previous enzyme geranium ten hydroxyl he says also applies to the side it's also those two enzymes on our or in close proximity to each other which is what we would expect and vince a burlap showed that this. and time is localized to a type of cell called i pad app again. the geranium ten hydroxyl he said time is also localized of the cell type so it's just a very nice piece of data that sort of all the enzymes that we would expect to be working together and coax press together are actually in the same cellular compartments in the same cell types which is which was kind of a nice confirmation of ok so on can we think about the mechanism in a little bit more detail and i'm draw. during the mechanism like this mostly because this is how my heart sank to the mechanism in his papers where we have the high dry coming in adding and then this five member during forming and then we could imagine that this black hole would form just by the in all adding into this this album hide on and match. in the post oct in my lab really spent days with molecular models and i'm not sure for really reproducing it very well on the sly but this is nats in mind best attempt to show this and essentially what we're showing is ok we've got to get this style to hide substrate this ten oxygen or e-mail folded correct. early in the presumably in the act of sight of of the cyclades on we don't know what the act of side of the of the cyclists looks likely because it's not crystallize jet that's that's r r r r goal to do that but but but but we have done it yet so this is just based on on on chemistry so this is we could imagine that for example these this dial. i could fold up like this and then we would have to imagine that the hydride from the any b.p.h. co-factor would come in and attacked here and get bond formation and then subsequently all formation and really what i'm trying to show on the slide is we've tried to fold up this does dial to hide in a variety of different ways and in all cases we get the wrong a stereo i summer. and the only way that not could get the right stereo why some reform was really if if we can talk or did the the structure of the energy ph co-factor relative to this to this dial to hide substrate where the the the the hybrid would essentially have to be kind of inside the fold of the substrate and is the. the hydra it was just very hard to imagine in a three dimensional model how if you add in that hydride you could in a concerted down or form this carbon carbon bond form on and so this an issue this is so we thought ok we should think about this a little bit more carefully and we went back to the proposed mechanism of the purchase to invade iraq case. this and it's actually as i mentioned earlier on this mechanism proceeds via formation of a needle and then there is believed to be spontaneous of auto timers ation backs to the quito pointy and so we said ok you know that actually probably let's work by. analogy and let's say to ourselves ok let's let's assume that the cyclades of mechanism invokes an eagle intermediate so here are just showing a sort of a of i think a typical he known reduction so you'd form this species here and then we could imagine that cyclists ation would take place subsequently so this is a i guess i'm calling. in a non concerted mechanism were first we form the e-mail and then we have a cycle is a shunt mechanism to form this five member during and then we could imagine the lack call for me and i also just one a highlight that if you look at this in all substrate you can see that i think it's actually set up very nicely for ahead. deals all the reaction so i think it's very interesting to speculate that this cyclades and signed might actually be catalyzing deals all the reaction we have absolutely no experimental proof for this is just one possible mechanisms were thinking about potential probes to maybe understand whether or not this mechanism plays are so for example. all a net is currently trying to synthesize the mythos the ether analog of this that we would hope would be a good substrate for deals alder but not such a good substrate for that michael type addition mechanism but again we were just not really sure whether or not this mechanism hold but i will point out that there. i have been a number of synthetic studies done on looking at your do it's an eric jacobson for example has shown that the year to its scaffold can be accessed us synthetically using an inverse electron demand had are deals older so again i think it's chemically reasonable to think that maybe this mechanism might be play but we.
absolutely don't know that at this point for sure.
ok i just want to again coming back to this question now where did this this on and sign come from and we can really look a little bit more carefully at the products specificity so i mention that we did a large scale isolation of this major on black hole the major product the slack tall but we also found three minor side. products that we could isolate instructor a structurally characterized in this large scale and smack reaction and you can see that one is just a reduction products of this is just the one for reduction product here and then we can also see all one two reduction products are just the reduction of this out to hide here and also what we believe. it is just a rearranged of a product of of this ten oxo draining all product here and i just want to note that the purchased from data are docked aces an enzyme that catalyze a one for reduction and it's an enzyme that is classified as belonging to a super family of rock faces that are specialized in one. into production reactions so we kind of like to think that the side products represent kind of this digital and some magic activities that may be. we had so nature evolves and productive uses that could reduce alba hides and from that this purchase from data ruddock taste was involved that could do a one for reduction and then from the protest or later doctors we could evolve.
the cyclades that to one for reduction but consequently also managed to a site was a shun as well so that's just kind of our thinking about. about how that and time work so again i just want to highlight we're really excited about this because it is a completely different type of turpan cyclades so if you can think of it because i don't know i think if you can think of a good chemical reactions there's probably an enzyme out there thought that that that nature has has developed ought to take to catalyze that reaction. ok and then just sort of one last point that i want to make about this on and sign is that yes i know you will know this this this dialled to hide a substrate ten oxygen rail it's an extremely reactive substrate and are we experimentally prove that by when we silence the cyclades that are good that. so that that dialled high got metabolized into twenty two different products and so what we wondered was is this a good substitute for generating different of for for for generating chemical diversity and what would happen if we subjected to incubated the substrate with a couple of with different productive uses and again we.
he happens to have been hand another on ruddock taste this is a reductionist it's actually involved in or got alkaloid bile synthesis and again this was an enzyme that we just happen to have in the laboratory and ease a isn't any the p. h. dependent and sign which reduces this double bond here so again you can really see the similarities of in the substrate. rates on and then this allows rotation around the sun now single bond and the subsequent formation of of this and medium species so this is the the reaction that easy a normally catalyze is in a fungus called aspergillus whom have got as it has absolutely nothing to do it your voice been blast implants anything else and what on. net and fernando did was they took our ten oxygen rainy all and incubated it with ease a in any the ph and not surprisingly they saw the reduced products not i'm i buy that kind of what we expected that if we incubated the substrate with a random murdoch case we would just see reduction but also we saw a very unusual type. of psych lies product that that name die quiet dial on in actually are respectable ratio of five to a ratio and again we can characterize the structure of this compound we could do a large scale and the magic reaction scale it up and characterize the go to the cop out by and more and so we kind. speculate that on nature might have come up with a way to make this style to hide and maybe by accident and then on this style to hide was so toxic that the plan just recruited any available ruddock taste to detox a fight this ten opposite during all and perhaps this ten protest from data. doctors was was was just the one that that happened to be that happened to be chosen on and i would guess that if the cyclic product had some favorable by activity that nature really would have pushed for the aleutian of of on of this enzyme to really favor the structure of the sickly product and were very. interested in sort of looking more closely at not just use a but lots and lots of other ruddock taste is and incubating them with this ten oxygen rainy all and really trying to explore how different and signs different ruddock tasers can be incubated with this reactive substrate to see what what what types of structure could could potentially be generated and whether those.
structures exist in nature not or what whether they have any potential bio bio activity or not and i just want to kind of highlight just where we're going on you know there's a whole lot more to buy a synthesis than just the blast and you can imagine that if you could genetically manipulate these pathways you could offer him.
meant the production of any number of the blast and analogues for example were other natural product analogues that may or may not have new and interesting bile activity on and if we couple this with an enzyme engineering and i think this is one of the reasons why identifying new enzymes and buys and that path. please is so important because if you identify them if you understand the structure of them if you have of can really and study them in a detailed mechanistic way you can change them.
and what west glenn a student l. long with ricky run the fan who's earlier work i won't i won't mention were able to do is they were able to say ok there have their these halogen aging and signs that had been identified from bacterial bio synthetic pathways that are known to halliburton eight the indulge rings of trip to fan. trip to fan as you know is the direct precursor to trip to mean and so what west did was he said ok it might be a valuable thing to put koreans and bromine is on a variety of natural products the addition of halogens is known to perturbed the pharmacological activity of many many different natural prada. and it would be really good to have the tools in place the ends of magic tools in place to be able to insert these types of bought of occasions into these biases that pathways and so just kind of is a proof of principle west said ok we're going to look at the scripps the structure of red age and he we were lucky that can be drawn in chris walsh had solved the crystal structure of this.
thousand any and simon and and had a detailed mechanistic understanding of it and we looked at how trip to fan bound in its active site and we said ok we really don't want trip to fan to be the substrate we really would prefer trip to mean the direct precursor to finish last in another monitor been in the local its to be favored is a substrate. and so west noted that this residue right here was actually pretty close to this car box league group which distinguishes trip to fan from trip to mean and west made a whole series of mutations at this position to try to switch the selective any of this sends i'm from trip to fan trip to mean and in fact he was able to do this so this is just a competition as they were we. for said the enzyme of the one to one mixture of trip to fan and trip to mean and we can see in the wild type on the halogen ending enzyme we really don't see any halogen ageing of trip to been but in this mutant that a west discovered we can see that there's virtually no halogen nation. of the native substrate trip to fan but he does see pretty good our conversion of trip to mean into chlorinated a trip to mean and west can go and he can transform that mutant back into katherine's this ruse a us on and actually observe the formation of halogen a doubtful its from as a result of that experiment. and so i just sort of mention that is kind of an example of the power of what you can do when you can sort assembling in manipulating these pathways and what we're doing in tandem with these enzymes discovery an enzyme engineering experiments is really trying to assemble the whole pathway together so that we can do these types of experiments and generate these compounds. much more efficiently and this is just a very sort of early simple example of kind of where we're going where we're taking sort of the enzymes involved in second logan virus and the system and coming up with ways to put them together into pathways and had are all basically expressing them in various different production platforms like on a kosher an event that yama for example and various. abele organism so that we can try to do these enzyme engineering experiments on much much more efficiently. ok so i think i'm going to stop there and i again i want to thank you very much on for your attention and the invitation and if you have any questions i'd be happy to try to answer them.