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Applied and Environmental Microbiology, October 1999, p. 4301-4312, Vol. 65, No. 10
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Halomethane:Bisulfide/Halide Ion Methyltransferase, an Unusual Corrinoid Enzyme of Environmental Significance Isolated from an Aerobic Methylotroph Using Chloromethane as the Sole Carbon Source

Catherine Coulter,1,2 John T. G. Hamilton,3 W. Colin McRoberts,3 Leonid Kulakov,2 Michael J. Larkin,2,4 and David B. Harper1,2,3,*

Microbial Biochemistry Section, School of Agriculture and Food Science,1 The QUESTOR Centre,2 and School of Biology and Biochemistry,4 The Queen's University of Belfast, and Food Science Division, Department of Agriculture for Northern Ireland,3 Belfast, United Kingdom

Received 9 April 1999/Accepted 20 July 1999

A novel dehalogenating/transhalogenating enzyme, halomethane:bisulfide/halide ion methyltransferase, has been isolated from the facultatively methylotrophic bacterium strain CC495, which uses chloromethane (CH3Cl) 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 CH3Cl; then it catalyzed methyl transfer from CH3Cl, CH3Br, or CH3I to the following acceptor ions (in order of decreasing efficacy): I-, HS-, Cl-, Br-, NO2-, 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, N2O, and Hg2+ to affect the methyltransferase suggests significant differences. During CH3Cl 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 CH3Br-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.

* Corresponding author. Mailing address: Microbial Biochemistry Section, School of Agriculture and Food Science, The Queen's University of Belfast, Newforge Lane, Belfast, BT9 5PX, United Kingdom. Phone: 44-1232-255343. Fax: 44-1232-669551. E-mail: D.Harper{at}qub.ac.uk.

Applied and Environmental Microbiology, October 1999, p. 4301-4312, Vol. 65, No. 10
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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