Ribosomal biosynthesis of the cyclic peptide toxins of Amanita
non ribosomal peptide synthesis
Transcript of 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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