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.