Identifying Substrate Specificity of Adenylation Domains in Methanogen NRPS Enzymes

Abstract

Inhibition of methane-producing archaea in ruminant animals has been an active area of research for many years, primarily motivated by pressures to minimise the carbon footprint of ruminant-based agriculture. To date, approaches to reduce methane production in ruminants have been only partially successful. Nonribosomal peptide synthetase (NRPS) enzymes (mru_0068, mru_0351, sm9_0755, and sm9_0760) from Methanobrevibacter ruminantium M1 and Methanobrevibacter millerae SM9 have been identified as potential molecular targets with which to develop methanogen reduction strategies. In bacteria and fungi, NRPS enzymes are involved in the synthesis of important secondary metabolites and as such are expected to confer significant survival advantage to producing methanogens. Crucial to understanding the biological role of these NRPS enzymes in their native hosts is the ability to synthesise their products in vitro, which in turn requires an understanding of substrate selection by the NRPS adenylation (A) domains. In this research project, attempts were made to determine the substrate selectivity of three different A domains in the NRPS enzymes. Internal A domain constructs for mru_0068-A1, mru_0068-A2, and mru_0351-A3 from M. ruminantium M1 were subcloned from the full-length NRPS enzymes with the aim of creating constructs amenable to purification into monomeric, soluble form. Attempts were made to purify the domains mru_0068-A2 and mru_0351-A3, however significant aggregation was observed for mru_0068-A2, reflecting ongoing challenges within the field for the expression of internal A domain truncations. Successful purification of soluble, monomeric mru_0351-A3 protein supported functional and structural studies into its substrate selectivity. Results from a biochemical PPi-release assay revealed that the A domain preferentially selects amino acid substrates on the basis of hydrophobic sidechain moieties, specifically valine, leucine, norvaline, and 2-aminobutyric acid. Attempts to crystallise the protein and determine its structure were not successful. Nevertheless, isolation of mru_0351-A3 in soluble, monomeric form has provided an experimental pathway by which to continue characterising its specificity through structural approaches and enzyme kinetics studies. Conditions for soluble expression of the mru_0068-A1 domain were also identified.