The role of zinc in the methylation of the coenzyme M thiol group in methanol:coenzyme M methyltransferase from Methanosarcina barkeri

Abstract

Methanol:coenzyme M methyltransferase from methanogenic archaea is a cobalamin-dependent enzyme composed of three different subunits: MtaA, MtaB and MtaC. MtaA is a zinc protein that catalyzes the methylation of coenzyme M (HS-CoM) with methylcob(III)alamin. We report zinc XAFS (X-ray absorption fine structure) results indicating that, in the absence of coenzyme M, zinc is probably coordinated by a single sulfur ligand and three oxygen or nitrogen ligands. In the presence of coenzyme M, one (N/O)-ligand was replaced by sulfur, most likely due to ligation of the thiol group of coenzyme M. Mutations in His237 or Cys239, which are proposed to be involved in ligating zinc, resulted in an over 90% loss in enzyme activity and in distinct changes in the zinc ligands. In the His237 fi Ala and Cys239 fi Ala mutants, coenzyme M also seemed to bind efficiently by ligation to zinc indicating that some aspects of the zinc ligand environment are surprisingly uncritical for coenzyme M binding. Keywords:1 1 zinc enzymes; methanogenic archaea; methyl transferases; thiol group alkylation; EXAFS.Methanosarcina barkeri and other Methanosarcina species can grow on methanol as carbon source which is disproportionated to CH 4 and CO 2 [1]. The first step in this metabolic pathway is the formation of methyl-coenzyme M (CH 3 -S-CoM) from methanol and coenzyme M (HS-CoM) [2].The reaction is catalyzed by methanol:coenzyme M methyltransferase which is composed of the three subunits MtaA (35.9 kDa), MtaB (50.7 kDa) and MtaC (27.9 kDa), of which MtaC is a corrinoid protein. They catalyze the following partial reactions [3][4][5][6][7].MtaA is a zinc protein [3,7,8] that also catalyzes the methylation of coenzyme M with methylcob(III)alamin [9]. Several isoenzymes of MtaA, designated MtbA and MtsA have been found [9][10][11].The methylation of coenzyme M to methyl-coenzyme M is a reaction in which a thiol group is alkylated. Enzymes catalyzing alkyl transfers to thiols have all been found to be zinc proteins [12] [19]. The postulated role of zinc in these enzymes is that of a Lewis acid that activates the thiol group to be alkylated. Coordination of the thiol group to the active site zinc has been shown by extended X-ray absorption fine structure (EXAFS) spectroscopy [14,15], by UV spectroscopy [20] and in the case of protein farnesyl transferase by crystal structure analysis [19]. It results in a decrease in the pK value of the thiol group as shown by the release of a proton upon binding of the substrate to the zinc enzyme [21].MtaA does not share sequence similarity to any of the other zinc enzymes catalyzing thiol group alkylation [8,22]. Abbreviations: HS-CoM, coenzyme M; CH 3 -S-CoM, methyl-coenzyme M; EXAFS, extended X-ray absorption fine structure; Mta, methanol:coenzyme M methyltransferase; MtaA, protein subunit of Mta; XANES, X-ray absorption near edge structure; XAS, X-ray absorption spectroscopy.

Connection between Multimetal(loid) Methylation in Methanoarchaea and Central Intermediates of Methanogenesis

“…(III) to the metal(loid)s. The mechanism of CoM methylation catalyzed by MtaA has been described as an activation of CoM through formation of a nucleophilic zinc-thiolate complex (16). This complex attacks the methyl group of CH 3 Cob(III), resulting in the transfer of a methyl carbocation from CH 3 Cob(III) to CoM and the formation of highly reduced Cob(I) (7,18).…”

Regulation of putative methyl‐sulphide methyltransferases in Methanosarcina acetivorans C2A

“…S2). The amino terminal domain of each protein contains a corrinoid-binding motif (GDVH-DIGKNLV) with the conserved active site histidine, whereas the C-terminal methyltransferase domain of each has the potential to co-ordinate Zinc by virtue of a conserved HXCX nC motif Gencic et al, 2001;Kruer et al, 2002). This motif is also conserved in the MtaA1, MtaA2 and MtbA methyltransferase 2 enzymes from all three Methanosarcina spp.…”

A Cysteine-Rich CCG Domain Contains a Novel [4Fe-4S] Cluster Binding Motif As Deduced from Studies with Subunit B of Heterodisulfide Reductase from Methanothermobacter marburgensis

“…The primary sequence of HdrA indicates that it contains the FAD binding site and four canonical binding motifs for [4Fe-4S] clusters. HdrB contains no sequence motif characteristic for the binding of known cofactors but has two unique cysteine-rich sequence motifs (CX [31][32][33][34][35][36][37][38][39] CCX [35][36] CXXC) of unknown function, designated as the CCG domain in the Pfam protein families database (accession number PF02754) (8). The ferredoxin-like subunit HdrC contains two canonical binding motifs for [4Fe-4S] clusters (9).…”