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ELIZABETH NOLAN: We're going to end the unit on synthesis

today.

And the focus of today's lecture will really

be looking at one system in detail

and the types of experiments that

are done to elucidate a biosynthetic pathway

for a non-ribosomal peptide.

And so just to recap from last time,

if we think about studying assembly lines in lab,

and we're thinking about this for a non-ribosomal peptide

synthetase, what needs to be done?

So first, it's necessary to, typically,

overexpress and purify domains, didomains or modules.

And so on Monday, it came up that often, these proteins

are enormous and it's not possible or feasible to express

entire modules, or entire proteins that

have multiple modules.

So oftentimes, people will look at individual domains,

or didomains, which are smaller and more amenable,

to overexpression in an organism like E. coli.

Then it's necessary to assay for A domain activity.

So we're called the A domains through the adenylation

domains.

And the question is, what monomer

is selected and activated?

And so the ATP-PPi exchange assay comes up here.

There needs to be assays for loading

of the T domain, or carrier protein, with the monomer.

Assay for peptide bond formation, which

is the condensation domain.

And then often, some assay for chain

released by the thioesterase domain.

OK, so assay for TE activity.

Chain release.

And so in terms about of thinking about these T domains,

we learned that these T domains need to be post-translationally

modified with the Ppant arm, which means we

need an enzyme called a PPTase.

And so in many cases, we don't know what the PPTase

is for a given gene cluster.

And what's done, often in the lab,

is that a PPTase from B. subtilis, named Sfp,

is used in order to post-translationally modify

the T domains with the Ppant arm.

So there is a serine residue in these T domains

that gets modified.

We looked over that in a prior lecture.

So this one is very useful.

And if you don't know the enzyme to use,

people will use recombinant Sfp And just recall,

we have the T domain.

There's a serine moiety.

We have a PPTase.

That's going to stick on the P pant arm here.

So we call this apo, holo, and then the amino acid,

or aryl acid monomer, in the case of NRPS

gets loaded here via a thioester.

And so Sfp can be used to get us here.

And even what people have done is make modified analogs,

where there's some R group.

So you can imagine using chemical synthesis

to load a monomer, or even some other type of group

that, for some reason, you might want to transfer here.

And this Sfp is very promiscuous and it can do that.

And so the take-home here is if you need a PPTase, overexpress,

purify, and utilize Sfp.

Here's just an example for review,

where we have a carrier protein, so a T domain,

and we have the PPTase activity here, Sfp,

attaching this Ppant arm.

And here, it's described with an R group.

And just to give you an example of possibilities here,

there have been many reports of CoA analogs being transferred

to T domains by Sfp.

And these can range from things like an isotope label

to peptides to steroids to some non-ribosomal peptide

derivative, or a fluorophore.

So this has been used as a tool.

And you might ask, why is this possible?

And if we just take a look at the structure of Sfp from B.

subtilits with coASH and magnesium bound.

What we see is that this end of the coA

is extended out into the solvent.

And at least in this structure here, it's

not interacting with regions of the protein.

So you can imagine that it's possible to attach

some group, even a bulky group, here

and be able to transfer it there.

So where we're going to focus the rest of the lecture

is on an assembly line responsible

for the biosynthesis of a natural product called

enterobactin, and this is a siderophore.

And so in thinking about this, what I would just

like to first note is that when we talk about these assembly

lines, we can group them into two types, which

are non-iterative and iterative assembly.

And so what does this mean?

So we've seen examples of non-iterative assembly

last time on Monday with the ACV tripeptide and the vancomycin

synthetase.

So in these non-interative assembly lines,

effectively, each step has its own module.

So each carrier protein, T domain, each condensation

or catalytic domain, is used only once as the chain grows.

And we see the chain passed along from module

to module here.

So also, the PKS we looked at for synthesis of DEB

is one of these non-iterative assembly lines.

So in contrast, in the example we're

going to look at today with the enterobactin synthetase is

an iterative assembly line, and this

is similar to what we saw in fatty acid synthase.

So in these iterative assembly lines,

effectively, only one module is employed over and over again.

So you can have the same carrier protein

and same catalytic domain used for multiple cycles of chain

elongation.

And that's what we saw in fatty acid synthase,

where there are multiple cycles in addition

of a C2 unit via the same domains.

And so what we're going to see today

is this type of iterative assembly

is responsible for the synthesis of this molecule here.

So first, just an overview of building blocks.

And then we'll talk about why organisms

want to make this molecule, and then

focus on the biosynthetic logic and experiments.

So this molecule, enterobactin, is produced from two monomers.

So we have 2, 3 dihydroxybenzoic acid, or DHB,

and we have serine here.

And there is a two-module assembly line

responsible for the synthesis of this natural product.

And that assembly line is shown here.

So we see that there's three proteins, EntE, EntB, and EntF.

We have an initiation module, elongation module, and this TE

domain for termination.

So overall, three separate proteins, two modules,

and seven domains.

So this NRPS is quite small.

And this is an example of a non-ribosomal peptide that's

produced by E. coli.

So E. coli makes this molecule, as well as

some other gram-negative bacteria.

So this is iterative.

We have three of each of these monomers,

yet only two T domains here, so imagine

one responsible for each.

So before we get more into this biosynthetic logic,

let's just take a moment to think about why

this molecule is produced.

So this is a case where we actually

have very good understanding about why an organism is

producing a natural product.

And this actually gives a segue into JoAnne's section

on metal homeostasis, which will come up after cholesterol

after spring break.

So many bacteria use non-ribosomal peptide synthesis

machinery in order to make chelators

in order to acquire iron.

And that's because iron is an essential nutrient

and it's actually quite scarce.

So if you imagine an organism in the soil,

maybe it needs to obtain iron from a rock.

Somehow it needs to get iron from our pool,

and concentrations are very tightly regulated,

and most iron is tightly bound.

And we can also think about this from a standpoint

of solubility, so simple KST type things.

We all know that iron 3, which is the predominant oxidation

state in aerobic conditions, is very insoluble.

So our cars rust up here in the Northeast

because they sit outside on the road in the winter,

and that's no good.

So we can think about 10 to the minus 18 molar.

And then if we think about free ion in human serum,

for instance, the concentration is even lower

because there's inherent toxicity associated

with free iron.

And you'll hear about that from JoAnne in more detail later.

So these organisms have a predicament

because for metabolism, they need

iron on the order of micromolar concentrations.

So how does some organism obtain micromolar iron

when in environments where, say, that's 10

to the minus 24 molar?

And there's a number of strategies that come up,

but one of the strategies is the biosynthesis

of non-ribosomal peptides that act as metal scavengers

and metal chelators.

And so I just show you two examples here.

And we have enterobactin, which we're going to focus on today.

And this is really just a wonderful molecule.

Yersiniabactin-- and I put this up here, in part,

because there were some questions

about those cyclization domains in the bleomycin gene cluster,

that we looked at that assembly line on Monday.

And this is another example where

cyclization of cysteine residues occurs in order

to give the final natural product via those modified

condensation domains here.

So if we think about enterobactin

for a moment longer, what happens,

effectively, this molecule can bind

iron 3 with higher affinity.

And the iron bound form is shown here.

So these aryl acids, these catechol groups,

provide six oxygen donors to the iron center

to get a structure like this.

So in terms of the organism in production,

what happens when these organisms are confronted

with iron limitations?

So essentially, they're starved for essential nutrients.

They'll turn on biosynthesis.

So they'll express the enterobactin synthetase,

which will allow for production of enterobactin.

So this is happening in the cytoplasm.

So we have those three proteins that

comprise the assembly line that use the HDML serine to produce

the natural product.

And then in addition to that biosynthetic machinery,

the organism needs to also express and use

a whole bunch of transport machinery.

So what happens is that this natural product is exported

into the extracellular space.

So this is a gram-negative organism,

so it has an inner membrane and an outer membrane.

And it's in the extracellular space

that enterobactin will scavenge iron 3.

So there's formation of the coordination

complex, shown in cartoon here.

And then there's a dedicated receptor

on the outer membrane that will recognize that iron bound form

and bring that into the cell.

And then through transport and through breakdown

of the natural product, this iron

can be released and then used.

So iron is a co-factor of many types of proteins and enzymes

here.

So a whole lot is going on.

We're going to focus on the biosynthetic part.

And so in thinking about this, from the standpoint

of a non-ribosomal peptide synthetase,

what's something interesting?

So in the examples we saw last time,