Atomic resolution structures of key enzyme MCR in anaerobic methanotrophy reveal novel and extensive post-translational modifications.

Anaerobic methanotrophic archaea (ANME) are crucial to planetary carbon cycling. They oxidise methane in anoxic niches by transferring electrons directly to nitrate or metal oxides and alternatively to sulfate-reducing bacteria. Due to their physiological complexity, none of the ANME species have been isolated, hampering the biochemical investigation of the enzymatic processes involved in anaerobic methane oxidation. Here, we deciphered the methane-capturing enzyme through native purification directly from microbial enrichments. Freshwater ANME-2d reducing nitrate, grown in bioreactors, and marine microbial enrichment containing ANME-2c, along with its bacterial syntrophic partners, were used as model systems. The crystal structures of the Methyl-Coenzyme M Reductases (MCRs), refined to true atomic resolution, captured its most precise image to date. 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. An exceptional content of seven post-translational modifications was observed in cristallo . Among them was a novel 3( S )-methylhistidine on the γ-chain of the ANME-2d MCR. Krypton gassing did not lead to the detection of an internal channel that would facilitate alkane diffusion to the active site, contrary to the ethane-activating homologue. Our results corroborate that MCRs from freshwater ANME-2d and marine ANME-2c should follow the conserved radical reaction mechanism of the methane-generating counterpart. The described pattern of post-translational modifications, different to ANME-1, highlights the importance of native purification as an unbiased approach to discovering intrinsic enzymatic features in uncultivated microorganisms existing in nature.