Methyl-coenzyme M reductase (MCR), found in strictly anaerobic methanogenic and methanotrophic archaea, catalyzes the reversible production and consumption of the potent greenhouse gas methane. MCR displays an α2β2γ2 protein domain architecture and contains two molecules of coenzyme F430, a nickel porphinoid cofactor which is essential for catalysis. The α subunit of MCR (McrA) contains several post-translational modifications (PTM), including a rare but universal thioamidation of glycine. Previously, thioglycine has been proposed to have a role in the reaction mechanism of MCR, however, the enzymes responsible for installing the thioamide moiety remained unknown. Based on the proposed function of the homologous genes involved in the biosynthesis of thioviridamide, a thioamide containing ribosomal natural product, we hypothesized that the archaeal tfuA and ycaO genes would be responsible for post-translational installation of thioglycine into McrA. Mass spectrometric characterization of McrA from the methanogenic archaeon Methanosarcina acetivorans lacking tfuA and/or ycaO revealed the presence of glycine, rather than thioglycine, supporting this hypothesis. Phenotypic characterization of the deletion mutant showed a severe growth rate defect on substrates with low free energy yields and at higher temperatures. Contrary to the previous proposals, our analysis supports a role for thioglycine in stabilizing the protein secondary structure near the active site and refutes its role in the catalytic mechanism of MCR. In addition, in-vitro biochemical and structural studies confirmed the involvement of these two enzymes in the thioamide formation and showed that it is an ATP dependent process. Moreover, our biochemical studies have also elucidated the factors governing the regiospecific installation of the thioamide into McrA as well as provided mechanistic insights for this interesting transformation. This work clarified a long-standing question regarding the function of this universal PTM found in MCR, a critical enzyme involved in the global carbon cycle.

Post-translational thioamidation of methyl-coenzyme M reductase, a key enzyme in the global carbon cycle

255th National Meeting (2018)

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