Halomethane:bisulfide/halide ion methyltransferase, an unusual corrinoid enzyme of environmental significance isolated from an aerobic methylotroph using chloromethane as the sole carbon source

Abstract

A novel dehalogenating/transhalogenating enzyme, halomethane:bisulfide/halide ion methyltransferase, has been isolated from the facultatively methylotrophic bacterium strain CC495, which uses chloromethane (CH(3)Cl) as the sole carbon source. Purification of the enzyme to homogeneity was achieved in high yield by anion-exchange chromatography and gel filtration. The methyltransferase was composed of a 67-kDa protein with a corrinoid-bound cobalt atom. The purified enzyme was inactive but was activated by preincubation with 5 mM dithiothreitol and 0.5 mM CH(3)Cl; then it catalyzed methyl transfer from CH(3)Cl, CH(3)Br, or CH(3)I to the following acceptor ions (in order of decreasing efficacy): I(-), HS(-), Cl(-), Br(-), NO(2)(-), CN(-), and SCN(-). Spectral analysis indicated that cobalt in the native enzyme existed as cob(II)alamin, which upon activation was reduced to the cob(I)alamin state and then was oxidized to methyl cob(III)alamin. During catalysis, the enzyme shuttles between the methyl cob(III)alamin and cob(I)alamin states, being alternately demethylated by the acceptor ion and remethylated by halomethane. Mechanistically the methyltransferase shows features in common with cobalamin-dependent methionine synthase from Escherichia coli. However, the failure of specific inhibitors of methionine synthase such as propyl iodide, N(2)O, and Hg(2+) to affect the methyltransferase suggests significant differences. During CH(3)Cl degradation by strain CC495, the physiological acceptor ion for the enzyme is probably HS(-), a hypothesis supported by the detection in cell extracts of methanethiol oxidase and formaldehyde dehydrogenase activities which provide a metabolic route to formate. 16S rRNA sequence analysis indicated that strain CC495 clusters with Rhizobium spp. in the alpha subdivision of the Proteobacteria and is closely related to strain IMB-1, a recently isolated CH(3)Br-degrading bacterium (T. L. Connell Hancock, A. M. Costello, M. E. Lidstrom, and R. S. Oremland, Appl. Environ. Microbiol. 64:2899-2905, 1998). The presence of this methyltransferase in bacterial populations in soil and sediments, if widespread, has important environmental implications.

FIG. 1

Growth of strain CC495 on chloromethane. Symbols: ■, chloromethane (measured in millimoles); ⧫, chloride (in millimoles); ▴, growth measured as absorbance at 560 nm.

FIG. 2

Phylogenetic analysis of the 16S rRNA gene from strain CC495. The tree was obtained by the neighbor-joining approach using the TREECON program. Similar phylogenesis was obtained when parsimony analysis of the same data was conducted. Bootstrap values (in percentages) are given at the nodes. Bar, 0.02 base substitutions per site. The 16S rRNA gene sequence from Methylocystis parvus OBBp was used as the outgroup. The GenBank accession numbers of the organisms are as follows: strain CC495, AF107722; strain DSM 7048T, AF011759; strain DSM 7051T, AF011760; strain DSM 6450T, AF011762; strain ER2, L20802; strain IMB-1, AF034798; Rhizobium loti R88b, U50165; R. loti LMG4284, X67230; Rhizobium ciceri UPM-Ca7, U07934; Rhizobium mediterraneum UPM-Ca36, L38825; R. loti IAM13588, D12791; Rhizobium huakuii IAM14158, D12797; Bartonella clarridgeiae CIP104772, X97822; Rhizobium fredii LMG6217, X67321; Rhizobium leguminosarum USDA2370, U29386; Rhodobium orientis MB312, D30792; M. extorquens, M29027; M. parvus OBBp, M29026.

FIG. 3

Coomassie blue-stained gels after SDS-PAGE of cell extracts of strain CC495 grown on CH₃Cl, CH₃Cl-methylamine, or methylamine. Lanes 1, 2, and 3 contain 2.5 μg protein of extracts from cells grown on CH₃Cl, CH₃Cl-methylamine, and methylamine, respectively. Lane 4 contains protein standards with molecular masses (in kilodaltons) as indicated.

FIG. 4

CH₃Cl utilization and formate formation by cell extracts of strain CC495. Cell extracts were incubated at 25°C with 50 mM phosphate buffer (pH 7.8) containing 1 mM NADH, 0.5 mM DTT, and 17.2 mM CH₃Cl. Formate concentrations (■) were determined by GC-MS, and CH₃Cl concentrations (●) were determined by gas chromatography.

FIG. 5

CH₃I formation from CH₃Cl and I⁻ by dialyzed cell extracts of strain CC495. Dialyzed extracts were incubated at 25°C with 3 mM I⁻, 0.5 mM DTT, and 0.5 mM CH₃Cl in 50 mM phosphate buffer (pH 7.8). CH₃I formation was determined by gas chromatography.

FIG. 6

SDS-PAGE of protein fractions from stages in the purification of methyltransferase from strain CC495. The gel was stained with Coomassie blue. Lane 1, 0.55 μg of protein from the gel filtration stage; lane 2, 0.69 μg of protein from the anion-exchange stage; lane 3, 0.46 μg of protein from crude cell extracts; lane 4, protein standards with molecular masses (in kilodaltons) as indicated.

FIG. 7

Effects of preincubation with DTT and CH₃Cl on the activation of methyltransferase. (a) Effect of preincubation for 60 min at 25°C with 0.5 mM CH₃Cl and different concentrations of DTT in 50 mM phosphate buffer (pH 7.0) on the activity of methyltransferase in the standard assay. (b) Effect of preincubation for 60 min at 25°C in 50 mM phosphate buffer (pH 7.0) with 0.5 mM CH₃Cl, 5 mM DTT, or 0.5 mM CH₃Cl plus 5 mM DTT on the activity of methyltransferase in the standard assay.

FIG. 8

Absorbance spectra of methyltransferase in unactivated and activated states: purified enzyme (7.5 μM) in 50 mM phosphate buffer (pH 7.0) (solid line), after addition of 5 mM DTT (dashed-and-dotted line), and after addition of 5 mM DTT and 10 mM CH₃Cl (dashed line).

FIG. 9

Scheme showing postulated mechanism of activation and catalysis for halomethane:bisulfide/halide ion methyltransferase.