O-demethylation by the homoacetogenic anaerobe Holophaga foetida studied by a new photometric methylation assay using electrochemically produced cob(I)alamin.
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 isobestic wavelength of the cob(II)alamin/cob(I)alamin redox couple (delta epsilon 540 = 4.40 nM-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 synringate-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: Km = 1.2 mM, Vmax = 220 nmol min-1.(mg protein)-1, and Vmax/Km = 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.
Kreft
J U
JU
Fakultät für Biologie, Universität Konstanz, Germany.
Schink
B
B
D000840:Anisoles / Q000378:metabolism; D004563:Electrochemistry; D006089:Gram-Negative Anaerobic Bacteria / Q000378:metabolism*; D007700:Kinetics; D008745:Methylation; D010084:Oxidation-Reduction; D010783:Photometry; D013763:Tetrahydrofolates / Q000378:metabolism / Q000494:pharmacology; D014641:Vanillic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D014805:Vitamin B 12 / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology
D000840:Anisoles; D013763:Tetrahydrofolates; C091385:3,5-dihydroxyanisole; C030371:5,6,7,8-tetrahydrofolic acid; C045317:isovanillic acid; C019476:mecobalamin; D014641:Vanillic Acid; D014805:Vitamin B 12