Importance of tetrahydrofolate and ATP in the anaerobic O-demethylation reaction for phenylmethylethers

Abstract

DL-Tetrahydrofolate (THF) and ATP were necessary for the anaerobic 0-demethylation of phenylmethylethers in cell extracts of the type strain (ATCC 29683) of the homoacetogen Acetobacterium woodii. The reactants for this enzymatic activity have not been previously demonstrated in any system, nor has the mediating enzyme been studied. An assay using reaction mixtures containing 1 mM THF, 2 mM ATP, and 2 mM hydroferulate (i.e., 4-hydroxy,3-methoxyphenylpropionate) was developed and was performed under stringent anaerobic conditions. Pyridine nucleotides and several other possible cofactors were tested but had no effect on the activity. After centrifugation of disrupted cells at 27,000 x g, the activity was found primarily in the supernatant, which had a specific activity of 14.2 + 0.5 nmol/min/mg of protein. At saturating levels of each of the other two substrates, apparent Km values for the variable substrate were 0.65 mM hydroferulate, 0.27 mM ATP, and 0.17 mM THF. Activity was significantly decreased when extract was preincubated at 60°C and was completely lost after preincubation in air for 30 min. Thus, the soluble anaerobic 0-demethylating enzyme system of A. woodii is oxygen sensitive. The THF-and ATP-dependent activity measurable in the soluble fraction of cell extracts constituted about 34% of the activity seen with intact cells.

Biotransformation of the isoflavonoids biochanin A, formononetin, and glycitein by Eubacterium limosum

“…The extracellular fractions of the bacterial cultures did not have O-demethylating activities. Previously, the association of this enzyme with the bacterial cell was shown [33]. Our observation has extended the broad range of substrates to be demethylated by E. limosum.…”

“…The oxygen atom in the ether bridge is intact after removal of the methyl group from the methoxybenzoic acid [18]. The acetogens growing on methoxylated compounds are expected to convert some of the methyl substitutes to the methyl carbon of acetate, while oxidizing others to carbon dioxide [33]. It has been shown that E. limosum growth with methoxylated aromatic compounds is proportional to the number of mol of methoxyl group present in the compound [24].…”

Recent advances in elucidation of biological corrinoid functions

“…In addition, two homomeric 14C-labelled corrinoid proteins were detected from which [lac_methyl]CH3_corrinoid has been isolated [119]. These findings are consistent with the tetrahydrofolate and ATP stimulations in vitro [120], propyl iodide inhibition [121] and [14C]acetic acid formation from radioactive methoxyl groups in vivo [122].…”

Anaerobic Metabolism of Aromatic Compounds

“…These bacteria only utilize the methyl group of the substrate for growth and excrete the demethylated aromatic scaffold into the medium (reviewed by Stupperich, 1993;White et al, 1996). O-demethylation of 3,4-dimethoxybenzoate and other methoxy-substituted aromatic compounds has been demonstrated in cell-free extracts of S. ovata and A. woodii (Berman and Frazer, 1992;Stupperich and Konle, 1993;Stupperich et al, 1996). The methyl group is transferred onto tetrahydrofolate (H,folate) by a corrinoid enzyme (Berman and Frazer, 1992;Stupperich and Konle, 1993;Eqn 18); the oxygen atom remains at the aromatic ring (De-Weerd et al, 1988).…”

“…O-demethylation of 3,4-dimethoxybenzoate and other methoxy-substituted aromatic compounds has been demonstrated in cell-free extracts of S. ovata and A. woodii (Berman and Frazer, 1992;Stupperich and Konle, 1993;Stupperich et al, 1996). The methyl group is transferred onto tetrahydrofolate (H,folate) by a corrinoid enzyme (Berman and Frazer, 1992;Stupperich and Konle, 1993;Eqn 18); the oxygen atom remains at the aromatic ring (De-Weerd et al, 1988). The catalytic presence of ATP and a strong reducing agent such as titanium(II1) citrate are required for the reaction in vitro (Fig.…”