Boswinkle, Kaleb Storm2022-07-062022-07-062022-07-05vt_gsexam:35171http://hdl.handle.net/10919/111130Methanogenesis is the biological production of methane and is utilized by methanogenic archaea (methanogens) to generate energy. This process is responsible for 70% of total atmospheric methane, a potent greenhouse gas and an important energy source (natural gas). In the future, reversing methanogenesis in an engineered methanogenic strain could be realized to efficiently convert natural gas into liquid fuels. Methyl coenzyme M reductase (Mcr) catalyzes the final reaction of methanogenesis in methanogens and the first reaction in the anaerobic oxidation of methane (AOM) carried out by the anaerobic methanotrophs (ANME). Cofactor F₄₃₀, a unique nickel-containing tetrapyrrole, serves as the prosthetic group and catalytic component of Mcr. Recently, multiple F₄₃₀ variants have been discovered in several methanogenic species, including Methanococcus maripaludis, Methanosarcina acetivorans, and Methanocaldococcus jannaschii. A novel variant reported here has an exact mass of 1008.3478, a similar absorption spectrum as unmodified F₄₃₀, and associates with purified Mcr from M. acetivorans. Based on the exact mass, this molecule is likely modified with a mercaptopropamide moiety. In some conditions, this modified F₄₃₀ comprises 30-50% of the total F₄₃₀ pool. We also report upon our work to identify the sulfur insertion enzyme required to produce methylthio-F₄₃₀ that functions with Mcr in ANME-1. We hypothesized that the insertion of the methylthio moiety is likely catalyzed by a methylthiotransferase (MTTase) homolog present in ANME. However, purified ANME MTTase does not appear to catalyze this reaction, and instead catalyzes the methylthiolation of N6-threonylcarbamoyladenosine (t6A) in tRNA.ETDenIn CopyrightmethanogenesisArchaearadical SAMThesis