Genetic engineering of anaerobic O-demethylases

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O-Demethylases of homoacetogenic bacteria mediate the demethylation of phenyl methyl ethers. They consist of four distinct proteins. The methyl transferase I catalyses the transfer of the methyl group to a corrinoid protein. Acetobacterium dehalogenans contains several inducible O-demethylases with different substrate spectra. This proposal follows three aims: 1.) to elucidate and understand the molecular basis of the methyl transfer reaction by methyl transferase I and its substrate specificity; 2.) to apply the results obtained for optimization of the catalytic properties; 3.) to adapt the substrate spectrum to the requirements for application in chemical syntheses and/or detection and quantification of phenyl methyl ethers. To achieve these goals, cross linking experiments of the different O-demethylase components and site directed mutagenesis will be used to identify and analyze the catalytically active and substrate binding domains of the methyl transferases I. Fusion proteins consisting of the O-demethylase components necessary for the methyl transfer will be constructed to unravel the interaction of these proteins. The data will be the basis for further mutagenesis to modify the enzymes with respect to substrate specificity and reaction rate.