, 2010). All putative zinc-binding partners of both methyltransferases are located in the catalytic domains of the enzymes (Fig. 3). Although the MT I mediate a similar reaction in A. dehalogenans, the putative zinc-binding amino acids as well as their position in the primary protein structure are different. Both MT I do not have the common binding motifs described for enzymes with similar functions such as the methionine synthases of E. coli (Peariso et al., 1998; Zhou et al., 1999). Usually, the distance between two of the three binding ligands is not larger than three amino acid residues and the third binding partner is separated

from these two amino acids by a longer distance (Vallee & Auld, 1990a). Both MT I of A. dehalogenans show unique zinc-binding motifs: E-X14-E-X20-H for MT Ivan

Decitabine research buy and D-X27-C-X39-C for MT Iver. Cysteine does not seem to be involved in zinc binding in MT Ivan. All other corrinoid-dependent methyltransferases investigated so far involve cysteine as a ligand for zinc (Peariso et al., 1998; Krüer et al., 2001; Hagemeier et al., 2006). MT Ivan only contains Selleckchem INK128 one cysteine residue (C286). When this residue was exchanged to alanine, zinc was still present and the enzyme was active. In principle, it cannot be excluded that the exchange of an amino acid might result in a conformation change of the protein and thus may be responsible for the loss of the zinc and activity. However, the controls performed by exchanging adjacent amino acids or shifting the position of the putative binding amino acid cysteine (MT Iver) by ±1 are in favor of the proposed zinc-binding sites. In the methanol methyltransferase MtaB of M. barkeri, zinc is bound to two cysteine residues and one glutamate residue (Hagemeier et al., 2006). The zinc-binding motif also differs from the common motifs and is described as E-X55-C-X48-C. It is therefore feasible that the corrinoid-dependent, zinc-containing methyltransferases have in common that they contain zinc-binding motifs different from those of other zinc enzymes. Besides the zinc-binding

amino acids, acidic amino acids were exchanged to alanine in both MT I to investigate their influence on the catalysis (Fig. 2). The restricted activities of the mutants obtained suggest an involvement of these negatively charged amino acids in the demethylation of the substrate and/or the transfer of the methyl group to the CP. For MtaB of M. barkeri, the analysis of the crystal structure also exhibits acidic amino acids close to the zinc-binding motif (Hagemeier et al., 2006). For these residues, a polarization of methanol and an enhancement of the charge density of zinc have been proposed, which supports cleavage of the substrate (Hagemeier et al., 2006). A similar function is suggested for the acidic amino acid residues of the MT I of A. dehalogenans. This work was supported by grants from the Deutsche Forschungsgemeinschaft.