Genetic engineering of anaerobic O-demethylases

同型产乙酸细菌的O-脱甲基酶介导苯甲醚的脱甲基作用。它们由四种不同的蛋白质组成。甲基转移酶I催化甲基转移至类咕啉蛋白。脱卤醋酸杆菌含有几种具有不同底物谱的诱导型O-脱甲基酶。该提案遵循三个目标：（1）。阐明和理解甲基转移酶I的甲基转移反应的分子基础及其底物特异性; 2.）将所获得的结果用于优化催化性能; 3.）以使底物光谱适应苯甲基醚的化学合成和/或检测和定量的应用要求。为了实现这些目标，将使用不同O-脱甲基酶组分的交联实验和定点诱变来鉴定和分析甲基转移酶I的催化活性和底物结合结构域。将构建由甲基转移所必需的O-脱甲基酶组分组成的融合蛋白，以解开这些蛋白质的相互作用。这些数据将是进一步诱变的基础，以修饰酶的底物特异性和反应速率。

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.