Post-translational thioamidation of methyl-coenzyme M reductase, a key enzyme in methanogenic and methanotrophic Archaea.

Abstract

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. The α subunit of MCR (McrA) contains several unusual post-translational modifications, including a rare thioamidation of glycine. Based on the presumed function of homologous genes involved in the biosynthesis of thioviridamide, a thioamide-containing natural product, we hypothesized that the archaeal <i>tfuA</i> and <i>ycaO</i> genes would be responsible for post-translational installation of thioglycine into McrA. Mass spectrometric characterization of McrA from the methanogenic archaeon <i>Methanosarcina acetivorans</i> lacking <i>tfuA</i> and<i>/</i>or <i>ycaO</i> revealed the presence of glycine, rather than thioglycine, supporting this hypothesis. Phenotypic characterization of the ∆<i>ycaO-tfuA</i> mutant revealed a severe growth rate defect on substrates with low free energy yields and at elevated temperatures (39°C - 45°C). Our analyses support a role for thioglycine in stabilizing the protein secondary structure near the active site.