Ether cleaving methyltransferases of the strict anaerobe Acetobacterium dehalogenans: Controlling the substrate spectrum by genetic engineering of the N-terminus

Abstract

The anaerobic cleavage of ether bonds of methoxylated substrates such as vanillate or veratrol in acetogenic bacteria is mediated by multi-component enzyme systems, the O-demethylases. Acetobacterium dehalogenans harbours different inducible O-demethylases with various substrate spectra. Two of these enzyme systems, the vanillate- and the veratrol-O-demethylases, have been characterized so far. One component of this enzyme system, the methyltransferase I (MT I), catalyses the cleavage of the substrate ether bond and the subsequent transfer of the methyl group to a corrinoid protein. For the C-termini of the methyltransferases I of the vanillate- and the veratrol-O-demethylases, a TIM barrel structure of the enzymes was predicted, whereas the N-termini are not part of this conserved structure. The deletion of the N-terminal regions led to a significant increase of activity (up to 20-fold) and an extended substrate spectrum of the mutants, which also comprised non-aromatic compounds such as the thioether methionine and diethylether. The exchange of the N-termini of the two methyltransferases I resulted in chimeric enzymes whose substrate specificities were those of the enzymes from which the N-termini were derived. This demonstrated the crucial role of the N-termini for the substrate specificity of the methyltransferases. © 2010 Blackwell Publishing Ltd.