Characterization of Non-Ribosomal Peptide Synthetase (NRPS) in Pseudoalteromonas SANK, the natural producer of thiomarinol antibiotic

Non-ribosomal peptide synthetases are large multimodular megaproteins with repeated catalytic domains. In linear (type A) NRPS, each module is responsible for incorporating one amino acid into the product, so the number of amino acids in the product can be predicted by the number of NRPS modules (Mootz et al, 2002; Hur et al, 2012).

The iterative NRPS types, in which the catalytic domain is reused repeatedly, are common in many fungal species. Siderophores, a high affinity iron chelators, and Enniatins, a cyclohexadepsipeptides with various biological activities, are among the compounds that produced by iterative NRPS in fungi (Bushley, Ripoll and Turgeon, 2008; Sy-Cordero, Pearce , Oberlies, 2012).

Thiomarinol produced by Pseudoalteromonas bacteria contains pyrrothine consisting of two cysteine molecules which are proposed to be produced by an NRPS gene cluster. The intriguing feature of Hol A gene, which is responsible for pyrrothine biosynthesis, is encoding only one Adenylation(A) domain, Condensation(C) domain and Peptidyl carrier protein(PCP) while pyrrothine should require two domains. This leads to the suggestion that Hol A is a dimeric protein and works as an iterative NRPS in a similar manner to fungal NRPSs (Fukuda, 2011).

The study includes cloning, expressing and purification of HolA (unusual NRPS) to allow biochemical characterisation and eventually X-ray crystallography. Studies of dimerization both in vivo (using bacterial two hybrid system) and in vitro (using crosslinking agent & AUC) will be reported as well as functional assays for ATPase activity and amino acid loading.