Gendron, Aleksei2023-01-252023-01-252023-01-24vt_gsexam:36306http://hdl.handle.net/10919/113412Methyl-coenzyme M reductase (MCR) is the key enzyme involved in the production of methane by methanogenic archaea and its consumption by anaerobic methanotrophs (ANME). MCR is a multimeric complex composed of six different subunits arranged in a 2α, 2β, 2γ configuration that requires two molecules of its nickel-containing tetrapyrrole prosthetic group, coenzyme F430. Additionally, the α subunits of MCR house a variety of different post-translational modifications across both methanogens and ANME. In methanogens, MCR is encoded in a conserved mcrBDCGA gene cluster, which encodes accessory proteins McrD and McrC. These are believed to be involved in the assembly and activation of MCR, respectively. However, one or both accessory proteins are often omitted from the operon in other MCR-containing archaea as is the case in ANME. MCR knowledge is mostly limited to methanogens due to difficulties associated with large-scale cultivation of ANME and other MCR-containing archaea. Due to the complexity of MCR, studies on this enzyme are also largely limited to native enzymes. Developing methods for the detailed biochemical characterization ANME MCRs would be highly desirable since these enzymes are proposed to be optimized for methane oxidation and thus have immense potential for bioenergy and greenhouse gas mitigation applications. In addition to containing the necessary machinery for the production of an assembled and active MCR, model methanogens are easier to culture and have established genetic manipulation techniques, making them ideal candidates for the development of heterologous expression systems. Thus, here we sought to generate such a system for the study of various ANME MCRs in the methanogen, Methanococcus maripaludis. We report the successful expression and purification of an ANME-2d MCR, marking a significant step toward the development of a heterologous MCR expression system. Additionally, our attempts to purify various recombinant MCRs revealed the importance of including accessory proteins, particularly McrD, within expression constructs. Therefore, we also sought to functionally characterize McrD, which we show is likely an MCR chaperone that plays a key role in MCR maturation. Taken together, our work has provided key insights into MCR assembly as well as provided a foundation for the eventual development of MCR based biocatalytic systems to be used for methane mitigation strategies and bioenergy platforms.ETDenCreative Commons Attribution 4.0 InternationalMethaneMethanogensMethyl-coenzyme M ReductaseMethanogenesisANMEOxidation of MethaneAssemblyHeterologous ExpressionDissertation