0-Demethylation by the homoacetogenic anaerobe Holophaga foetida studied by a new photometric methylation assay using electrochemically produced cob(1)alamin

Abstract

The previously studied complete methyl transfer sequence of tetrahydrofolate-dependent 0-demethylation catalyzed by Holophaga foetida strain TMBS4 extracts was separated into two steps using cobalamins as non-physiological substrates : electrochemically produced cob(1)alamin served as methyl acceptor for phenyl methyl ether demethylation, yielding methylcob(1II)alamin (reaction I), and methylcob(1II)alamin served as donor for tetrahydrofolate methylation, yielding 5-methyl tetrahydrofolate (reaction 11). Both reactions were measured with a new and direct photometric assay of cob(1)alamin methylation (or the reverse reaction) at 540 nm, the isosbestic wavelength of the cob(II)alamin/cob(I)alamin redox couple = 4.40 mM-' . cm-I). The rates of reactions I and I1 were proportional to protein concentration, unlike the complete reaction sequence. Small components of cell extract did not affect activity of reactions I and 11. Isovanillate demethylation by extracts of syringate-grown cells (reaction I) required reductive activation by cob(1)alamin and was inhibited and inactivated by cob(II)alamin, indicating that the reaction mechanism was a nucleo-philic attack of an enzyme-bound corrinoid in the reduced Co(1) 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-' . (mg protein)-' [0.76 mM cob(I)alamin] and apparent kinetic constants for cob(1)alamin were: K, = 1.2 mM, V,,, = 220 nmol . min-' . (mg protein)-', and V,,,,,/K,,, = 180 nmol . min-' . (mg protein)-' . mN-'. 3,5-Dihydroxy-anisole demethylation by extracts of 3,5-dihydroxyanisole-grown cells (also reaction I) was much slower. Reaction I1 did not require activation; specific activity and the specificity constant for methylcob(II1)alamin were much lower.