Studies on methyl chloride dehalogenase and O-demethylase in cell extracts of the homoacetogen strain MC based on a newly developed coupled enzyme assay |
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Authors: | Michael Meßmer Simone Reinhardt Gert Wohlfarth G. Diekert |
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Affiliation: | Institut für Mikrobiologie, Universit?t Stuttgart, Allmandring 31, D-70550 Stuttgart, Germany Tel. +49-0711-6855483; Fax +49-0711-6855725 e-mail: gabriele.diekert@po.uni-stuttgart.de, DE
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Abstract: | An enzyme assay was developed to determine the activities of methyl chloride dehalogenase and O-demethylase of the homoacetogen strain MC. The formation of methyl tetrahydrofolate from tetrahydrofolate and methyl chloride or from tetrahydrofolate and vanillate was coupled to the oxidation of methyl tetrahydrofolate to methylene tetrahydrofolate mediated by methylene tetrahydrofolate reductase purified from Peptostreptococcus productus (strain Marburg) and to the subsequent oxidation of methylene tetrahydrofolate to methenyl tetrahydrofolate catalyzed by methylene tetrahydrofolate dehydrogenase purified from the same organism. To drive the endergonic methyl tetrahydrofolate oxidation with NAD+ as an electron acceptor, the NADH formed in this reaction was reoxidized in the exergonic lactate dehydrogenase reaction. The formation of NADPH and methenyl tetrahydrofolate in the methylene tetrahydrofolate dehydrogenase reaction was followed photometrically at 350 nm; ε350 was about 29.5 mM–1cm–1 (pH 6.5). Using the coupled enzyme assay, the cofactor requirements, the apparent kinetic parameters, the pH and temperature optima of both enzymes, and the effect of inhibitors were determined. The activity of methyl chloride dehalogenase and of O-demethylase was dependent on the presence of ATP; arsenate severely inhibited both enzyme activities in the absence of ATP. The coupled enzyme assay described allows purification and characterization of methyl chloride dehalogenase and O-demethylase and is also appropriate for the enzymatic determination of methyl tetrahydrofolate. Received: 2 August 1995 / Accepted: 28 September 1995 |
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Keywords: | Strain MC Methyl chloride Methyl chloride dehalogenase Methoxylated aromatic compounds O-Demethylase Phenylmethyl ethers Methyl tetrahydrofolate Methylene tetrahydrofolate reductase Methylene tetrahydrofolate dehydrogenase Enzyme activation Homoacetogenic bacteria |
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