Posttranslational Methylation of Arginine in Methyl Coenzyme M Reductase Has a Profound Impact on both Methanogenesis and Growth of Methanococcus maripaludis

Abstract

Catalyzing the key step for anaerobic production and/or oxidation of methane and likely other short-chain alkanes, methyl coenzyme M reductase (Mcr) and its homologs play a key role in the global carbon cycle. The McrA subunit possesses up to five conserved posttranslational modifications (PTMs) at its active site. It was previously suggested that methanogenesis marker protein 10 (Mmp10) could play an important role in methanogenesis. To systematically examine its physiological role, mmpX (locus tag MMP1554), the gene encoding Mmp10 in Methanococcus maripaludis, was deleted with a new genetic tool, resulting in the complete loss of the 5-C-(S)-methylarginine PTM of residue 275 in the McrA subunit. When the ΔmmpX mutant was complemented with the wild-type gene expressed by either a strong or a weak promoter, methylation was fully restored. Compared to the parental strain, maximal rates of methane formation by whole cells were reduced by 40 to 60% in the ΔmmpX mutant. The reduction in activity was fully reversed by the complement with the strong promoter. Site-directed mutagenesis of mmpX resulted in a differential loss of arginine methylation among the mutants in vivo, suggesting that activities of Mmp10 directly modulated methylation. R275 was present in a highly conserved PXRR275(A/S)R(G/A) signature sequence in McrAs. The only other protein in M. maripaludis containing a similar sequence was not methylated, suggesting that Mmp10 is specific for McrA. In conclusion, Mmp10 modulates the methyl-Arg PTM on McrA in a highly specific manner, which has a profound impact on Mcr activity. IMPORTANCE Mcr is the key enzyme in methanogenesis and a promising candidate for bioengineering the conversion of methane to liquid fuel. Our knowledge of Mcr is still limited. In terms of complexity, uniqueness, and environmental importance, Mcr is more comparable to photosynthetic reaction centers than conventional enzymes. PTMs have long been hypothesized to play key roles in modulating Mcr activity. Here, we directly link the mmpX gene to the arginine PTM of Mcr, demonstrate its association with methanogenesis activity, and offer insights into its substrate specificity and putative cofactor binding sites. This is also the first time that a PTM of McrA has been shown to have a substantial impact on both methanogenesis and growth in the absence of additional stressors.

Methanosarcina acetivorans: A Model for Mechanistic Understanding of Aceticlastic and Reverse Methanogenesis

“…A unique radical SAM methyltransferase was shown required for methylation of the active-site arginine and concluded important for stability under imposed oxidative and heat stress (Deobald et al, 2018;Radle et al, 2019). Deletion of a homolog essential for arginine methylation in the obligate CO 2 -reducing methanogen Methanococcus maripaludis resulted in a 40-60% loss in the rate of methanogenesis consistent with partial loss of Mcr activity (Lyu et al, 2020). Deletion of two genes essential for thioglycine synthesis in McrA of Ms. acetivorans produced mutants severely impaired in the rate of growth with acetate and when exposed to thermal and oxidative stress, results supporting a role for thioglycine in stabilizing the McrA active-site although not essential.…”

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

“…The core modified amino acids are widely conserved and include N 1 -methylhistidine (or 3-methylhistidine), 5-(S)-methylarginine, and thioglycine ( Fig 1B). The widespread conservation of these core modifications is exemplified by the recently discovered enzymes responsible for installation of the thioglycine and 5-(S)-methylarginine modifications, whose encoding genes were originally designated as methanogenesis marker genes because of their universal occurrence in the genomes of sequenced methanogens [26][27][28][29][30]. Given that 5-(S)methylarginine and thioglycine are extremely rare in nature, yet universally conserved in MCR, it was speculated that these residues must be essential for catalysis [24].…”

Methyl-Coenzyme M Reductase and Its Post-translational Modifications

“…This study was the first to link MCR activity and a specific PTM. It also firstly demonstrates that a PTM could affect both methanogenesis and cell growth without the addition of stressor ( Lyu et al, 2020 ). Furthermore, this study also indicated a high specificity of Mmp10 to McrA and the binding sites of cofactors, SAM, and cobalamin, in Mmp10 ( Lyu et al, 2020 ).…”

“…It also firstly demonstrates that a PTM could affect both methanogenesis and cell growth without the addition of stressor ( Lyu et al, 2020 ). Furthermore, this study also indicated a high specificity of Mmp10 to McrA and the binding sites of cofactors, SAM, and cobalamin, in Mmp10 ( Lyu et al, 2020 ). For glutamine methylation, although the putative cobalamin-dependent radical SAM enzyme has been proposed ( Deobald et al, 2018 ), there has been no solid evidence.…”

“…The functions of methylation in MCRs are not fully addressed with only few studies. Histidine methylation is theoretically thought to serve to position the imidazole ring that coordinates CoB ( Grabarse et al, 2000 ), while arginine methylation at the active site of MCR could play multiple roles in the catalysis, assembly, or stability of the enzyme ( Deobald et al, 2018 ; Lyu et al, 2020 ).…”