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Properties of the methylcobalamin:H4folate methyltransferase involved in chloromethane utilization by Methylobacterium sp. strain CM4
- Source :
- European Journal of Biochemistry. 264:242-249
- Publication Year :
- 1999
- Publisher :
- Wiley, 1999.
-
Abstract
- Methylobacterium sp. strain CM4 is a strictly aerobic methylotrophic proteobacterium growing with chloromethane as the sole carbon and energy source. Genetic evidence and measurements of enzyme activity in cell-free extracts have suggested a multistep pathway for the conversion of chloromethane to formate. The postulated pathway is initiated by a corrinoid-dependent methyltransferase system involving methyltransferase I (CmuA) and methyltransferase II (CmuB), which transfer the methyl group of chloromethane onto tetrahydrofolate (H4folate) [Vannelli et al. (1999) Proc. Natl Acad. Sci. USA 96, 4615-4620]. We report the overexpression in Escherichia coli and the purification to apparent homogeneity of methyltransferase II. This homodimeric enzyme, with a subunit molecular mass of 33 kDa, catalyzed the conversion of methylcobalamin and H4folate to cob(I)alamin and methyl-H4folate with a specific activity of 22 nmol x min-1 x (mg protein)-1. The apparent kinetic constants for H4folate were: Km = 240 microM, Vmax = 28.5 nmol x min-1 x (mg protein)-1. The reaction appeared to be first order with respect to methylcobalamin at concentrations up to 2 mM, presumably reflecting the fact that methylcobalamin is an artificial substitute for the methylated methyltransferase I, the natural substrate of the enzyme. Tetrahydromethanopterin, a coenzyme also present in Methylobacterium, did not serve as a methyl group acceptor for methyltransferase II. Purified methyltransferase II restored chloromethane dehalogenation by a cell free extract of a strain CM4 mutant defective in methyltransferase II.
- Subjects :
- Methyltransferase
Stereochemistry
Molecular Sequence Data
Biochemistry
Catalysis
Cofactor
chemistry.chemical_compound
medicine
Amino Acid Sequence
DNA Primers
chemistry.chemical_classification
Base Sequence
Gram-Negative Aerobic Bacteria
Sequence Homology, Amino Acid
biology
Chloromethane
Temperature
Tetrahydromethanopterin
Hydrogen-Ion Concentration
Chromatography, Ion Exchange
Protein O-Methyltransferase
biology.organism_classification
Recombinant Proteins
Enzyme
chemistry
Methylcobalamin
Methyl Chloride
biology.protein
Methylobacterium
Electrophoresis, Polyacrylamide Gel
Energy source
medicine.drug
Subjects
Details
- ISSN :
- 14321033, 00142956, and 46154620
- Volume :
- 264
- Database :
- OpenAIRE
- Journal :
- European Journal of Biochemistry
- Accession number :
- edsair.doi.dedup.....3988909beeb76c11ced715d4f22f6e48