non ribosomal peptide synthesis

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Inigo.J01109151013


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How many amino acids are there in nature?

Ans : 22 ??


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There are 22 proteinogenic amino acids.

Proteinogenic amino acids are those that are

recognised by ribosomes for proteinsynthesis.

There can be more than million other aminoacids as we know any compound with an amine

and carboxylic acid group attached to a carbonis an amino acid .

This is the basis of NRPS


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Without the assistance of ribosomes* Allows non proteinogenic Amino Acids

(D-isomers,carboxy acids, and

N- methylated residues, etc)

* Only small oligopeptides (3-22 peptides)

* Cyclic, Branched peptides

* NRPs assist in non growth activities

* Constrained peptide structure(for easy activity) Catalyzed by Large Multienzyme complexes called

NON RIBOSOMAL PEPTIDE SYNTHETHASES..


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Large enzymes Contains several modules

Each module is formed by several domains

The genes encoding for these synthetases arearranged as operons.

Mechanism of synthesis : MulticarrierThiotemplate Mechanism


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Each module is responsible forthe specificincorporation of one dedicated substrate intothegrowing oligopeptide chain.

1 MODULE 1 AMINO ACID (colinearity rule)


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Each module has several domains Primary and secondary domains

Primary domains are very essential domains

in all synthetases Secondary domains are utility domains

The subdomains of domains operate inrotatory motion for them to be stable.


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Primary Domains :Adenylation Domain( gate keepers)

Thiolation or (PCP) Domain

Condensation DomainTermination Domain( thio esterase)


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Secondary Domains: Epimerisation domain

Methylation domain

Oxidation domain Reduction domain, etc


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Thiotemplate mechanism Steps:

INITIATION

ELONGATIONTERMINATION


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First substrate recognition by some ~10 aminoacids(signature motif) in adenylation(A) domainby lock and key method.

the A domain catalyzes the activation of asubstrateas aminoacyladenylate through the

Mg2+-dependent hydrolysisof ATP and therelease of pyrophoshate

ATP + aa AMP~aa + Ppi

This AMP~aa binds non covalently to the enzyme

SHENZYME+ AMP~aa SHENZYME..AMP~aa


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T domains must be post-translationally modified with a4'-Ppant group a conserved seryl residue attaches to 4-Ppant to

convert it into holo enzyme. The SH group (thiol) belongs to the phosphopantetheine

arm attached to an invariant serineresidue of the apo-T(thiolation) domain by a dedicated 4'-phosphopantetheine(ppan) transferase that usescoenzyme A (CoA) as a substrate

The amino acid has more affinity to the thiol group thanAMP, so binds covalently to it.

SHENZYME..AMP~aa

aa-SENZYME

Amino acyl AMP amino acyl thioester


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A state, H state, and an intermediate A/H state T domains play a central role in the function of

NRPSs as they must interact not only with Adomains in the context of aminoacylthioester

formation but also with other catalytic domainsinvolved in peptide bond formation, peptidemodification, or peptide release from the NRPS

This requires the T domain to interact with multiple

partners in an exquisitely timed sequence


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Now, the A domain of the next modulerecognizes the next amino acid and binds to it

Then, a peptide bond is formed between thesetwo amino acids with the help of the enzyme

peptidyl ligase by the nucleophilic attack of

the -amino group of module 2 aa on the thioester-activated carboxygroup of aa of module 1 to givea dipeptide

It is then translocatedto module 2 by peptidyl

transferase enzyme This process repeats to elongate the peptide

chain


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EPIMERISATION:

Biosynthesis very specific to Epimers. Hence, epimerization domain carries the role

of epimerizing. It is found before condensation domain. Thus, condensation domain also could

contain substrate recognition residues torecognise these epimerised residues.

Peptide synthesis is also specific to oxidative

compounds, reduced residues, methylatedresidues,etc for which respective domains takethe responsibility.


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The termination domain is found on theN-terminal end of the last module of theenzyme complex.

Here, the peptide chain cyclizes, hydrolyses,etc to yield the oligopeptide.


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:Location of termination

domain

CYCLIZATION


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MACRO CYCLISATION


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NRPS + Poly Ketide synthesis .. ( a topic youshould refer)


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Gramicidin s Tyrosidine


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A paralogous version of the methionyl-tRNA synthetase,which is widespread in bacteria, and present evidenceusing contextual information that it might functionindependently of protein synthesis as a peptide ligase inthe formation of a peptide- derived secondary metabolite.This metabolite is likely to be heavily modified through

multiple reactions catalyzed by a metal-binding cupindomain and a lysine N6 monooxygenase that are strictlyassociated with this paralogous methionyl-tRNAsynthetase (MtRS).

An analogous system wherein the MtRS has been replacedby more typical peptide ligases with the ATP-grasp or

modular condensation-domains.(Predicted class-I aminoacyl tRNA synthetase-like proteins innon-ribosomal peptide synthesis L Aravind*, Robson F deSouza, Lakshminarayan M Iyer)


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The peptide synthesis stops instantaneously if anyintermediate amino acid is missing and would notcontinue even if the next step amino acids arepresent

The peptide chain that has not terminated properly or

has missed the termination step remains on themodular enzyme and would not cleave off.

The synthesis starts only if the initial amino acid ispresent and is recognized.

The peptide synthesis is very specific toepimerisation and other structural changes to theamino acid.

Can contain non proteinogenic amino acids too.


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Researches on these topics took place evenbefore 1970s ( lippmann, rosowkki, et.al)

So, how far are we updated??

All that is in this PowerPoint is just anintroduction.. For extensive studies , goto pubmed , pnas and biomed sites


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non ribosomal peptide synthesis

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