NON RIBOSOMAL PEPTIDE SYNTHESIS

Click to edit Master subtitle style 01109151013 Inigo.J

4/28/12

How many amino acids are there in nature?

Ans : 22 ??

4/28/12

WRONG!!!!!!
There

are 22 proteinogenic amino acids.

Proteinogenic amino acids are those that are recognised by ribosomes for protein synthesis. There can be more than million other amino acids as we know any compound with an amine and carboxylic acid group is an amino acid . This is the basis of NRPS
4/28/12

Non Ribosomal?
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..
4/28/12

NRP synthetases
Large

enzymes Contains several modules Each module is formed by several domains The genes encoding for these synthetases are arranged as operons. Mechanism of synthesis : Multicarrier Thiotemplate Mechanism

4/28/12

MODULES ??
Each

module is responsible for the specific incorporation of one dedicated substrate into the growing oligopeptide chain. 1 MODULE 1 AMINO ACID (colinearity rule)

4/28/12

DOMAINS??
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 in rotatory motion for them to be stable.

4/28/12

4/28/12

 Primary

Domains : Adenylation Domain Thiolation or (PCP) Domain Condensation Domain Termination Domain

4/28/12

Secondary Domains: Epimerisation domain Methylation domain Oxidation domain Reduction domain, etc

4/28/12

4/28/12

BIOSYNTHESIS
Thiotemplate Steps:

mechanism

INITIATION ELONGATION TERMINATION

4/28/12

INITIATION ADENYLATION
First

substrate recognition by some ~10 amino acids(signature motif) in adenylation(A) domain by lock and key method. the A domain catalyzes the activation of a substrate as aminoacyladenylate through the Mg2+-dependent hydrolysis of ATP and the release of pyrophoshate ATP + aa AMP~aa + Ppi This AMP~aa binds non covalently to the
4/28/12

4/28/12

THIOLATION
The

SH group (thiol) belongs to the phosphopantetheine arm attached to an invariant serine residue of the apo-T (thiolation) domain by a dedicated 4'-phosphopantetheine (ppan) transferase that uses coenzyme A (CoA) as a substrate The amino acid has more affinity to the thiol group than AMP, so binds covalently to it. SHENZYME..AMP~aa aa-SENZYME

4/28/12

4/28/12

ELONGATION CONDENSATION
Now,

the A domain of the next module recognizes the next amino acid and binds to it Then, a peptide bond is formed between these two 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 carboxy group of aa of module 1 to give a dipeptide It is then translocated to module 2 by peptidyl transferase enzyme This process repeats to elongate the peptide 4/28/12 chain

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 to recognise these epimerised residues. Peptide synthesis is also specific to oxidative compounds, reduced residues, methylated residues,etc for which respective domains take the responsibility.

4/28/12

4/28/12

TERMINATION
The

termination domain is found on the N-terminal end of the last module of the enzyme complex. Here, the peptide chain cyclizes, hydrolyses, etc to yield the oligopeptide.

4/28/12

:Location of termination domain

CYCLIZATION

4/28/12

MACRO

4/28/12

NRPS + Poly Ketide synthesis .. ( a toipc you should refer)

4/28/12

Eg. Of non ribosomal peptides:

Gramicidin Tyrosidine

4/28/12

aatRNA in nrps
A

paralogous version of the methionyl-tRNA synthetase, which is widespread in bacteria, and present evidence using contextual information that it might function independently of protein synthesis as a peptide ligase in the formation of a peptidederived secondary metabolite. This metabolite is likely to be heavily modified through multiple reactions catalyzed by a metal-binding cupin domain and a lysine N6 monooxygenase that are strictly associated with this paralogous methionyl-tRNA synthetase (MtRS). 4/28/12

SALIENT FEATURES OF NRP SYNTHESIS:

The

peptide synthesis stops instantaneously if any intermediate amino acid is missing and would not continue even if the next step amino acids are present The peptide chain that has not terminated properly or has missed the termination step remains on the modular enzyme and would not cleave off. The synthesis starts only if the initial amino acid is present and is recognized. The peptide synthesis is very specific to epimerisation and other structural changes 4/28/12 to the amino acid.

All these topics seem new??

Researches

on these topics took place even before 1970s ( lippmann, rosowkki, et.al) So, how far are we updated?? All that is in this PowerPoint is just an introduction.. For extensive studies , go to pubmed , pnas and biomed sites

4/28/12

THANK YOU

4/28/12