Name: 18.PK和NRP合成酶 4 (18. PK and NRP Synthases 4)
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基礎被動語態

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

And the focus of today's lecture will really

are done to elucidate a biosynthetic pathway

if we think about studying assembly lines in lab,

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

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

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

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

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

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

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

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

is used in order to post-translationally modify

So there is a serine residue in these T domains

people will use recombinant Sfp And just recall,

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

And even what people have done is make modified analogs,

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,

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

and we have the PPTase activity here, Sfp,

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

And these can range from things like an isotope label

to peptides to steroids to some non-ribosomal peptide

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

subtilits with coASH and magnesium bound.

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

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

for the biosynthesis of a natural product called

And so in thinking about this, what I would just

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18.PK和NRP合成酶 4 (18. PK and NRP Synthases 4)

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