Investigations into Methyl-coenzyme M Reductase Behavior: Expression in a Heterologous Host, Putative Post-Translational Modification Genes, and Molecular Dynamics of MCR Homologues Bound to F430 Variants

Abstract

Methanogens are microorganisms widely found in wetlands and the digestive tracts of animals, which produce methane as a metabolic byproduct. The organism possesses the key methane-forming enzyme methyl-coenzyme M reductase (MCR) which is a dimer of heterotrimers comprised of McrA (α), McrB (β), and McrG (γ) subunits. The enzyme uses a unique nickel-containing coenzyme F430 for activity and contains several unprecedented post-translational modifications (PTMs). The six PTMs located in α subunit of MCR are 2-(S)-methylglutamine, 5-(S)-methylarginine, 3-methylhistidine, S-methylcysteine, didehydroaspartate, and thioglycine residues. Homologues of MCR have been identified in anaerobic methanotrophic archaea (ANME) which operates the anaerobic oxidation of methane (AOM), and in Candidatus Ethanoperedens thermophilum which is an ethane oxidizer. While the MCR homologues share the same structural composition, they differ in the composition of their PTMs, with a 172-methylthio F430 and a 17,172-dimethyl F430 contained in ANME-1 and in Ca E. thermophilum respectively. The exact roles of these PTMs are unknown though several hypotheses have been proposed including their role in improving MCR stability under mesophilic conditions. Processes involved in the maturation and activation of the active enzyme are not yet fully understood. A better understanding of the assembly and activation of MCR may enable its application in natural gas conversion strategies and the development of inhibitors to reduce natural greenhouse gas emissions. Current investigation and progress in some of the PTMs and the expression of MCR in a heterologous, non-methanogenic host is described. Comparative genomics and homology studies were used to identify target genes suspected to be responsible for the PTMs. Putative genes reported (mcmA) and suspected (mm4) to be responsible for the methylcysteine and methylhistidine PTMs respectively, were studied using a combination of computational tools, in vitro and in vivo methods. Molecular dynamics (MD) methods and distance calculations were utilized to investigate the effects of the PTMs on MCR and its homologues. MD simulations were carried out on MCR homologues with and without their PTMs. Results show that MCR exhibits half sites reactivity, and that the PTMs may play a role in coordinating MCR catalytic activity within its two active sites. MCR homologues without PTMs exhibited less dynamism and this could be an indicator that the PTMs evolved as an adaptation of thermophilic proto-MCR to mesophilic growth, enabling proper enzyme dynamics at lower temperature. The requirement of zinc as an accessory factor for the activity of McmA in the methylcysteine modification was inferred from the results. Soluble McrA and McrG proteins were successfully expressed from Escherichia coli cell lines. McrG was coeluted with several coenzyme F430 biosynthetic (Cfb) proteins. Previous work by the Mansoorabadi group showed that CfbE interacts with McrD and could be activated by it. The coelution of McrG with Cfb proteins suggests that the Cfb proteins may form a larger coenzyme F430 synthase complex which interacts with McrG and McrD to coordinate the insertion of coenzyme F430 into the MCR active site. CfbE interaction with McrG indicates that CfbE might be the direct F430 chaperone that delivers the coenzyme to MCR. Considering MCR’s strategic role in methane recycling, understanding these processes are pivotal for the enzyme’s application in strategies for natural gas conversion and reduction.