Isolation of O-demethylase, an ether-cleaving enzyme system of the homoacetogenic strain MC
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© Springer-Verlag 1997
O R I G I N A L PA P E R
Franz Kaufmann · Gert Wohlfarth · Gabriele Diekert
Isolation of O -demethylase, an ether-cleaving enzyme system of the homoacetogenic strain MC
Received: 5 February 1997 / Accepted: 17 April 1997
Abstract The O-demethylase of the methylotrophic homoacetogenic bacterium strain MC was purified to apparent homogeneity. The enzyme system consisted of four different components that were designated A, B, C, and D according to their elution sequence from the anionic-exchange chromatography column. All four components were essentially required for catalysis of the transfer of the methyl group from phenyl methyl ethers to tetrahydrofolate. According to gel filtration and SDS-PAGE, components A and B were monomers with apparent molecular masses of approximately 26 kDa (subunit 25 kDa) and 36 (subunit 41 kDa), respectively; component C appeared to be a trimeric protein (195 kDa, subunit 67 kDa); and component D was probably a dimer (64 kDa, subunit 30 kDa). Component A contained one corrinoid per monomer. In crude extracts, component D appeared to be the rate-limiting protein for the complete methyl transfer reaction. Additional requirements for the reaction were ATP and low-potential reducing equivalents supplied by either titanium(III) citrate or H2 plus hydrogenase purified from strain MC.
aromatic acids. Under anoxic conditions, the latter reaction was shown to be catalyzed by methylotrophic homoacetogenic bacteria (for recent reviews, see Frazer 1994; White et al. 1996). In their energy metabolism, the bacteria convert the methyl group to acetate via the acetyl-CoA pathway (= carbon monoxide dehydrogenase pathway; Ragsdale 1991; Diekert 1992; Diekert and Wohlfarth 1994). The key reaction in phenyl methyl ether degradation is the cleavage of the ether bond, followed by the transfer of the methyl group to the acceptor tetrahydrofolate (Berman and Frazer 1992; Meßmer et al. 1993; Stupperich and Konle 1993; El Kasmi et al. 1994; Kreft and Schink 1994). In this reaction, which is catalyzed by the O-demethylase, methyl tetrahydrofolate is formed. Methyl tetrahydrofolate is, on the one hand, oxidized to CO2 to provide reducing equivalents for the reduction of CO2 to carbon monoxide; on the other hand it is combined with carbon monoxide and CoA to acetyl-CoA, which is converted to acetate. A simplified scheme of the pathway is depicted in Fig. 1. Studies on the O-demethylase reac-
Key words Corrinoid protein · Ether cleavage · Hydrogenase · Methyltransferase · O-demethylase · Strain MC · Tetrahydrofolate Abbreviation FH4 Tetrahydrofolate
Introduction Phenyl methyl ethers such as syringate and vanillate are degradation products of lignin, one of the major components of wood. Removal of the O-methyl moiety is an important step in the further degradation of the methoxylated
F. Kaufmann · G. Wohlfarth · G. Diekert (Y) Institut für Mikrobiologie, Universität Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany Tel. +49-711-6855483; Fax +49-711-6855725 e-mail: [email protected]
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