Atomic resolution structures of the methane-activating enzyme in anaerobic methanotrophy reveal extensive...

Anaerobic methanotrophic archaea (ANME) are crucial to planetary carbon cycling. They oxidise methane in anoxic niches by transferring electrons to nitrate, metal oxides, or sulfate-reducing bacteria. No ANMEs have been isolated, hampering the biochemical investigation of anaerobic methane oxidation. Here, we obtained the true atomic resolution structure of their methane-capturing system (Methyl-Coenzyme M Reductase, MCR), circumventing the isolation barrier by exploiting microbial enrichments of freshwater nitrate-reducing ANME-2d grown in bioreactors, and marine ANME-2c in syntrophy with bacterial partners. Despite their physiological differences, these ANMEs have extremely conserved MCR structures, similar to homologs from methanogenic Methanosarcinales, rather than the phylogenetically distant MCR of ANME-1 isolated from Black Sea mats. The three studied enzymes have seven post-translational modifications, among them was a novel 3(S)-methylhistidine on the γ-chain of both ANME-2d MCRs. Labelling with gaseous krypton did not reveal any internal channels that would facilitate alkane diffusion to the active site, as observed in the ethane-specialised enzyme. Based on our data, the methanotrophic MCRs should follow the same radical reaction mechanism proposed for the methane-generating homologues. The described pattern of post-translational modifications underscores the importance of native purification as a powerful approach to discovering intrinsic enzymatic features in non-isolated microorganisms existing in nature.