O-demethylase from Acetobacterium dehalogenans--cloning, sequencing, and active expression of the gene encoding the corrinoid protein.
The ether-cleaving O-demethylase from the strictly anaerobic homoacetogen Acetobacterium dehalogenans catalyses the methyltransfer from 4-hydroxy-3-methoxy-benzoate (vanillate) to tetrahydrofolate. In the first step a vanillate :corrinoid protein methyltransferase (methyltransferase I) mediates the methylation of a 25-kDa corrinoid protein with the cofactor reduced to cob(I)alamin. The methyl group is then transferred to tetrahydrofolate by the action of a methylcorrinoid protein:tetrahydrofolate methyltransferase (methyltransferase II). Using primers derived from the amino-terminal sequences of the corrinoid protein and the vanillate:corrinoid protein methyltransferase (methyltransferase I), a 723-bp fragment was amplified by PCR, which contained the gene odmA encoding the corrinoid protein of O-demethylase. Downstream of odmA, part of the odmB gene encoding methyltransferase I was identified. The amino acid sequence deduced from odmA showed about 60% similarity to the cobalamin-binding domain of methionine synthase from Escherichia coli (MetH) and to corrinoid proteins of methyltransferase systems involved in methanogenesis from methanol and methylamines. The sequence contained the DXHXXG consensus sequence typical for displacement of the dimethylbenzimidazole base of the corrinoid cofactor by a histidine from the protein. Heterologous expression of odmA in E. coli yielded a colourless, oxygen-insensitive apoprotein, which was able to bind one mol cobalamin or methylcobalamin/mol protein. Both of these reconstituted forms of the protein were active in the overall O-demethylation reaction. OdmA reconstituted with hydroxocobalamin and reduced by titanium(III) citrate to the cob(I)alamin form was methylated with vanillate by methyltransferase I in an irreversible reaction. Methylcobalamin carrying OdmA served as methyl group donor for the methylation of tetrahydrofolate by methyltransferase II. This reaction was found to be reversible, since methyltranSferase II also catalysed the methylation of cob(I)alamin containing OdmA with methyltetrahydrofolate.
Kaufmann
F
F
Institut für Mikrobiologie der Universität Stuttgart, Germany.
Wohlfarth
G
G
Diekert
G
G
D000091:Acetobacter / Q000201:enzymology*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D008780:Methyltransferases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010090:Oxidoreductases, O-Demethylating / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D010090:Oxidoreductases, O-Demethylating; D008780:Methyltransferases