Methyl-coenzyme M reductase (MCR) is an archaeal enzyme that catalyzes the final step of methanogenesis and the first step in the anaerobic oxidation of methane, the energy metabolisms of methanogens and anaerobic methanotrophs (ANME), respectively. Variants of MCR, known as alkyl-coenzyme M reductases, are involved in the anaerobic oxidation of short-chain alkanes including ethane, propane, and butane as well as the catabolism of long-chain alkanes from oil reservoirs. MCR is a dimer of heterotrimers (encoded by mcrABG) and requires the nickel-containing tetrapyrrole prosthetic group known as coenzyme F430. MCR houses a series of unusual post-translational modifications within its active site whose identities vary depending on the organism and whose functions remain unclear. Methanogenic MCRs are encoded in a highly conserved mcrBDCGA gene cluster, which encodes two accessory proteins, McrD and McrC, that are believed to be involved in the assembly and activation of MCR, respectively. The requirement of a unique and complex coenzyme, various unusual post-translational modifications, and many remaining questions surrounding assembly and activation of MCR largely limit in vitro experiments to native enzymes with recombinant methods only recently appearing. Production of MCRs in a heterologous host is an important step toward developing optimized biocatalytic systems for methane production as well as for bioconversion of methane and other alkanes into value-added compounds. This review will first summarize MCR catalysis and structure, followed by a discussion of advances and challenges related to the production of diverse MCRs in a heterologous host.

Many methanogenic archaea synthesize β-amino acids as osmolytes that allow survival in high salinity environments. Here, we investigated the radical S-adenosylmethionine (SAM) aminomutases involved in the biosynthesis of Nε-acetyl-β-lysine and β-glutamate in Methanococcus maripaludis C7. Lysine 2,3-aminomutase (KAM), encoded by MmarC7_0106, was overexpressed and purified from Escherichia coli, followed by biochemical characterization. In the presence of l-lysine, SAM, and dithionite, this archaeal KAM had a k cat = 14.3 s–1 and a K m = 19.2 mM. The product was shown to be 3­(S)-β-lysine, which is like the well-characterized Clostridium KAM as opposed to the E. coli KAM that produces 3­(R)-β-lysine. We further describe the function of MmarC7_1783, a putative radical SAM aminomutase with a ∼160 amino acid extension at its N-terminus. Bioinformatic analysis of the possible substrate-binding residues suggested a function as glutamate 2,3-aminomutase, which was confirmed here through heterologous expression in a methanogen followed by detection of β-glutamate in cell extracts. β-Glutamate has been known to serve as an osmolyte in select methanogens for a long time, but its biosynthetic origin remained unknown until now. Thus, this study defines the biosynthetic routes for β-lysine and β-glutamate in M. maripaludis and expands the importance and diversity of radical SAM enzymes in all domains of life.

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Methyl‐coenzyme M reductase (MCR) is the key rate‐determining enzyme of methanogenesis as well as the anaerobic oxidation of methane, the essential energy metabolisms of methanogenic archaea and anaerobic methanotrophs (ANME), respectively. MCR is a dimer of heterotrimers with a 2α, 2β, 2γ configuration, and requires the nickel tetrapyrrole prosthetic group, coenzyme F430. The requirement of a unique cofactor, various unusual post translational modifications, and many remaining questions surrounding assembly and activation of MCR has so far largely limited in vitro experiments to native enzymes. To allow further investigation into the catalytic properties and mechanistic aspects of different MCRs, as well as facilitate the development of optimized biocatalytic systems to convert methane to more usable liquid fuels and other valuable compounds, we are developing methods for the heterologous expression of recombinant MCRs in Methanococcus maripaludis, a model methanogen with robust genetic tools. In methanogens, MCR is encoded in the highly conserved MCR gene cluster mcrBDCGA, which encodes two accessory proteins (McrD and McrC) in addition to the MCR‐encoding genes (McrA, McrB, and McrG). The accessory proteins are proposed to be involved in the assembly and activation of MCR. Interestingly, most ANME lack one or more accessory proteins in their MCR gene clusters. We have created a series of MCR expression constructs containing the MCR operons from several ANME organisms as well as several methanogens, with and without accessory protein(s). All constructs contain a his‐tag on the C‐terminus of McrA which allows the purification and determination of proper assembly. We have successfully purified a recombinant ANME‐2d MCR that is assembled and binds F430, whereas in the case of the ANME‐1 MCR, we can only recover the his‐tagged McrA. Since the ANME‐2d MCR operon contains McrD and the ANME‐1 operon does not, this result supports the importance of McrD for proper assembly in vivo. Combined with other results from the expression of recombinant methanogenic MCRs, our results indicate that MCR accessory proteins are organism specific. Current work is focused on demonstrating the role of McrD through in vitro binding studies as well as further elucidating the importance of mcrDand mcrC for recombinant expression of diverse MCRs.

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