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 mcrD and mcrC for recombinant expression of diverse MCRs.
