Connection between multimetal(loid) methylation in methanoarchaea and central intermediates of methanogenesis

Abstract

In spite of the significant impact of biomethylation on the mobility and toxicity of metals and metalloids in the environment, little is known about the biological formation of these methylated metal(loid) compounds. While element-specific methyltransferases have been isolated for arsenic, the striking versatility of methanoarchaea to methylate numerous metal(loid)s, including rare elements like bismuth, is still not understood. Here, we demonstrate that the same metal(loid)s (arsenic, selenium, antimony, tellurium, and bismuth) that are methylated by Methanosarcina mazei in vivo are also methylated by in vitro assays with purified recombinant MtaA, a methyltransferase catalyzing the methyl transfer from methylcobalamin [CH₃Cob(III)] to 2-mercaptoethanesulfonic acid (CoM) in methylotrophic methanogenesis. Detailed studies revealed that cob(I)alamin [Cob(I)], formed by MtaA-catalyzed demethylation of CH₃Cob(III), is the causative agent for the multimetal(loid) methylation observed. Moreover, Cob(I) is also capable of metal(loid) hydride generation. Global transcriptome profiling of M. mazei cultures exposed to bismuth did not reveal induced methyltransferase systems but upregulated regeneration of methanogenic cofactors in the presence of bismuth. Thus, we conclude that the multimetal(loid) methylation in vivo is attributed to side reactions of CH₃Cob(III) with reduced cofactors formed in methanogenesis. The close connection between metal(loid) methylation and methanogenesis explains the general capability of methanoarchaea to methylate metal(loid)s.

Fig. 1.

Scheme of CoM methylation in the methanol-utilizing methanogenic pathway of M. mazei. The methyl group of methanol is cleaved off by MtaBC and transferred to the reduced cofactor Cob(I). Then, MtaA catalyzes the methyl transfer from CH₃Cob(III) to CoM by deprotonating the thiol group of CoM (26).

Fig. 2.

Metal(loid) volatilization patterns obtained in the presence of crude extracts of noninduced M. mazei (column A), recombinant MtaA (column B), and electrochemically formed Cob(I) (column C). Shown are PT-GC-ICP-MS chromatograms of the volatile metal(loid) species produced under the following reaction conditions: for columns A and B, 1 mM CH₃Cob(III) and CoM in 50 mM HEPES, pH 7, were incubated with 100 μg ml⁻¹ M. mazei crude extract or 10 μg ml⁻¹ purified recombinant MtaA in the presence of 0.1 μM As, 10 μM Se, 0.1 μM Sb, 10 μM Te, and 0.1 μM Bi at 30°C for 10 min prior to headspace analyses; for column C, 5 ml of 0.5 mM CH₃Cob(III) in 50 mM phosphate buffer, pH 7, was incubated with 0.25 mM Cob(I) and a 0.25 mM concentration of either As, Sb, Bi, Se, or Te at 37°C for 30 min prior to headspace analyses.

Fig. 3.

Reactions of arsenite with equal amounts of CH₃Cob(III) and Cob(I) (A and C in both panels; all references to the graphs apply to both panels I and II), with an excess of Cob(I) over CH₃Cob(III) (B) or Cob(I) alone (D) in the presence of MtaA and CoM (A and B), or without MtaA and CoM (C and D), analyzed by UV/Vis spectra (I) and PT-GC-ICP-MS (II). MtaA at 10 μg ml⁻¹ was added to assay mixtures containing 0.1 mM CH₃Cob(III) (A and B) and either 0.05 mM CoM (A) or 0.14 mM CoM (B). The reaction was allowed to proceed until no further demethylation of CH₃Cob(III) was observed photometrically (see Fig. S2 in the supplemental material). For the assays shown in graphs C and D, the mixture contained 0.05 mM (C) and 0.1 mM (D) Cob(I) prepared by electrochemical reduction of aquocob(III)alamin. For the assays shown in the C graphs, the mixture was additionally amended by 0.05 mM CH₃Cob(III). Reaction volumes were 1 ml, and all reactions were conducted under strict anaerobic conditions in 50 mM HEPES (pH 7) at 30°C. UV/Vis spectra were measured in 1-min intervals for 10 min. Thereafter, headspace was sampled for analysis of volatile arsenic species.