Mass spectrometry and structural biology of methanogen non-ribosomal peptide synthetases

Abstract

Non-ribosomal peptide synthetases (NRPSs) are multi-modular enzymes that synthesize a wide range of bio-active peptides such as siderophores, toxins, and antibacterial and insecticidal agents. This thesis describes the mass spectrometric and structural investigation of the cryptic NRPS enzymes from the rumen methanogens, Methanobrevibacter ruminantium and Methanobrevibacter millerae. NRPSs use their adenylation domains for initial substrate selection and subsequently synthesize their products in an assembly line-like manner where the reaction intermediates remain covalently tethered to the enzyme via a phosphopantetheine (Ppant) group attached to the peptide carrier protein (PCP) domains. This thesis describes a novel mass spectrometry-based Ppant ejection assay to identify the covalently tethered NRPS reaction intermediates. The Ppant ejection assays showed that despite of a number of substrates being adenylated by methanogen NRPSs, only a few were identified tethered to Ppant, thereby providing further insights into substrate specificity. Furthermore, the Ppant ejection assays were able to pick the substrates from complex mixtures such as microbial lysates, demonstrating the utility of this assay as a substrate screening tool for cryptic NRPS enzymes. This Ppant ejection assay is a significant advance over the previously reported mass spectrometry-based methods by enabling unambiguous identification of the reaction intermediates tethered to full-length NRPSs rather than isolated PCP domains. NRPSs are characterized by extensive inter-domain communications as a consequence of their assembly-line mode of synthesis. One crucial yet unexplored interaction is that of the reductase (R) domain with the immediately upstream PCP domain. Reductase domains catalyse the reductive release of the mature peptide product from the terminal PCP domain of the NRPS. This thesis describes the crystal structure of a methanogen NRPS PCP-R di-domain construct. This is the first NRPS reductase domain structure to be determined together with the upstream PCP domain. The structure reveals that a helix-turn-helix motif plays a major role in the interface between the PCP and reductase domains. The information derived from the described PCP-R interface will aid in gaining further mechanistic insights into the peptide termination reaction catalysed by the reductase domains, and may have implications in engineering NRPSs to synthesize novel peptide products.