Team:Heidelberg/NRPS

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NRPS. Get to know the theory.

Overview

Everybody knows the dogma of molecular biology - DNA makes RNA makes protein, but it doesn't cover all nature's capabilities, as for example non-ribosomal peptide synthetases, or short NRPS. This alternative pathway for peptide formation is mainly found in bacteria and fungi, but can at also be functionally transferred to mamallian cells (pmid:22543310). Natural NRPs can have various functions from simple dyes up to metal chelators or antibiotics. NRPSs are large protein complexes adding amino or arylic acids, taken from a pool of more than 500 monomers, to a growing peptide chain without the need for a template. The molecular structure of NRPS is based on modules, each incorporating exactly one monomer and comprising several domains of different functions. The most important domains are the adenylation (specific substrate activation), the thiolation (peptide / monomer carrier) and the condensation domain (peptide bond formation), which are needed to form basic peptide chains. Besides these various other domains are known to introduce secondary modifications. Thanks to their remarkable modularity and the wide range of substrates, NRPSs bear the impressive biosynthetic potential to create novel non-ribosomal peptides of various functions in vivo or in vitro. The range of possible applications exceeding natural functions remains unforseen.

Figure 1: The three levels of modularity in NRPS are the gene products, modules each incorporating one amino acid and the domains in every module.

Modular Structure

The biggest benefit of NRPS for synthetic biology is it's very modular structure. This starts at the gene level and goes as deep as domain or even single residue level. This modular organisation can be exploited by reorganisation of the modules in order to achieve different products.

Biosynthetic gene cluster
The genes necessary for the biosynthesis are normally organised in a gene cluster. This contains the genes coding for the non-ribosomal peptide synthetases, which can reach a size of up to 1.5 MDa (cyclosporin Fischbach), genes necessary for the monomers' biosynthesis, as well as tailoring enzymes introducing further modifications in the peptide. There is normally more than just one peptide synthetase. The proteins are often connected by communication domains in order to keep the structure of the assembly line. If one wants to transfer a whole pathway to a different host organisms one crucial apect, besides the successful cloning of the synthetases, is the monomer supply. Depending on the hosts's endogenous machinery one can leave out certain genes or has to include other pathways in order to keep up the supply.

One module - one amino acid
Each NRP synthetase is organised in so called modules, where every single module is responsible for incorporating one amino acid in the growing peptide chain. Since all modules have similar minimal structure components one can reorder them easily to achive a different product. These minimal structure components are NRPS specific protein domains each providing the assembly line with a different function. The three levels of modularity - proteins, modules and domains are shown in figure 1.(Fischbach)

Chain elongation

The mechansim of the peptide bond formation in NRPS is partially different from that in the ribosome. The biggest differences are the attachment of the growing peptide chain and the number of catalytic domains. In ribosomal synthesis one ribosome can add many amino acids, but in non-ribosomal synthesis the number of catalytic domains rises linearly with the number of amino acids incorporated. Thus the latter is only suitable for oligopeptide synthesis.

Figure 2: Phosphopantetheinyltransferases are essential for non-ribosomal peptide synthesis, as they are needed for posttranslational modification of the thiolation domains. (Modified from Fischbach)
Thiolation domain and PPTases
In the ribosome the growing peptide chain and the monomers to be incorporated are bound to tRNA, but never to the ribosome itself. NRP synthetases contain thiolation domains (T-domains) in every module, where the corresponding amino acid is covalently bound to the enzyme via a thioester bond. The essential amino acid residue of the T-domains is a serine which is posttranslationally modified to carry the sulfhydryl group required for the thioester bond. This modification is carried out by separate proteins - the phosphopantetheinyltranferases (PPTases), which use coenzyme A as cofactor. The reaction is shown in figure 2. As the functionality of the thiolation domains is essential for the peptide synthesis one should consider transferring a suitable PPTase to a host organism together with the synthetases.

Adenylation domain
The actual attachement of the amino acid to the already modified T-domain is carried out by the adenlytation domain (A-domain). Condensation domain
TErmination - ThioEsterase

Monomer range

Epimerisation and stereoselectivity
Non-proteinogenic amino acids

Modifying domains

N-Acylation
O-Acylation
Heterocyclisation

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