Specificity of O-demethylation in extracts of the homoacetogenic Holophaga foetida and demethylation kinetics measured by a coupled photometric assay

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 ⬚meta⬚-hydroxylated compounds 3,5-dihydroxyanisole and 3-hydroxyanisole (the ⬚meta⬚ system) and lacked a decarboxylase. H4folate-dependent demethylation produced CH3-H4folate. 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.