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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