Didehydroaspartate Modification in Methyl‐Coenzyme M Reductase Catalyzing Methane Formation

Abstract

All methanogenic and methanotrophic archaea known to date contain methyl‐coenzyme M reductase (MCR) that catalyzes the reversible reduction of methyl‐coenzyme M to methane. This enzyme contains the nickel porphinoid F430 as a prosthetic group and, highly conserved, a thioglycine and four methylated amino acid residues near the active site. We describe herein the presence of a novel post‐translationally modified amino acid, didehydroaspartate, adjacent to the thioglycine as revealed by mass spectrometry and high‐resolution X‐ray crystallography. Upon chemical reduction, the didehydroaspartate residue was converted into aspartate. Didehydroaspartate was found in MCR I and II from Methanothermobacter marburgensis and in MCR of phylogenetically distantly related Methanosarcina barkeri but not in MCR I and II of Methanothermobacter wolfeii, which indicates that didehydroaspartate is dispensable but might have a role in fine‐tuning the active site to increase the catalytic efficiency.

Post-Translational Modifications of Protein Backbones: Unique Functions, Mechanisms, and Challenges

“…A suite of interesting, enzymatically installed bbPTMs has been discovered in archaeal methyl-coenzyme M reductase, a key enzyme in methane metabolism (Figure 3d). 23−25 Didehydroaspartate, 25 α-methylglutamine, 24 and thioglycine 24 (substitution of the carbonyl oxygen of a glycine residue with a sulfur atom) have been detected in several homologues near the 29 and by protein isoaspartate methyltransferase (PIMT), 30 respectively. (b) Proposed mechanism 13,27 for thioamide formation in methyl-coenzyme M reductase via a kinase that targets the backbone amide, YcaO, and an auxiliary protein of unknown function, TfuA, which may be involved in substrate recognition or sulfur transfer.…”

“…active site, attesting to their importance. A candidate oxidoreductase for didehydroaspartate formation has been postulated, 25 and it has been demonstrated that the C α -methyl group of α-methylglutamine originates from the cofactor Sadenosylmethionine, but a specific methyltransferase has not been identified. 26 Recently, the enzymes responsible for thioglycine formation, a backbone kinase (YcaO) and an auxiliary protein of unknown function (TfuA), have been identified through homologues involved in natural product thioamide biosynthesis pathways (Figure 4a).…”

“…(c) Isoaspartate-containing hairpin in MurA from Enterobacter cloacae (UDP-N-acetylglucosamine 1carboxyvinyltransferase, PDB entry 1EJC) 22. (d) bbPTMs discovered in the active site region of methyl-coenzyme M reductase from the methanogenic archaeon Methanothermobacter marburgensis (left, α-methylglutamine; center, thioglycine; right, dehydroaspartate; PDB entry 5A0Y) 23,25. A potential n → π* interaction 28 involving thioglycine is indicated by a dashed line.…”

The Biological Methane‐Forming Reaction: Mechanism Confirmed Through Spectroscopic Characterization of a Key Intermediate

“…Furthermore,M CR contains many modified amino acids near the active site,namley,four methylated amino acids,d idehydroaspartate, [9] and thioglycine,t he main-chain carbonyl oxygen of which is exchanged with sulfur. Fore xample,t he crystal structure of the active MCR red1 form has to be solved.…”

“…To date, all proposed mechanisms are based on crystal structures of the inactive forms of MCR. Furthermore,M CR contains many modified amino acids near the active site,namley,four methylated amino acids,d idehydroaspartate, [9] and thioglycine,t he main-chain carbonyl oxygen of which is exchanged with sulfur. [10] Thec atalytic mechanism should explain why these modifications are present.…”