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

Dipti D Nayak,Douglas A Mitchell,Andi Liu,Neha Agrawal,Satish K Nair,Shi-Hui Dong,William W Metcalf,Roy Rodriguez-Carerro,Henrik Sass

doi:10.1371/journal.pbio.3000507

Dipti D Nayak, Douglas A Mitchell + Show 7 more

Open Access

https://doi.org/10.1371/journal.pbio.3000507

Copy DOI

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.

Highlights

Methyl-coenzyme M (CoM) reductase (MCR) is an unusual and important enzyme, which to date has only been observed in strictly anaerobic, methane-metabolizing archaea [1,2]
We previously described the addition of an affinity tag that allowed for the facile purification of MCR from M. acetivorans cells [34]
The active site of MCR from Methanosarcina acetivorans (MaMCR) shows electron density features corresponding to the factor 430 (F430) cofactor, as well as for the CoM and methyl-coenzyme B (CoB), showing that the affinity tag does not disrupt the association of these molecules

Summary

Introduction

Methyl-coenzyme M (CoM) reductase (MCR) is an unusual and important enzyme, which to date has only been observed in strictly anaerobic, methane-metabolizing archaea [1,2]. In these organisms, MCR catalyzes the reversible interconversion of CoM (2-methylmercaptoethanesulfonate) and coenzyme B (CoB, 7-thioheptanoylthreoninephosphate) to methane and a CoB-CoM heterodisulfide: CH3 À S À CoM þ HS À CoB ⇄ CH4 þ CoM ÀSÀSÀ CoB. Members of the larger ACR family play pivotal roles in archaeal evolution, climate change, and the carbon cycle They offer new opportunities for the development of bio-based solutions for the production of methane and other renewable fuels [15]. Many fundamental properties of this important enzyme remain poorly understood

Methods

Results

Conclusion