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  • ELIZABETH NOLAN: --by talking about ClpX.

  • And then we're going to move into module 4--

  • which is the last module before spring break--

  • on synthases and assembly line biosynthesis.

  • So basically last time, where we left off is,

  • we went over experiments that were

  • done to look at denaturation, translocation,

  • and degradation by ClpXP.

  • And closed with a question about what actually is going on

  • in ClpX with this ATP binding and hydrolysis

  • to allow for these condemned protein

  • substrates to be unfolded and translocated

  • into the degradation chamber.

  • And I left you just with the statement that although we

  • think about ClpX as this hexamer that has six identical

  • subunits, what studies have shown is that there's some

  • inherent asymmetry within this AAA+ ATPase.

  • And that's what we're going to talk about a little bit.

  • So this is just a slide from a few lectures ago

  • that's showing the top view and side view of ClpX

  • and how we've thought about this.

  • And I think these studies just really

  • highlight how complicated these machines are

  • and that there's still a lot more we

  • need to figure out here.

  • So as I said last time, this asymmetry

  • comes from whether or not each ClpX subunit

  • is bound to nucleotide.

  • And so basically, from looking at many different crystal

  • structures, what can be done is that the ClpX subunits

  • can be divided into two different types based

  • on conformation here.

  • And so in thinking about this, we

  • want to first think about the ClpX domain organization.

  • And if we just think about this, what ClpX has is

  • an end domain followed by a domain

  • that's called the large domain and then followed

  • by a small domain.

  • So 633 amino acids, just to give you a sense of size,

  • and about 69 kilodaltons per subunit.

  • And so what we're going to focus on

  • are the large and the small subunits

  • and what's observed from many different crystal structures.

  • And so these two different types of subunit

  • have been described as loadable and unloadable,

  • and that depends on whether or not nucleotide is bound.

  • So if we consider of these two types,

  • just thinking about the large and small domains,

  • we have this loadable arrangement

  • which binds nucleotide.

  • And in cartoon, something like this.

  • So we have the large subunit.

  • We have the small subunit.

  • And we have this region that's called a hinge.

  • So this is one ClpX.

  • So ATP binds.

  • And so the other type is described as unloadable.

  • And this type of subunit does not bind

  • nucleotide when in this unloadable conformation.

  • And so we can draw this.

  • Here, again, we have the large subunit.

  • And there's a change in conformation.

  • And here's the small subunit here.

  • So what's been found from looking at many crystal

  • structures is that within the ClpX hexamer,

  • there's an arrangement of these loadable and unloadable

  • subunits.

  • So in many crystals, what's found

  • is that there's four loadable--

  • I'm just going to do with "L"--

  • plus two unloadable subunits arranged with about

  • two-fold symmetry, so LULLUL.

  • So there's some asymmetry in the subunits.

  • And so also from these crystal structures,

  • there's some more observations that we

  • don't see with just these cartoons of the 6-mer.

  • So we can learn about how subunits interact, of course,

  • and this is what's shown.

  • So if we look at these structures

  • and consider how these subunits interact, what we find

  • is that the small subunit of one ClpX--

  • or sorry, the small domain of one ClpX subunit interacts

  • with the large domain of the adjacent ClpX subunit.

  • And so we can draw this.

  • Basically, if we consider a large domain--

  • and let's say this is subunit 2--

  • then what we find is that there's

  • the small domain and then the large domain

  • of subunit next door.

  • So let's call this subunit 1.

  • So here's our hinge of subunit 1,

  • and ATP binding happens in here.

  • So we can think about this arrangement.

  • And then what's been defined is something called a rigid body.

  • And so this rigid body is comprised

  • of the large domain of one subunit

  • and the small domain of the next.

  • Rigid body.

  • So large domain of one ClpX and small domain of another subunit

  • that's adjacent.

  • So in thinking about this, we can consider the ClpX hexamer

  • in another way.

  • So how I've initially presented it to you

  • when we introduced these oligomers is just as a 6-mer,

  • right?

  • 6 subunits.

  • But another way to think about ClpX

  • is that it's actually six rigid bodies that

  • are connected by hinges, where each rigid body has

  • a component from two subunits, a large domain from one

  • and a small from another.

  • And so the hinges are within a single subunit

  • based on this cartoon where ATP binds.

  • And so the thinking is that ATP binding and hydrolysis

  • results in changes in the hinge geometry

  • and that this change in confirmation

  • in the hinge with ATP binding and hydrolysis

  • allows for conformational change in another subunit here.

  • So six rigid bodies connected by six

  • hinges, effectively, as opposed to just six

  • standalone subunits.

  • Each subunit's communicating with one another here.

  • So this is pretty complicated, right?

  • It's another level of sophistication

  • within this hexamer here.

  • So what about these loadable and unloadable conformations?

  • I've told you that in these crystal

  • structures, what's seen often are these four

  • loadable and two unloadable subunits

  • with a particular arrangement.

  • So we can ask the question, do these individual subunits

  • maintain the same conformation during these attempts

  • to denature and translocate polypeptides?

  • So is one subunit just committed to being

  • loadable and another subunit committed to being unloadable?

  • Or did they switch dynamically?

  • And so recently, there were a number

  • of studies looking at that.

  • And effectively, as of a few years ago,

  • many studies suggest--

  • or support switching by a given subunit.

  • And they also indicate that every ClpX subunit

  • must bind to ATP at some point during these cycles

  • for unfolding and translocation.

  • But they're not all doing it at the same time.

  • So the way to think about this is

  • that there's some dynamic interconversion

  • between these loadable and unloadable subunits

  • within the hexamer, and somehow--

  • yep?

  • AUDIENCE: I just want to ask you--

  • ELIZABETH NOLAN: Going to make trouble?

  • JOANNE STUBBE: --a question.

  • Yeah.

  • So when you have all these structures,

  • are they all with an ATP analogue?

  • ELIZABETH NOLAN: I don't know the answer to that.

  • JOANNE STUBBE: OK.

  • Because ATP analogues have wide--

  • you guys have already seen ADPNP or ADPCH2P They really

  • have very different properties when you study these, the ATPs.

  • So if these-- and probably they don't have ATP because they

  • probably--

  • ELIZABETH NOLAN: Right.

  • They want to get a stable--

  • JOANNE STUBBE: So anyhow, that's something to keep

  • in the back of your mind.

  • ELIZABETH NOLAN: So just is this an artifact is what

  • JoAnne's suggesting from use of a non-hydrolysable ATP

  • analogue.

  • JOANNE STUBBE: And there's many examples

  • of that in the literature.

  • Everybody uses it.

  • It's just something you need to keep in the back of your mind.

  • That's the best we can do.

  • ELIZABETH NOLAN: So next week, someone

  • should ask during recitation there, for that.

  • So what about the mechanical work?

  • How this is often depicted, in terms of grabbing and pulling

  • on a polypeptide substrate, is via these rigid bodies.

  • And we're not going to go into details about this,

  • but just to describe the typical cartoon picture effectively,

  • imagine we have some polypeptide that needs to enter

  • the degradation chamber.

  • So those pore loops we heard about that

  • are involved in substrate binding

  • are in the large domain of ClpX.

  • So here we have one large domain,

  • and then we can have the small domain of the adjacent subunit

  • here.

  • And just imagine here we have another large domain

  • with its pore loop.

  • And then we'd have the adjacent subunit here.

  • So effectively, it's thought that these pore loops

  • in the large domains grip the substrate and help

  • drag the substrate to allow for translocation

  • into the degradation chamber.

  • So this would be going to the chamber, that direction

  • here for that.

  • So somehow the ATP binding and hydrolysis

  • is allowing this to occur--

  • so to ClpP here for that.

  • So next week in recitation, you're

  • going to have a real treat because an expert, Reuben, will

  • be discussing some single molecule methods that

  • have been applied to studying this degradation chamber.

  • So bring your questions to him because he really

  • knows what is state of the field right now for this.

  • So we've talked a lot about how the substrate needs to get in.

  • We have the SSRI tag.

  • We have all of this ATP consumption unfolding,

  • translocation by ClpX.

  • And then we talked about the serine protease mechanism

  • in terms of how peptides are degraded in the chamber.

  • So then the final question just going to touch upon is,

  • how does the polypeptide that's been degraded

  • get out of the chamber?

  • So ClpXP will give products that are

  • 7 to 8 amino acids in length, so short polypeptides.

  • So how are they released?

  • And we can think about two possibilities

  • for how these polypeptides are released.

  • One is that they're released through the axial pores.

  • So somehow those pores that allow polypeptide substrate

  • to go in also allow product fragments to go out.

  • And then the second option is that there's release

  • through transient side pores between the ClpP 7-mers.

  • So effectively, if we imagine coming back to our ClpP,

  • we have a 7-mer--

  • back-to-back 7-mers, do the fragments

  • come out, say, of the hole?

  • Or somehow do they come out from this region here?

  • To the best of my knowledge, this is a bit unclear,

  • and I don't think they're mutually exclusive.

  • So questions have come up.

  • If they're to come out of an axial pore,

  • does that mean ClpX has to be dissociated?

  • In terms of this equator region, there

  • are structures showing that this degradation

  • chamber can breathe.

  • And there's a picture of that in the posted notes from Friday

  • where you can see opening here.

  • And there has been some experiments

  • done where people have put cysteines in this region

  • by site-directed mutagenesis.

  • So you can imagine, for instance,

  • if you have a cysteine here and a cysteine here,

  • and you oxidize to form a disulfide such that those two

  • 7-mers are locked together.

  • You can ask, if we load the chamber with small polypeptides

  • and we have these effectively cross-linked by disulfides,

  • can the polypeptides get out?

  • And then if we reduce this to have them no longer attached

  • to one another, do those polypeptides

  • stay put or not there?

  • Those experiments gave some evidence

  • for release of peptides through this region here,

  • but there's also evidence for release of peptides

  • through the pore.

  • And in terms of cartoon depictions.

  • In the lecture notes, if you take a close look,

  • you'll see that both come out there.

  • So I'd say if you're curious about that,

  • you can read some of the literature

  • and come to some own conclusions.

  • One last point on the Clp system before we move on to module 4,

  • you should just be aware that there's other Clp family

  • members.

  • So not only ClpX and P. And so in the Clp system--

  • actually, I'm going to make one other point after this

  • too, about degradation chambers.

  • So there are players ClpA, ClpB, in addition to ClpX.

  • So these are all three different AAA+ ATPases.

  • And you've actually encountered ClpB last week.

  • So this is HSP 100, which came up in question 2 on the exam

  • there by another name.

  • And then in addition to ClpP, there's

  • also ClpS and some other players here for that.

  • They each have their own personality within protein

  • quality control here for that.

  • And then we've only looked at this degradation chamber

  • from bacteria.

  • You might want to ask the question,

  • what happens in other organisms?

  • And the answer is that the complexity varies and systems

  • become tremendously more complex as you move from bacteria

  • into eukaryotes there.

  • And so if we consider the different degradation chambers,

  • what do we see?

  • So we find these proteasomes in all forms of life.

  • And as I just said, the level of complexity

  • varies depending on the organism.

  • And so what we've seen with ClpP is the most simple system

  • where we have two rings that have only one type of subunit.

  • So just say E. coli.

  • One type of subunit.

  • What happens if we go to archae?

  • We find that we have four rings, each of which is 7-mer.

  • And these four rings include two different types of subunits.

  • So I'll call these alpha and beta.

  • So what we find is that there's a 7-mer of 7 alpha subunits,

  • then 7-mers that have 7 beta subunits, and here

  • a 7-mer with alpha.

  • So we see two types of subunit and four rings.

  • So then what about yeast?

  • Tremendously complex.

  • So we have this architecture again

  • of four rings, an organized alpha, beta, beta, alpha.

  • But what we find in this case is that in each of these--

  • I'm not going to draw it like that, but each of these

  • have seven different subunits.

  • There's a depiction of this in the notes.

  • So just imagine-- how does this get assembled?

  • I have no clue.

  • But somehow each of these heptamers

  • has to be assembled with seven different subunits.

  • And then they're put together in this series of four rings.

  • And then as you'll see after spring break in JoAnne's

  • section, the eukaryotic proteasome

  • has this 19S regulatory particle that's

  • involved in recognizing condemned proteins that

  • have polyubiquitin chains.

  • And compared to the ClpX ATPase, it's much, much more complex.

  • So there's many different proteins

  • that constitute this necessary part of the machine.

  • But there is a hexamer, ATPase hexamer, within there

  • to facilitate translocation of the polypeptide

  • into the degradation chamber.

  • So some of this will come back again

  • in the latter half of the class.

  • So with that, we're going to close on degradation and move

  • into module 4, which is focused on macromolecular machines that

  • are involved in the biosynthesis of natural products,

  • specifically polyketides and nonribosomal peptides.

  • And so we're completely taking a loop back

  • to thinking about a biological polymerization,

  • like what we were thinking with the ribosome

  • from the process of breaking down a polypeptide.

  • And so where are we going?

  • We can think about assembly lines,

  • although this is a helpful way on the board

  • to think about these systems.

  • But it's not really what they look like.

  • And you'll learn about that in recitation this week.

  • Yeah?

  • AUDIENCE: Could you explain the interaction

  • between ATP and the hinge area?

  • ELIZABETH NOLAN: OK.

  • So the ATP binding site is just rewinding here

  • in that hinge region.

  • And there's going to be conformational change

  • in the hinge with ATP binding and hydrolysis there.

  • And that's sufficient in terms of the level of detail

  • for this.

  • But the main thing to keep in mind, each subunit binds ATP.

  • But on the basis of the information gathered

  • with the caveats JoAnne brought up,

  • different subunits bind ATP at different times in the cycle.

  • AUDIENCE: OK.

  • Thank you.

  • ELIZABETH NOLAN: And changes in this subunit,

  • conformational changes that result from that,

  • can be translated to the next door subunit here.

  • AUDIENCE: OK.

  • ELIZABETH NOLAN: OK.

  • So where are we going?

  • By a week from now, you should have a good handle

  • on how to think about the biosynthesis of structures

  • like erythromycin, of penicillin.

  • These are products of assembly lines.

  • And so where we'll go is with a brief overview of fatty acid

  • biosynthesis and then look into polyketide synthase

  • and nonribosomal peptide synthetase assembly lines here.

  • And then some case studies.

  • So on the topic of ATP, where we just went back to with ClpX,

  • just taking a look here, what do you

  • know about these systems in ATP by the names?

  • This is just a little language use and definition.

  • So there's a subtle difference here.

  • What's the difference?

  • AUDIENCE: Synthase versus synthetase?

  • ELIZABETH NOLAN: Yeah.

  • And what does that tell you right off the bat?

  • About ATP.

  • So it's a subtlety, right?

  • Synthase is a general term.

  • Synthetase indicates ATP is involved.

  • So as we'll see, these nonribosomal peptide

  • synthetases employ ATP.

  • And we're going to see chemistry very similar to what

  • you saw with the aminoacyl-tRNA synthetases

  • in terms of activating amino acid monomers.

  • But in this case, the machine is forming a nonribosomal peptide

  • rather than a ribosomal peptide here.

  • If you are not familiar with fatty acid biosynthesis,

  • I highly encourage you to go do some review,

  • either from your 5.07 notes last term if you were in the class

  • or from a biochemistry book.

  • And there'll be some additional slides of overview information

  • posted online.

  • So we'll just touch upon it today but not

  • go into tremendous detail here.

  • So what are our questions for this module?

  • I think for most everyone in the room,

  • this module will contain the most new information

  • from the standpoint of a new system

  • compared to what we've talked about so far.

  • So what are polyketides and how are these molecules

  • biosynthesized by polyketide synthases?

  • What are nonribosomal peptides and how are they

  • made by these machines called nonribosomal peptide

  • synthetases?

  • And what we're going to look at is the assembly line

  • organization, so effectively the organization of domains that

  • provide these linear polymers.

  • So what is the assembly line organization and logic for PKS?

  • And likewise for NRPS.

  • And then we can ask, how can a given assembly

  • line for a given PKS or NRPS natural product

  • be basically predicted from the structure

  • of the natural product?

  • So you should be able to work back and forth in terms

  • of looking at a structure and coming up

  • with a biosynthetic prediction and also seeing

  • biosynthetic machinery and getting a sense as to what

  • that small molecule metabolite's backbone might look like.

  • How are these studied experimentally?

  • And we'll look at the biosynthesis

  • of a molecule called enterobactin as a case study.

  • And so one thing I'll just point out right now

  • is that these synthases and synthetases do not

  • look like an assembly line.

  • And we'll draw domains in a linear order which really

  • facilitates thinking about the chemistry,

  • but the structures are not just a line of domains or proteins

  • next door to one another.

  • And this week in recitation, you'll

  • get to see some cryo-EM studies on fatty acid synthase and

  • related machines there which will give you

  • a sense of their dynamics.

  • So just a review.

  • If we think about template-dependent

  • polymerizations in biology, we're

  • all familiar with DNA replication, transcription,

  • and translation.

  • And what you'll see in this unit is

  • that these template-driven polymerizations

  • occur in the biosynthesis of natural products here.

  • And effectively, these assembly lines, in a way,

  • provide this template.

  • So they're small molecules being biosynthesized

  • by microbes using some pretty amazing machinery.

  • So when we think about template-driven

  • polymerizations, we think about an initiation process,

  • elongation process, and termination.

  • We saw that with a translation cycle.

  • And we'll see the same type of systems here.

  • So what does some of these structures look like?

  • Here are just some examples on the top

  • of polyketides, two examples.

  • They look very different at first glance, and they are.

  • So we have tetracycline.

  • We have four fused 6-membered rings.

  • It's an aromatic polyketide, an antibiotic.

  • We have this erythromycin here, which is a macrolide.

  • We encountered macrolines in the translation section

  • because they bind the ribosome, another type of antibiotic.

  • If we look at some nonribosomal peptides, all of that

  • can be used clinically.

  • We see the penicillins.

  • So we have a 4 or 5 fused ring system here, a beta-lactam.

  • This comes from three amino acid building blocks initially.

  • We have vancomycin.

  • This is an antibiotic of last resort.

  • And this structure looks really quite complicated,

  • but what we'll see is that it's based on seven

  • proteogenic amino acids.

  • So it's the 7-mer peptide backbone

  • that gives rise to this structure here for that.

  • And then we see there's some sugars,

  • so these can be put on by other enzymes here.

  • So on top we see a lot of ketones and OH groups.

  • Those are good hints that maybe polyketide logic is being used.

  • Here we see a number of peptide bonds,

  • amide bonds, a good indicator of NRPS at play.

  • And here, just to point out, these systems get very complex.

  • And there's natural products out there

  • that are biosynthesized from a combination

  • of polyketide synthase logic and nonribosomal peptide synthetase

  • logic here.

  • These include molecules like yersiniabactin.

  • This is an iron chelator produced by Yersinia pestis,

  • and some pathogenic E. coli, this immunosuppressant

  • rapamycin as examples.

  • So as we move forward, I put a lot

  • of structures of small molecule metabolites in the slides.

  • You can go back and use them as a way

  • to study and try to make predictions

  • about what is the machinery at play,

  • for instance, to give all of these heterocycles?

  • How are those made?

  • We'll see the assembly line does that.

  • So what organisms produce these molecules?

  • Largely, bacteria and fungi.

  • And there are some correlations out there, I'll just point out,

  • related to genome size and the number

  • of metabolites being made.

  • So bioinformatics guides a lot of current studies

  • of the biosynthesis of these types of molecules.

  • So you can imagine that you sequence a genome.

  • You have some information about gene clusters.

  • So these are groups of genes where

  • the proteins work together to biosynthesize the molecule.

  • And often, the genes that encode proteins in these metabolites

  • are clustered.

  • And so bioinformatics approaches can help find these.

  • What's found is that for bacteria, some phyla

  • are more prolific producers of these molecules than others.

  • And what's been shown in a general way

  • is that organisms with small genomes--

  • so something like E. coli--

  • produce fewer of these metabolites.

  • That's not to say none.

  • So enterobactin, which we'll look at for a case study,

  • is made by E. coli.

  • But they don't make as many.

  • And effectively, organisms with larger genomes produce more.

  • And so here is just a correlation

  • between the number of genes.

  • And the genome size of the organism

  • where they see around 3 Mb, there's a switch here.

  • Often, these molecules-- yeah?

  • AUDIENCE: Is there any hypotheses

  • about an evolutionary driving factor

  • for the development of this machinery

  • and why it correlates to genome size?

  • ELIZABETH NOLAN: If there is, I don't know.

  • I don't think about evolution very well, quite frankly.

  • What is thought is that many of these molecules

  • are thought to be involved in defense

  • and that an organism with a smaller genome size

  • uses other strategies.

  • And so for instance, E. coli, which

  • I cited as a small genome, will use

  • a number of ribosomal peptides as defense molecules

  • that get post-translationally modified after the fact.

  • But why that organism chooses to do that versus say something

  • like Streptomyces that produces many, many different natural

  • products, I'm not sure about that.

  • So let's look at an example of a gene cluster,

  • just so you get a sense of how much machinery

  • is required to do the full biosynthesis of a molecule.

  • So this is for a nonribosomal peptide shown here.

  • It has some structural similarities

  • to the vancomycin we saw on a prior slide,

  • and it is a member of the vancomycin family.

  • So this gene cluster for the biosynthesis of this metabolite

  • contains 30 different genes and is depicted here.

  • So each one of these arrows indicates an open reading

  • frame.

  • So each one begins with a start, ends with a stop codon.

  • And it's assumed to be the coding sequence of the gene.

  • And so what is encoded in these 30 genes?

  • Well, first there are the genes for what

  • we call the assembly line.

  • And if it isn't clear what assembly line means,

  • as we move forward through this week, it will be.

  • So there's genes required to make the 7-mer polypeptide

  • backbone.

  • There's genes required for modification of the backbone.

  • So how do these sugars get attached, for instance?

  • Those are going to be some tailoring enzymes.

  • And then if you take a close look,

  • there's a number of non-proteinogenic amino acids

  • in this molecule, and that means they

  • have to come from somewhere.

  • And so this gene cluster also includes

  • genes that are required for the biosynthesis of those monomers.

  • So there's a lot of effort going in to making

  • this molecule by some organism.

  • And so presumably, under some set of conditions,

  • it's important.

  • So moving towards the chemistry, with that background in hand,

  • what are some points to make?

  • So what we'll learn and see is that the assembly lines that

  • produce the polyketides and nonribosomal peptides

  • are macromolecular machines.

  • So there's dedicated macromolecular machines

  • for the biosynthesis of these secondary metabolites.

  • And so what are secondary metabolites

  • versus a primary metabolite?

  • So what's a primary metabolite?

  • AUDIENCE: I'm not even totally sure how to define metabolites.

  • Isn't metabolites what goes in?

  • Or what comes out?

  • ELIZABETH NOLAN: Rebecca?

  • AUDIENCE: Or easily produced directly from the materials

  • the cell's consuming?

  • ELIZABETH NOLAN: So presumably, the cell

  • needs to get materials to biosynthesize

  • the secondary metabolites too, right?

  • Somewhere, these amino acid monomers

  • or the monomers that are used for polyketide synthetase

  • need to--

  • they'd have to come from somewhere, right?

  • So are primary metabolites important for growth?

  • AUDIENCE: Yes.

  • ELIZABETH NOLAN: Yes.

  • Development?

  • Reproduction?

  • AUDIENCE: Yes.

  • ELIZABETH NOLAN: Yeah, right.

  • Under normal conditions, right?

  • We're in trouble if we don't have our primary metabolites

  • there, whether they're ingested or biosynthesized.

  • What about a secondary metabolite?

  • Just taking that--

  • AUDIENCE: I'm guessing it's not necessary.

  • AUDIENCE: --something we can make from primary metabolites?

  • ELIZABETH NOLAN: No.

  • Well, you can.

  • You can.

  • So a secondary--

  • AUDIENCE: --necessary?

  • ELIZABETH NOLAN: Yeah.

  • A secondary metabolite is not required for normal growth,

  • development, reproduction.

  • So for some reason, under some circumstances of need,

  • these secondary metabolites get produced.

  • So for some of these antibiotic molecules, maybe

  • the organism needs to defend itself.

  • In the case of enterobactin or yersiniabactin,

  • maybe that organism needs iron.

  • And so it's producing a molecule that

  • will help it obtain that there.

  • So what is going on?

  • We've seen some pretty complex molecules.

  • What we're going to see is that these assembly lines convert

  • simple acid monomers, if it's a polyketide synthase

  • or amino acid monomers for a nonribosomal peptide

  • synthetase, into linear polymers.

  • So we're going to look at template-driven polymerizations

  • that initially give linear polymers.

  • And in the case of PKS, this is very similar

  • to fatty acid biosynthesis.

  • What we see is that the assembly lines

  • allow for iterative additions of malonyl and methylmalonyl

  • units.

  • And they catalyze carbon-carbon bond formations.

  • In the case of nonribosomal peptide synthetases,

  • what we'll see is that these allow for condensations

  • of amino acids to form peptide bonds

  • and effectively form nonribosomal polypeptides.

  • So polypeptide synthesis without the ribosome.

  • So even though the PKS and NRPS are forming a different type

  • of bond and that requires different chemistry, what

  • we'll see is that they use very similar logic.

  • And just getting the logic sorted out initially

  • makes life much easier down the road.

  • So take some time to look over the depictions in the notes

  • outside of class as we go forward.

  • So these assembly lines use acyl or aminoacyl thioesters

  • as the activated monomer units.

  • So then how do we get from this linear polypeptide

  • to some more complex structure?

  • The short message on that is that the, quote, "polymers"

  • that are produced--

  • and they may be short, right?

  • We just saw-- they are short, 7 amino acids for vancomycin.

  • They can undergo further elaboration

  • to give these complex structures.

  • So there can be tailoring enzymes

  • that work on the products of the assembly line.

  • Or there can be domains in the assembly line that

  • give additional activities that allow for methylation

  • or cyclization here.

  • So we can think about fatty acid synthase as a paradigm here.

  • And so if we think about fatty acid

  • biosynthesis making some molecule like this oil here,

  • just as brief overview in the last few minutes of class.

  • Fatty acids are synthesized by FAS.

  • And what happens is that there's elongation

  • by one unit at a time.

  • And each unit provides two carbons.

  • So there's two carbon atoms per elongation.

  • And so hopefully you're all familiar with two ways

  • to form a carbon-carbon bond, at least related

  • to biochemistry, one of which is Claisen condensations.

  • So Claisen condensations allow for carbon-carbon bond

  • formation and join the units.

  • To keep in mind, the monomers are always

  • thioesters, not oxoesters.

  • And for fatty acid biosynthesis, the two monomer units

  • are shown here.

  • So we have a starter and an extender, acetyl CoA or malonyl

  • CoA here.

  • And here we have coenzyme A.

  • So just as a brief review, if we think about these monomer

  • units--

  • so here we have acetyl CoA.

  • So what can we say about this guy here, in this thioester?

  • So is this acidic or not?

  • Compared to an oxoester.

  • How many of you have heard about fatty acid biosynthesis?

  • AUDIENCE: [INAUDIBLE]

  • ELIZABETH NOLAN: So why are thioesters used and not

  • oxoesters?

  • AUDIENCE: [INAUDIBLE] use the other end?

  • ELIZABETH NOLAN: OK.

  • So we'll go into a little more detail on Friday

  • to make sure the chemistry is straight here

  • because I'm not certain it is.

  • So--

  • AUDIENCE: Is oxoester referring to not that [INAUDIBLE]----

  • ELIZABETH NOLAN: OK.

  • So for Friday, think about a thioester versus an oxoester,

  • and how do properties differ?

  • And why might we want to be using thioesters?

  • And also review the Claisen condensation

  • because that's the chemistry that's

  • going to be happening to form the carbon-carbon bonds

  • in the fatty acid synthase and in the polyketide synthases.

  • And what we're going to see is that the monomers in each case,

  • they're tethered as thioesters.

  • So why is that?

  • And I will turn around and point at somebody,

  • and you can let us know.

  • Are you excited?

  • OK.

  • So you're off the hook for Wednesday.

  • I need to be out of town, and I'll see you on Friday.

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B2 中高級

15.PK和NRP合成酶1 (15. PK and NRP Synthases 1)

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    林宜悉 發佈於 2021 年 01 月 14 日
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