O ‐Demethylation by the Homoacetogenic Anaerobe Holophaga Foetida Studied by a New Photometric Methylation Assay Using Electrochemically Produced Cob(I)Alamin

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 (Δɛ₅₄₀= 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‐Dihydroxy‐anisole 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.