Abstract:
Non-ribosomal Peptide Synthetases (NRPSs) are large, modular, multi domain enzymes that function to synthesize a specific short peptide product in an assembly-line fashion. This is accomplished by the selection, loading and linking of amino or carboxylic acid monomers via peptide bonding. Many NRPS enzymes have been identified and studied in both bacterial and fungal genomes, and allow for the production of secondary metabolites. The ruminant methanogen, Methanobrevibacter ruminantium M1, has recently been shown to possess two NRPS encoding genes, representing the first to be discovered in Archaea. The non-ribosomal peptide products produced by the methanogen NRPS enzymes have not been identified. I have conducted research on one of these methanogen NRPS enzymes, mru_0351. Recombinant expression of both a single domain (mru_0351-A3L) and a multi domain (mru_0351-C2R) protein construct excised from the mru_0351 NRPS has allowed for their characterisation using a host of biochemical and biophysical techniques. Efforts have focused on interrogating the reactions catalysed by the NRPS adenylation domains, which are responsible for initial substrate selection, and thus serve a key gatekeeping role in committing a given substrate to the biosynthetic pathway. Adenylation domains sequentially catalyse two reactions: adenylation and thiolation. The adenylation reaction serves as a substrate filtering step, electively activating cognate monomers. The thiolation reaction then covalently tethers the activated substrate to a post-translationally attached 4’-phosphopantetheine group on the NRPS assembly. Crystallographic and small angle X-ray scattering (SAXS) techniques were used to provide structural insights into the mechanics of the methanogen mru_0351 NRPS. Diffracting protein crystals of proteolytic products from mru_0351-C2R and mru_0351-A3L were produced, indicating promising leads towards structural determination of these NRPS constructs. SAXS analysis revealed subtle conformational changes in the multidomain structure of mru_0351-C2R upon addition of substrates. Development of mass spectrometry methods to identify intermediates tethered to the 4’-phosphopantetheinyl post-translational modification revealed that the mru_0351 NRPS enzyme’s adenylation domains confer additional selectivity via the thiolation reaction. Thiolation reaction substrate preference has not previously been reported in NRPS enzymes, and this discovery may have broad implications for how module specificity is conferred in NRPS enzymes in general.