ANAEROBIC PATHWAY FOR CONVERSION OF THE METHYL-GROUP OF AROMATIC METHYL ETHERS TO ACETIC-ACID BY CLOSTRIDIUM-THERMOACETICUM

Clostridium thermoaceticum and other anaerobic acetogenic bacteria can utilize the methyl group of aromatic methyl ethers as a carbon and en ergy source. It has been unclear what pathway is used to metabolize th is methyl group. In the work reported here, the pathway was establishe d by identifying and quantitating the substrates, stable intermediates , and products of O-demethylation of syringic acid. By measuring the d ependence of the O-demethylation reaction on purified enzymes of the a cetyl-CoA pathway, it was established that CO dehydrogenase, the corri noid/iron-sulfur protein, and methyltransferase all were required for acetyl-CoA formation. By C-13-NMR spectroscopy it was shown that the O -demethylase from C. thermoaceticum converts the methyl group of syrin gate to methyltetrahydrofolate (CH3-H(4)folate). When the reaction was conducted in the presence of CO, H-2, or titanium(III), or in the abs ence of any electron donor, the rate of demethylation of syringic acid at pH 7.2 was similar to 15 nmol min(-1) mg(-1). In the absence of CO , CH3-H(4)folate accumulated as a stable product. When CO was added, ( CH3)-C-13-H(4)folate was converted to [2-C-13]acetyl-CoA, [2-C-13]acet yl phosphate, and [2-C-13]acetate. Therefore, the acetogenic O-demethy lase uses H(4)folate as acceptor of the methyl group of phenyl methyl ethers and catalyzes the formation of CH3-H(4)folate. The pathway of c onversion of CH3-H(4)folate, CO, and CoA to acetyl-CoA has been studie d previously. Methyltransferase catalyzes the reaction of CH3-H(4)fola te with the corrinoid/ iron-sulfur protein to form a methylcobalt spec ies. The nickel/iron-sulfur enzyme CO dehydrogenase then catalyzes the final steps in the formation of acetyl-CoA. Although C. thermoaceticu m is a strict anaerobe, the O-demethylase activity in cell extracts wa s found to be relatively oxygen insensitive and to not require reducti ve activation. It also was insensitive to propyl iodide, indicating th at it is not a corrinoid protein. Having a broad substrate range, the O-demethylase can demethylate lignin-derived methoxy phenolics such as syringic or hydroxyvanillic acids, plant hormones like acetosyringone , and herbicides such as dicamba to the corresponding phenolic product s.