Besides acetogenic bacteria, only Desulfitobacterium has been described to utilize and cleave phenyl methyl ethers under anoxic conditions; however, no ether-cleaving O-demethylases from the latter organisms have been identified and investigated so far. In this study, genes of an operon encoding O-demethylase components of Desulfitobacterium hafniense strain DCB-2 were cloned and heterologously expressed in Escherichia coli. Methyltransferases I and II were characterized. Methyltransferase I mediated the ether cleavage and the transfer of the methyl group to the superreduced corrinoid of a corrinoid protein. Desulfitobacterium methyltransferase I had 66% identity (80% similarity) to that of the vanillate-demethylating methyltransferase I (OdmB) of Acetobacterium dehalogenans. The substrate spectrum was also similar to that of the latter enzyme; however, Desulfitobacterium methyltransferase I showed a higher level of activity for guaiacol and used methyl chloride as a substrate. Methyltransferase II catalyzed the transfer of the methyl group from the methylated corrinoid protein to tetrahydrofolate. It also showed a high identity (ϳ70%) to methyltransferases II of A. dehalogenans. The corrinoid protein was produced in E. coli as cofactor-free apoprotein that could be reconstituted with hydroxocobalamin or methylcobalamin to function in the methyltransferase I and II assays. Six COG3894 proteins, which were assumed to function as activating enzymes mediating the reduction of the corrinoid protein after an inadvertent oxidation of the corrinoid cofactor, were studied with respect to their abilities to reduce the recombinant reconstituted corrinoid protein. Of these six proteins, only one was found to catalyze the reduction of the corrinoid protein.A cetogenic bacteria were the first anaerobes described to utilize phenyl methyl ethers as energy substrates (2). These organisms mediate the cleavage of the substrate ether bond and utilize the methyl group, which is oxidized to CO 2 in the oxidative part of catabolism. The reducing equivalents derived from methyl group oxidation are transferred to CO 2 upon the formation of an enzyme-bound carbon monoxide, which is then combined with further methyl groups to finally yield acetate (6). The key enzymes in the methylotrophic phenyl methyl ether metabolism of acetogens such as Acetobacterium dehalogenans (23) and Moorella thermoacetica (13) are the O-demethylases. These inducible enzyme systems mediate the ether cleavage and transfer of the methyl group to tetrahydrofolate (FH 4 ). Until now, three of these acetogenic O-demethylase systems were purified and characterized (7,14,25). In general, they consist of four protein components: two methyltransferases (MTs) (MT I and MT II), a corrinoid protein (CP), and an activating enzyme (AE). Both MTs and CP are involved in the catalytic cycle (Fig. 1) (Fig. 1). In A. dehalogenans, the genes encoding MT I, MT II, and CP are usually organized into an operon (31). Only one AE gene has been detected so far; this gene is not part of a...
The anaerobic veratrol O-demethylase mediates the transfer of the methyl group of the phenyl methyl ether veratrol to tetrahydrofolate. The primary methyl group acceptor is the cobalt of a corrinoid protein, which has to be in the +1 oxidation state to bind the methyl group. Due to the negative redox potential of the cob(II)/cob(I)alamin couple, autoxidation of the cobalt may accidentally occur. In this study, the reduction of the corrinoid to the superreduced [Co(I)] state was investigated. The ATP-dependent reduction of the corrinoid protein of the veratrol O-demethylase was shown to be dependent on titanium(III) citrate as electron donor and on an activating enzyme. In the presence of ATP, activating enzyme, and Ti(III), the redox potential versus the standard hydrogen electrode (E (SHE)) of the cob(II)alamin/cob(I)alamin couple in the corrinoid protein was determined to be -290 mV (pH 7.5), whereas E (SHE) at pH 7.5 was lower than -450 mV in the absence of either activating enzyme or ATP. ADP, AMP, or GTP could not replace ATP in the activation reaction. The ATP analogue adenosine-5'-(beta,gamma-imido)triphosphate (AMP-PNP, 2-4 mM) completely inhibited the corrinoid reduction in the presence of ATP (2 mM).
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