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  • ELIZABETH NOLAN: We're going to get started

  • and what we'll do today is continue

  • with fatty acid synthase.

  • Because that's the paradigm for these macromolecular machines,

  • like the PKS, and then we'll go over

  • the logic of polyketide synthases.

  • So we left off last time with this discussion

  • about some molecules that will be involved

  • and in particular thioesters, and I

  • asked about the alpha H. So just going back

  • to introductory organic chemistry, what

  • are the properties of this atom here?

  • AUDIENCE: [INAUDIBLE] acidic.

  • ELIZABETH NOLAN: Yeah.

  • OK, right.

  • So this is acidic.

  • So if you have--

  • OK?

  • So what that means is if there is a base that can deprotonate

  • that, we can get an enolate.

  • OK, and this is the type of chemistry

  • that's going to be happening with the thioesters that

  • are used in fatty acid synthase and also polyketide synthase.

  • And just to rewind a little bit more,

  • if we think about carbon-carbon bond forming reactions

  • in nature, which is what's happening in fatty acid

  • biosynthesis and in polyketide biosynthesis,

  • effectively, nature uses three different types of reaction.

  • OK, so one is the aldol, two are the Claisen,

  • and three [INAUDIBLE] transfer.

  • OK, and so we're going to see Claisen condensations in FAS

  • and PKS biosynthesis.

  • And then after spring break, when

  • Joanne starts with cholesterol biosynthesis,

  • that will involve [INAUDIBLE] transfers.

  • And hopefully, you've seen aldol reactions sometime

  • before within biochemistry here.

  • OK?

  • So we need to think about just what the general Claisen

  • condensation is that we're going to be seeing here

  • and the consequences of this acidic proton.

  • So also just keep in mind, rewinding a little more,

  • nature uses thioesters not esters,

  • and so the alpha H is more acidic.

  • The carbonyl is more activated for nucloephilic attack.

  • And there's some resonance arguments

  • and orbital overlap arguments that

  • can guide those conclusions, if you wish to do them here.

  • OK.

  • So let's imagine that we have a thioester.

  • We have a base.

  • OK, that's going to be [INAUDIBLE],, which

  • is going to get us to here.

  • So this is our nucleophile, and what you'll see coming forward

  • is an enolate.

  • So imagine we have that, and we add it with another thioester,

  • and here's our electrophile.

  • What do we get?

  • We get formation of a beta-keto thioester, which is the Claisen

  • condensation product.

  • OK, you have two thioesters.

  • OK?

  • So effectively, this acyl thioester is doubly activated,

  • so it can be--

  • did I lose it?

  • Oh no, problems.

  • Sorry about that.

  • It can be activated as an electrophile at the C1

  • position, so next door to the sulfur.

  • And it can be activated as a nucleophile at the C2 position

  • here.

  • So this is the general chemistry that's

  • going to be happening by FAS and PKS

  • in terms of forming carbon-carbon bonds

  • between monomers here.

  • OK?

  • So in fatty acid synthase, we have two monomer units.

  • OK?

  • So we have acetyl-CoA and malonyl-CoA.

  • Acetyl-CoA is the starter unit, sometimes called unit 0,

  • and then malonyl-CoA is the extender.

  • And so recall that in fatty acid biosynthesis,

  • each elongation event adds two carbons,

  • and if we look at malonyl-CoA, we have three here.

  • Right?

  • So there's decarboxylation of malonyl-CoA

  • to generate a C2 unit, and there's

  • details of that in the lecture 15 notes.

  • And SCoA is coenzyme A, here, and there's some information

  • as to the biosynthesis of these starter and extender units

  • in the notes.

  • We're not going to go over that in lecture here.

  • So in terms of using these monomers to obtain

  • fatty acids, first what we're going to go over

  • are the domains in FAS.

  • And so we can consider domains that

  • are required for extension of the fatty acid chain

  • and then domains that are required

  • for tailoring of that effectively to reduce

  • the carbonyl, as shown.

  • And we're going to go through these,

  • because what we're going to find is

  • that with polyketide biosynthesis,

  • the same types of domains are used.

  • So this logic extends there.

  • OK.

  • So first, we have domains required

  • for elongation of the fatty acid chain by one two-carbon unit.

  • OK.

  • So these include domains that may be abbreviated

  • as AAT or MAT, and they can be grouped as AT

  • and stand for acetyl or malonyltransferase.

  • OK.

  • We have an Acyl Carrier Protein, ACP,

  • and this carries the growing chain between the domains

  • of fatty acid synthase.

  • And so in recitation this week, you're

  • going to see how these domains move around

  • and talk about the length of this acyl carrier protein.

  • We also have the ketosynthase.

  • So what the ketosynthase does is it accepts the growing

  • chain from the acyl carrier protein,

  • and it catalyzes the Claisen condensation

  • with the next monomer.

  • And what we'll see is that this ketosynthase

  • uses covalent catalysis, and via a cysteine thiolate residue.

  • So these are the key domains required

  • for elongation of the chain.

  • OK?

  • And then what we also need are domains required for tailoring,

  • and just to clarify, I'm defining domain here

  • as a polypeptide with a single enzymatic activity.

  • So domains can be connected to one another,

  • or they can be standalone in different types of synthases,

  • but domain means polypeptide with

  • a single enzymatic activity.

  • So what are the domains required for tailoring?

  • And these work after addition of the C2 unit

  • to the growing chain.

  • So first, there's a ketoreductase.

  • And as indicated, what this enzyme does

  • is it reduces the carbonyl of the previous unit to an OH

  • and uses an NADPH H plus.

  • We also have the dehydratase here,

  • and this forms an alpha, beta-alkene from the product

  • of the ketoreductase action.

  • And then we have an enoyl reductase

  • that reduces this alpha, beta-alkene,

  • and this also requires NADPH H plus here.

  • And then some fatty synthases use

  • a domain called a thioesterase for chain release,

  • and that's noted as TE.

  • And we'll see thioesterases in the PKS and in our PS sections

  • here.

  • So one comment regarding the acyl carrier protein,

  • and then we'll just look at the fatty acid synthase cycle

  • and see how these domains are acting.

  • So in order for the acyl carrier protein

  • to carry this growing chain, it first

  • needs to be post-translationally modified

  • with what's called a PPant arm.

  • And that arm provides the ability

  • to have these monomers, or growing chains, linked

  • via a thioester.

  • And so just to go over this post-translational

  • modification, so post-translational modification

  • of acyl carrier protein with the PPant arm.

  • OK.

  • If we consider apo acyl carrier protein,

  • and apo means that the PPant arm is not attached.

  • There's a serine residue.

  • An enzyme called the PPTase comes along,

  • and it allows for post-translational modification

  • of this serine using CoASH, releasing 3', 5'-ADP to give

  • ACP post-translationally modified with the PPant arm.

  • OK?

  • And we'll look at the actual chemical

  • structures in a minute.

  • What I want to point out is that throughout this unit,

  • this squiggle, some form of squiggle here,

  • is the abbreviation for the PPant arm.

  • OK?

  • And this is very flexible and about 20 angstroms in length.

  • So what does this actually look like?

  • So here we have CoASH.

  • So PPant is an abbreviation for phosphopantetheine, here,

  • this moiety, and here's the 3', 5'-ADP.

  • And so effectively, what's shown on the board is repeated here.

  • Except for here, we're seeing the full structure

  • of the phosphopantetheinylated acyl carrier protein.

  • So this squiggle abbreviation indicates

  • this post-translational modification

  • onto a serine residue of the ACP.

  • Just as an example of structure, so here

  • is a structure of acyl carrier protein from E. coli.

  • It's about 10 kilodaltons, so not very big,

  • and we see the PPant arm here attached.

  • OK?

  • So if we think about fatty acids biosynthesis,

  • we can think about this in three steps, better iterated.

  • OK.

  • So first we have loading, so the acyl carrier proteins need

  • to be loaded with monomers.

  • Sometimes, this step the reactions

  • are described as priming reactions.

  • We have initiation and elongation

  • all grouped together here and, three, at some point,

  • a termination.

  • OK?

  • So we've thought about these before from the standpoint

  • of biological polymerizations.

  • So what about the FAS cycle?

  • Here's one depiction, and I've provided multiple depictions

  • in the lecture 15 notes.

  • Because some people find different cycles easier

  • than others, but let's just take a look.

  • So this charts out the various domains--

  • the starter and the extender and then the chemistry that

  • occurs on these steps.

  • And so what needs to happen is that there

  • needs to be some loading and initiation where

  • the acetyl-CoA is loaded onto an acyl carrier protein.

  • So that's shown here via transferase here,

  • and then, from the acyl carrier protein,

  • this monomer is loaded onto the ketosynthase.

  • If we look here, we have one of our extender units,

  • the malonyl-CoA, and the CO2 unit

  • that gets removed during decarboxylation,

  • as shown in this light blue.

  • OK?

  • We need to have this extender unit also transferred

  • to an acyl carrier protein via the action of an AT.

  • So we see lots of the CoA.

  • Here we have the acyl carrier protein with the PPant arm.

  • It's not a squiggle here.

  • It is the next one with this malonyl unit loaded.

  • There's a decarboxylation, and what do we see happening here?

  • We have a chain elongation event,

  • so Claisen condensation catalyzed

  • by the ketosynthase between the starter and the first extender

  • to give us this beta-keto thioester.

  • So once this carbon-carbon bond is formed to give us

  • the beta-keto thioester, there's processing of the beta carbon

  • via those tailoring domains--

  • the dehydratase and the enoyl reductase.

  • And so we see reduction of the beta ketone here,

  • we see formation of the alkene, and then we see reduction

  • to get us to this point.

  • And so this cycle can repeat itself until, at some point,

  • there's a termination event.

  • And in this case here, we see a thioesterase catalyzing

  • hydrolytic release of the fatty acid chain.

  • This is the depiction you'll see in recitation today,

  • or saw before.

  • And I guess what I like about this depiction is

  • that you see color coding separating

  • the elongation and the domains involved

  • in elongation with then the processing of the beta ketone

  • here and then termination.

  • OK.

  • So we get some fatty acid from this.

  • And so where we're going to go with this overview is looking

  • at the polyketides and to ask what similar and different

  • in terms of polyketide biosynthesis?

  • And so where we can begin with thinking

  • about that is asking what are the starters and extenders?

  • And so these are the starters and extenders

  • we saw for fatty acids, and here are

  • the starters and extenders for polyketides, so very similar.

  • Right?

  • We just see that there's some additional options,

  • so we also have this propionyl-CoA here.

  • In addition to malonyl-CoA as an extender,

  • we see that methylmalonyl-CoA can be employed.

  • So what are the core domains of the PKS?

  • They're similar to those of FAS, and we'll just focus

  • on the PKS side of this table.

  • So this is a helpful table when reviewing

  • both types of assembly lines.

  • So the core means that every module,

  • which I'll define in a moment, contains these domains.

  • So we see that there's a ketosynthase,

  • an acyltransferase, and a thiolation domain.

  • So this thiolation domain is the same

  • as the acyl carrier protein.

  • So there's different terminology used, and within the notes,

  • I have some pages that are dedicated

  • to these terminologies.

  • OK?

  • So for PKS, here, we have the ketosynthase,

  • we have acetyltransferase, and then

  • we have this T domain which equals acyl carrier

  • protein here.

  • OK?

  • So then what about these tailoring

  • domains that were required to produce the fatty acid?

  • What we see in polyketide biosynthesis

  • is that those domains are optional.

  • So one or more of these domains may be in a given module.

  • So that's an overview, and then we'll

  • look at an example of some domains and modules.

  • So we're going to focus on type 1 polyketide synthases.

  • And in these, what we're going to see

  • is that catalytic and carrier protein domains are fused,

  • and they're organized into what we'll term modules.

  • So a module is defined as a group of domains that's

  • responsible for activating, forming the carbon-carbon bonds

  • and tailoring a monomer.

  • So there is an individual module for every monomer

  • within the growing chain.

  • And the order of the modules in the polyketide synthase

  • determines the functional group status,

  • and that functional group status is determined by whether or not

  • these optional domains are there.

  • OK?

  • How do we look for modules?

  • The easiest way is to look for one of these thiolation or ACP

  • domains.

  • So each module has one of these.

  • So you can count your number of T domains,

  • and then you know, OK, there's 7T domains,

  • so there's 7 monomers, for instance.

  • So each Claisen condensation is a chain elongation and chain

  • translocation event.

  • Keep in mind, the starting monomer--

  • so whether that's acetyl-CoA or propionyl-CoA--

  • does not contain a CO2 group.

  • So there's no decarboxylation of the starting monomer,

  • but decarboxylation of malonyl-CoA

  • occurs, like in fatty acid synthase,

  • and if that's the case, it provides a C2 unit.

  • And if methylmalonyl-CoA is the extender,

  • this decarboxylation provides a C3 unit

  • because of that methyl group.

  • So key difference, as we just saw,

  • in fatty acid biosynthesis, we have complete reduction of that

  • beta-keto group in every elongation cycle

  • because of these three tailoring domains--

  • the KR, DH, and ER.

  • In PKS, what can happen is that reduction

  • of this beta-keto group may not happen at all,

  • or it may be incomplete in each elongation step.

  • So what that means is that polyketides

  • retain functional groups during chain elongation.

  • And if you look back at some of the structures that

  • were in the notes from last time,

  • you'll see that, in terms of ketones, hydroxyls,

  • double bonds, et cetera.

  • And also, the other point to note

  • is that there can be additional chemistry,

  • and that these assembly lines where polyketide synthases,

  • non-ribosomal peptide synthatases can contain what

  • are called optional domains.

  • So these are additional domains that

  • are not required for formation of the carbon-carbon bond

  • or amide bond in non-ribosomal peptide synthases.

  • But they can do other chemistry there, so

  • imagine a methyltransferase, for instance, or some cyclization

  • domain.

  • So how do we show these domains and modules?

  • So typically, a given synthase is depicted from left to right

  • in order of domain and bond-forming reactions here.

  • So let's just take a look.

  • So if we consider PKS domains and modules,

  • we're just going to look at a pretend assembly line.

  • OK?

  • So this I'm defining here as an optional domain.

  • So in this depiction, going from left to right,

  • each one of these circles is a domain, so

  • a polypeptide with a single enzymatic activity.

  • Note that they're all basically touching one

  • another which indicates in these types of notations

  • that the polypeptide continues.

  • It's not two different proteins, but we

  • have one polypeptide here.

  • I said that there's modules, and we can identify modules

  • by counting T domains.

  • So here, we have three T domains.

  • So effectively there's three modules.

  • So we have a module here, we have a module here,

  • and we have a module here.

  • What do we see?

  • Two of these modules have a ketosynthase,

  • so that's the domain that catalyzes the Claisen

  • condensation.

  • We have no ketosynthase here, in this first module.

  • Why is that?

  • We're all the way to the left.

  • This is effectively our starter or loading module.

  • So the propionyl-CoA or acetyl-CoA

  • will be here, as we'll see, and there's nothing

  • upstream to catalyze a condensate event with.

  • So there's no KS domain in the starting

  • module here or loading module.

  • OK.

  • So this is often called loading or starter.

  • So if we think about these optional domains for a minute

  • and think about how they work.

  • If we go back to fatty acid synthase, and let's

  • just imagine we have this species attached.

  • We have the action of the KR, the dehydratase,

  • and the ER to give us the fully-reduced species.

  • Where here, we have a CH2 to group

  • rather than the beta-ketone.

  • So what happens in PKS in terms of

  • the different optional domains?

  • So we could have this and have full reduction.

  • We can imagine maybe there's no enoyl reductase.

  • So the module has the ketoreductase

  • and the dehydratase but no enoyl reductase, and so as a result,

  • this polyketide ends up with a double bond here.

  • OK?

  • What if we have nobody dehydratase, like this?

  • OK.

  • We just work backwards from the FAS cycle.

  • We'd be left with this OH group at the beta position.

  • Right?

  • And if we have none of them, so no ketoreductase, dehydratase,

  • or enoyl reductase, the beta-ketone

  • will be retained, here.

  • So what this also means is that you can just

  • look at some polyketide and assess

  • what the situation is from the standpoint

  • of these optional domains.

  • So let's just take an example.

  • If we have three cycles of elongation, and let's

  • imagine we had an acetyl-CoA starter plus three malonyl-CoA.

  • So what do we end up with?

  • Let's imagine our chain looks like this.

  • What do we see?

  • So two carbons are added during each elongation cycle

  • to the chain here, and we can see those here, here,

  • here, and here.

  • OK?

  • So a total of four C2 units, one from the starter

  • and then three from these three extenders.

  • And then we can look at what the functional group status is

  • and say, OK, well here, we have no ketoreductase.

  • And here, there was ketoreductase action,

  • but there's no dehydratase.

  • And here, what do we see?

  • We see that there was a reduction of the beta-ketone

  • and then the action of the dehydratase,

  • but we're left at the alkene, so no enoyl reductase.

  • Right?

  • So just looking, you can begin to decipher

  • in a given module what optional domains are there.

  • So what we'll do is take a look at an actual PKS assembly line

  • and then look at the chemistry happening on it here.

  • These are just for your review.

  • This is a polyketide synthase responsible for making

  • this molecule here.

  • So D-E-B or DEB is a 14-membered macrolactone.

  • It's a precursor to the antibiotic erythromycin here,

  • and this is the cartoon depiction

  • of the polyketide synthase required for the biosynthesis

  • of this molecule.

  • So what do we see looking at this polyketide synthase?

  • So it's more complicated than this one here,

  • but the same principles apply.

  • And what we'll see is that it's comprised of three proteins.

  • There's seven modules, so one loading or starter module

  • and six elongation modules, and there's a total of 28 domains.

  • OK?

  • And I said before, the placement and the identity

  • of these domains dictates the identity of the growing chain.

  • So let's take a look.

  • So first, how do we know there's three proteins?

  • We know that in this type of cartoon

  • because we end up seeing some breaks

  • between different domains.

  • So here, for instance, the AT, the T, the KS, et cetera,

  • they're all attached to one another in the cartoon.

  • That means it's all one polypeptide chain,

  • but this one polypeptide chain has

  • many different enzymatic activities in it,

  • because it has different domains.

  • When we see a break--

  • so for instance here this T domain and this KS domain

  • are not touching one another.

  • That means we have two separate proteins.

  • So this T domain is at the terminus of DEBS 1,

  • and DEBS 2 begins with this ketosynthase.

  • OK?

  • Likewise, we have a break here, between the T domain

  • and this ketosynthase.

  • So three proteins make up this assembly line,

  • and so when thinking about this, these proteins

  • are going to have to interact with each other in one

  • way or another.

  • And so there's a lot of dynamics in protein-protein interactions

  • happening here.

  • How do we know there's seven modules?

  • And remember each module is responsible for one monomer

  • unit.

  • We count the T domains, so we have one, two, three, four,

  • five, six, seven T domains.

  • So like the acyl carrier proteins

  • of fatty acid synthase, these T domains

  • will be post-translationally modified with a PPant arm.

  • And that PPant arm will be loaded

  • with the acetyl-CoA or methylmalonyl-CoA or

  • malonyl-CoA monomers.

  • We have a loading module.

  • So the loading module has no ketosynthase,

  • because there's nothing upstream over here

  • for catalyzing a carbon-carbon bond formation event.

  • And then we see modules one through six,

  • so sometimes the loading module is module zero.

  • We see that each one has a ketosynthase,

  • so there'll be carbon-carbon bond formation

  • going along this assembly line.

  • And we see that the optional domains vary.

  • So for instance, module one has a ketoreductase

  • as does module two.

  • Look at module four.

  • We see all three domains required

  • for complete processing of that beta-keto group here.

  • Here, only a ketoreductase, and here only a ketoreductase.

  • OK?

  • So just looking at this, you can say,

  • OK well, we'll have an OH group here, here.

  • Here we have complete processing.

  • Just ignore this.

  • It's in lower case, because it's a non-functional reductase

  • domain.

  • It's not operating as annotated here.

  • So what happens?

  • So again, there's post-translational modification

  • of this T domain, so it has a serine.

  • The serine gets modified with the PPant arm, as shown here,

  • and we use that squiggle depiction,

  • as I showed for the acyl carrier protein of FAS.

  • So post-translational modification of these T domains

  • has to happen before any of the monomers

  • are loaded onto this assembly line.

  • And these PPant arms allow us to use bioesters as the linkages

  • and through the chemistry I showed earlier.

  • So here, what we're seeing in this cartoon, going from here,

  • this indicates that the T domains are not

  • post-translationally modified.

  • And here, we see the assembly line

  • after action of some [? phosphopentyltransferase ?]

  • loading these arms.

  • OK?

  • So each T domain gets post-translationally modified.

  • What happens next?

  • We have loading of monomers.

  • And we'll look at module zero and one on the board

  • and then look at how the whole assembly line goes.

  • AUDIENCE: Do you ever get selected

  • post-translational modification of the T domains

  • and if so, does that facilitate different modules being

  • like on or off, so to speak?

  • ELIZABETH NOLAN: I don't know.

  • I don't know in terms of the kinetics,

  • and say, does one T domain get loaded by a PPTase

  • before the other?

  • These enzymes are very complex, and there's

  • a lot we don't know.

  • But that would be interesting, if it's the case.

  • I wouldn't rule it out, but I just don't know.

  • One thing to point out too, these assembly lines are huge.

  • So this is something we'll talk about more the next time,

  • as we begin to discuss how do you experimentally study them?

  • But some are the size of the ribosome for the biosynthesis

  • of one natural product.

  • And what that means, from the standpoint

  • of in vitro characterization, is that often

  • you just can't express a whole assembly line,

  • let alone say one protein that has a few modules.

  • So often, what people will do is individually express domains

  • or dye domains and study the reactions

  • they catalyze in their chemistry there.

  • And so it would be very difficult even

  • to test that in terms of in vitro.

  • Is there an ordering to how the T domains are loaded?

  • And then there's question too, do you even

  • know what the dedicated PPTase is?

  • So there's some tricks that are done on the bench top

  • to get around not knowing that, which we'll talk about later.

  • So back to this assembly line to make DEB.

  • So we're just going to go over the loading module

  • and module 1 and look at a Claisen condensation catalyzed

  • by the KS.

  • And this chemistry pertains to the various other modules

  • and other PKS.

  • So we have our AT domain and our thiolation domain of module 0,

  • and then we have the ketosynthase, the AT domain,

  • the ketoreductase, and the T domain of module 1.

  • OK.

  • I'm drawing these a little up and down just

  • to make it easier to show the chemistry.

  • So sometimes you see them straight,

  • sometimes moved around here, but it's all the same.

  • So we have these PPant arms on the two T domains.

  • So what happens now, after these have been post-translationally

  • modified?

  • We need the action of the AT domains

  • to load the monomers onto the PPant arms

  • here, so action of the AT domain.

  • So what do we end up with?

  • In this case, the starter is a propionyl-CoA,

  • so we can see that here.

  • And we have a methylmalonyl-CoA as the extender, that

  • gets loaded, and I'm going to draw the cysteine thiolate

  • of the ketosynthase here.

  • So what happens next?

  • We need to have decarboxylation of the methylmalonyl-CoA

  • monomer to give us a C3 unit.

  • And it's C3 because of this methyl group,

  • but the growing chain will grow by two carbons.

  • And then we need to have transfer of this starter

  • to the ketosynthase.

  • So the ketosynthase is involved in covalent catalysis here.

  • So what happens, we can imagine here, we have attack,

  • and then here, we're going to have the decarboxylation.

  • We have chain transfer to the ketosynthase,

  • and here, decarboxylation leaves us this species.

  • OK?

  • OK.

  • So now, what happens?

  • Now, the assembly's set up for the Claisen condensation

  • to occur which is catalyzed by the ketosynthase.

  • Right?

  • So what will happen here?

  • You can imagine that, and as a result, where do we end up?

  • I'll just draw it down here.

  • And what else do we have?

  • We have a ketoreductase.

  • So this ketoreductase will act on the monomer

  • of the upstream unit, and that's how it always is.

  • So if there's optional domains in module 1,

  • they act on the monomer from module 0.

  • If there's optional domains in module 2,

  • they'll act on the monomer for module 1.

  • OK?

  • So we see here now we have reduction

  • of the ketone from module 1 to here via the ketoreductase.

  • OK?

  • So if we take a look at what's on the PowerPoint

  • here, what we're seeing is one depiction of this assembly line

  • to make DEB indicating the growing chain.

  • OK?

  • So as we walk through each module,

  • we see an additional monomer attached.

  • So the chain elongates, and then you

  • can track what's happening to the ketone group

  • of the upstream monomer on the basis of the optional domains

  • here.

  • If we look in this one, which I like this one because they

  • color code.

  • So they color code the different modules along with the monomer,

  • and so it's pretty easy to trace what's happening.

  • So for instance, here we have the loading module,

  • and we have the starter unit in red.

  • And here we see that it's been reduced by the ketoreductase

  • of the upstream blue module.

  • Here, we have the green module, here is its monomer,

  • and we see its ketoreductase acted on the blue monomer

  • from module 1, et cetera here.

  • So I encourage you all to just very systematically work

  • through the assembly lines that are provided in these notes,

  • and it's the same type of chemistry over and over again.

  • And if you learn the patterns, it

  • ends up being quite easy to work through, at least

  • the simple assembly line.

  • So as you can imagine, complexity increases,

  • and we'll look at some examples of more complex ones as well.

  • So where we'll start next time with this

  • is just briefly looking at chain release by the thioesterase.

  • And then we'll do an overview of non-ribosomal peptide

  • biosynthesis logic and then look at some example assembly lines.

  • So we have the exams to give back.

  • I'll just say a few things.

  • So the average was around a 68, plus or minus 10, 11,

  • 12 for the standard deviation.

  • I'd say, if you were in the mid 70s and above,

  • you did really well.

  • If you're into the low 60s, that is OK,

  • but we'd really like things to improve for the next one.

  • In terms of the exam and just some feedback-- and I'll

  • put feedback as well in the key which will be posted

  • later today or early tomorrow.

  • There wasn't one question that say the whole class bombed,

  • so that's good.

  • There were a few things for just general improvement,

  • and I want to bring this up, so you can also think about it

  • in terms of problem sets.

  • One involves being quantitative.

  • So there's certainly qualitative trends and data,

  • but there's also quantitative information there,

  • and that can be important to look at.

  • And one example I'll give of that involved question one.

  • If you recall, there was an analysis of GDP hydrolysis

  • and an analysis of peptide bond formation.

  • And quantitative analysis of the peptide bond formation

  • experiments will show that all of the lysyl-tRNAs

  • were used up in the case of the codon that was AAA.

  • Whereas, some of those tRNAs were not

  • used up when the codon contained that 6-methyl-A

  • in position one.

  • Right?

  • And if you linked that back to the kinetic model

  • along with the other data, what that indicates

  • is that proofreading is going on.

  • Right?

  • Some of those tRNAs are being rejected from the ribosome

  • there.

  • So that was one place where quantitiation, a fair number

  • of you missed that.

  • And another thing I just want to stress

  • is to make sure you answered the question being asked.

  • And where an example of that came up was in question one

  • with the final question asking about relating the data back

  • to the kinetic model.

  • And so if a question asks that you really do

  • need to go back to the model which was in the appendix

  • and think about that.

  • So many of you gave some very interesting answers

  • and presented hypotheses about perhaps the 6-methyl-A

  • is involved in regulation and controlling

  • like the timing of translation.

  • And that's terrific and interesting to think about,

  • but it wasn't the answer to the question.

  • Right?

  • Which was to go beyond the conclusions

  • from the experiments with GTP hydrolysis

  • and formation of that dipeptide, and ask

  • how can we conceptualize this from the standpoint

  • of the model we studied in class?

  • And then just the third point I'll make

  • is related to question two and specifically to GroEL.

  • But the more general thing is that if we

  • learn about a system in class, unless there's

  • compelling data presented in a question

  • to suggest the model is something other than what we

  • learned or its behavior is something other than what

  • we learned, stick with what you know.

  • So in the use of GroEL, the idea in that experiment

  • was that, if you recall, this question was looking at these J

  • proteins and asking, how do J proteins

  • facilitate disaggregation?

  • Right?

  • And so a GroEL trap was used that cannot hydrolyze ATP,

  • which means it's not active at folding any polypeptide.

  • But the idea there is that these J proteins end up

  • allowing monomers to come out of the aggregate,

  • and then GroEL can trap and unfold the monomer

  • to prevent reactivation.

  • And so a number of people came to the conclusion

  • that GroEL was binding that aggregate somehow

  • in its chamber.

  • And what we learned about GroEL is

  • that its chamber can't house a protein over 60 kilodaltons.

  • Right?

  • We saw that in terms of the in vitro assays

  • that were done looking at what its native substrates are.

  • Right?

  • So always go back to what you know, and then you

  • need to ask yourselves, are the data

  • suggesting some other behavior?

  • And if that were the case, like what

  • is your analysis of those data there?

  • So please, even if you did really well, look at the key

  • and see what the key has to say.

  • And if you have questions, you can make an appointment with me

  • or come to office hours or discuss with Shiva there.

  • OK?

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