Specificity of O-demethylation in extracts of the homoacetogenic Holophaga foetida and demethylation kinetics measured b
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© Springer-Verlag 1997
O R I G I N A L PA P E R
Jan-Ulrich Kreft · Bernhard Schink
Specificity of O -demethylation in extracts of the homoacetogenic Holophaga foetida and demethylation kinetics measured by a coupled photometric assay
Received: 8 November 1996 / Accepted: 13 January 1997
Abstract The kinetics and specificity of O-demethylation were studied in cell-free extracts of the strictly anaerobic, methanethiol- and dimethylsulfide-producing homoacetogen Holophaga foetida strain TMBS4 with methanethiol and tetrahydrofolate (H4folate) as methyl acceptors. Extracts of cells grown with 3,4,5-trimethoxybenzoate contained an enzyme system that demethylated various phenyl methyl ethers with at least one ortho-positioned hydroxyl or methoxyl group (the ortho system) and also contained a decarboxylase. Extracts of cells grown with 3,5-dihydroxyanisole contained an enzyme system with a novel specificity that demethylated only the metahydroxylated compounds 3,5-dihydroxyanisole and 3-hydroxyanisole (the meta system) and lacked a decarboxylase. H4folate-dependent demethylation produced CH3H4folate. For a photometric in vitro assay of the meta system, the NADPH-consuming phloroglucinol reductase (PR) reaction was coupled to the phloroglucinol-yielding demethylation of 3,5-dihydroxyanisole. The kinetics of the indicator enzyme PR were studied. The cell extract had a high and stable specific PR activity. PR was inhibited by phloroglucinol (substrate inhibition) and the substrate analogue 3,5-dihydroxyanisole. Doubling the PR activity of the coupled enzyme assay by additions of a PR-enriched fraction had no effect, showing that the PR activity supplied by cell extract did not limit reaction rates. Demethylation activity of the meta system with either methyl acceptor increased with the square of the protein concentration. With H4folate, the in vivo activity could be attained. Kinetic parameters for the methyl acceptors were determined.
Dedicated to Prof. Achim Kröger on the occasion of his 60th birthday J.-U. Kreft · B. Schink (Y) Fakultät für Biologie, Universität Konstanz, D-78434 Konstanz, Germany Tel. +49-7531-88-2140; Fax +49-7531-88-2966 e-mail: [email protected]
Key words Anaerobic degradation · Methoxylated aromatic compounds · Dimethylsulfide · Methyl transfer · Ether cleavage · Phloroglucinol reductase Abbreviations PR Phloroglucinol reductase · H4 folate Tetrahydrofolate
Introduction Anaerobic bacteria demethylate phenyl methyl ethers by O-demethylation (DeWeerd et al. 1988). In Acetobacterium woodii cell extracts, ATP and tetrahydrafolate (H4folate) are necessary for the air-sensitive demethylation reaction (Berman and Frazer 1992). In Sporomusa ovata cell extracts, demethylation is corrinoid-dependent, needs reductive activation by Ti3+ and ATP, and yields CH3-H4folate (Stupperich and Konle 1993). In strain MC cell extracts, demethylation yields CH3-H4folate and requires ATP substoichiometrically (Meßmer et al. 1993, 1996). CH3-H4folate production can be measured by a coupled enzyme assay (Meß
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