Functional interactions between posttranslationally modified amino acids of methyl-coenzyme M reductase in Methanosarcina acetivorans

Abstract

The enzyme methyl-coenzyme M reductase (MCR) plays an important role in mediating global levels of methane by catalyzing a reversible reaction that leads to the production or consumption of this potent greenhouse gas in methanogenic and methanotrophic archaea. In methanogenic archaea, the alpha subunit of MCR (McrA) typically contains four to six posttranslationally modified amino acids near the active site. Recent studies have identified enzymes performing two of these modifications (thioglycine and 5-[S]-methylarginine), yet little is known about the formation and function of the remaining posttranslationally modified residues. Here, we provide in vivo evidence that a dedicated S-adenosylmethionine-dependent methyltransferase encoded by a gene we designated methylcysteine modification (mcmA) is responsible for formation of S-methylcysteine in Methanosarcina acetivorans McrA. Phenotypic analysis of mutants incapable of cysteine methylation suggests that the S-methylcysteine residue might play a role in adaption to mesophilic conditions. To examine the interactions between the S-methylcysteine residue and the previously characterized thioglycine, 5-(S)-methylarginine modifications, we generated M. acetivorans mutants lacking the three known modification genes in all possible combinations. Phenotypic analyses revealed complex, physiologically relevant interactions between the modified residues, which alter the thermal stability of MCR in a combinatorial fashion that is not readily predictable from the phenotypes of single mutants. High-resolution crystal structures of inactive MCR lacking the modified amino acids were indistinguishable from the fully modified enzyme, suggesting that interactions between the posttranslationally modified residues do not exert a major influence on the static structure of the enzyme but rather serve to fine-tune the activity and efficiency of MCR.

Methyl-Coenzyme M Reductase and Its Post-translational Modifications

“…Histidine and cysteine are possibly catalyzed by SAM-dependent N- and S-methyltransferases, respectively, while arginine and glutamine seem to have different enzymatic methylation ( Selmer et al, 2000 ). The methylation of cysteine and arginine have been shown to be related to a dedicated SAM-dependent (encoding by mcmA ) and a potential radical SAM-dependent methyltransferase (Mmp10, encoding by mmpX ); however, the enzyme for histidine and glutamine modification still remains unknown ( Deobald et al, 2018 ; Nayak et al, 2020 ). YcaO and TfuA were found to be involved in the biosynthesis of thioglycine.…”

“…Moreover, it was indicated that the biosynthesis of 1- N -methylhistidine and S -methylcysteine was probably catalyzed by SAM-dependent N - and S -methyltransferases by which the methyl group is transferred to N - or S -nucleophiles of histidine or cysteine via a S N 2 reaction. The gene ( mcmA ) encoding the SAM-dependent methyltransferase for cysteine methylation has been recently identified, and it has been indicated that cysteine methylation might play a role in adaption to mesophilic conditions ( Nayak et al, 2020 ). However, 2-( S )-methylglutamine and 5-( S )-methylarginine could not be formed by this mechanism ( Liscombe et al, 2012 ; Nayak et al, 2020 ).…”

“…The gene ( mcmA ) encoding the SAM-dependent methyltransferase for cysteine methylation has been recently identified, and it has been indicated that cysteine methylation might play a role in adaption to mesophilic conditions ( Nayak et al, 2020 ). However, 2-( S )-methylglutamine and 5-( S )-methylarginine could not be formed by this mechanism ( Liscombe et al, 2012 ; Nayak et al, 2020 ).…”

“…Recently, to test the interactions between arginine methylation, cysteine methylation and thioamidation, Nayak et al established knock-out strains with all possible combination, and observed that the ΔycaO-tfuA / ΔmcmA double mutant strain had most severe growth, while the one with triple deletion showed a faster growth, suggesting that unmodified Arg 285 could alleviate changes brought by the absence of thioglycine and methyl-cysteine. In addition, by phenotypic analyses, they showed that interactions between PTMs could cause unpredictable changes of the thermal stability of MCR, which might influence the activity and efficiency of MCRs ( Nayak et al, 2020 ).…”

“…As one remarkable feature of MCRs, coenzyme F430 located in the active site has been proved to be crucial, and focuses have been put on its reaction mechanism and activation ( Wongnate and Ragsdale, 2015 ; Wongnate et al, 2016 ; Lyu et al, 2018a ). Besides coenzyme F430, unusual post-translational modification (PTM) is identified as another remarkable feature of MCR ( Kahnt et al, 2007 ; Mansoorabadi et al, 2017 ; Nayak et al, 2020 ). However, the biosynthesis and functions of MCR PTMs remain largely unclear.…”