O-Demethylation by the homoacetogenic anaerobe Holophaga foetida studied by a new photometric methylation assay using electrochemically produced cob(I)alamin

Jan-Ulrich Kreft, Bernhard Schink

Research output: Contribution to journalArticlepeer-review

35 Citations (Scopus)

Abstract

The previously studied complete methyl transfer sequence of tetrahydrofolate-dependent O-demethylation catalyzed by Holophaga foetida strain TMBS4 extracts was separated into two steps using cobalamins as non-physiological substrates: electrochemically produced cob(I)alamin served as methyl acceptor for phenyl methyl ether demethylation, yielding methylcob(III)alamin (reaction I), and methylcob(III)alamin served as donor for tetrahydrofolate methylation, yielding 5-methyl tetrahydrofolate (reaction II). Both reactions were measured with a new and direct photometric assay of cob(I)alamin methylation (or the reverse reaction) at 540 nm, the isosbestic wavelength of the cob(II)alamin/cob(I)alamin redox couple (Delta epsilon(540) = 4.40 mM(-1) . cm(-1)). The rates of reactions I and II were proportional to protein concentration, unlike the complete reaction sequence. Small components of cell extract did not affect activity of reactions I and II. Isovanillate demethylation by extracts of syringate-grown cells (reaction I) required reductive activation by cob(I)alamin and was inhibited and inactivated by cob(II)alamin, indicating that the reaction mechanism was a nucleophilic attack of an enzyme-bound corrinoid in the reduced Co(I) state on the methyl carbon of the ether, rather than a radical attack. Only phenyl methyl ethers were demethylated; demethylation rates were enhanced by ortho-hydroxyl or para-carboxyl groups, but reduced by additional meta substituents. The rate of isovanillate demethylation was 81 nmol . min(-1) . (mg protein)(-1) [0.76 mM cob(I)alamin] and apparent kinetic constants for cob(I)alamin were: K-m = 1.2 mM, V-max = 220 nmol min(-1) . (mg protein)-1, and V-max/K- m = 180 nmol . min(-1) . (mg protein)(-1) . mM(-1) . 3,5- Dihydroxyanisole demethylation by extracts of 3,5-dihydroxyanisole- grown cells (also reaction I) was much slower. Reaction II did not require activation; specific activity and the specificity constant for methylcob(III)alamin were much lower
Original languageEnglish
Pages (from-to)945-951
Number of pages7
JournalEuropean Journal of Biochemistry
Volume226
DOIs
Publication statusPublished - 1994

Fingerprint

Dive into the research topics of 'O-Demethylation by the homoacetogenic anaerobe Holophaga foetida studied by a new photometric methylation assay using electrochemically produced cob(I)alamin'. Together they form a unique fingerprint.

Cite this