, 2001) All components of both systems were heterologously produ

, 2001). All components of both systems were heterologously produced in Escherichia coli (Schilhabel et al., 2009). Both MT I are zinc-containing enzymes (Schilhabel et al., 2009). Zinc may have structural or catalytic functions in proteins (Vallee & Auld, 1990a, b). The metal is generally bound to the side chains of histidine, cysteine, aspartate or glutamate (Vallee & Auld,

1990a). In most cases, zinc is bound to three amino acid side chains and one water molecule when the metal has a catalytic function in enzymes (Auld, 2001). These zinc-binding motifs usually exhibit common characteristics with regard to the distances between the zinc-binding amino acids in the primary structure of the proteins (Auld, 2001). Two of these amino acids are separated by a short distance of one to three amino acids; the third ligand

is located at a distance of 20–120 amino acids to the other ligands Venetoclax clinical trial (Vallee & Auld, 1990a). Exceptions to this rule are the cofactor-dependent alcohol dehydrogenase (Vallee & Auld, 1990a) and the cobalamin-dependent methanol methyltransferase of Methanosarcina barkeri (Hagemeier et al., 2006). In this study, we report on the identification of the zinc-binding motifs of MT Ivan of the vanillate-O-demethylase and MT Iver of the veratrol-O-demethylase of A. dehalogenans using site-directed mutagenesis. Acetobacterium dehalogenans was cultivated anaerobically as described PF-562271 earlier (Traunecker et al., 1991). Syringate (20 mM) or fructose (20 mM) was used as a growth substrate. The production of the recombinant proteins and the purification of the methyltransferases and of CP were performed as described earlier (Schilhabel et al., 2009). For the activation reaction, crude extracts of E. coli containing the recombinant AE were used. These crude extracts did not exhibit methyltransferase MTMR9 activity. Cells of A. dehalogenans (0.2 g wet weight) were

suspended in 1 mL 10 mM Tris-HCl, pH 8.0, containing 10 mM EDTA. The genomic DNA was isolated according to Bollet et al. (1991). After incubation with 0.01% RNase (w/v) for 15 min at 37 °C, the DNA was stored at 4 °C. Expression cassettes of the mutated genes of MT Ivan and MT Iver (GenBank accession no. AF087018 and AY318856) as fusion proteins with a C-terminal Strep-tag and with restriction sites for the cloning in pET11a (Agilent Technologies, Böblingen, Germany) were constructed from PCR products. Point mutations of both enzymes were generated using overlap extension PCR essentially using the method described by An et al. (2005). The mutations were inserted using multistep PCR. In the first PCR step, two fragments were amplified: one by the combination of primer 1 (MT Ivan) or 3 (MT Iver) (Table 1) with the mutated reverse primer and the other fragment by the combination of the mutated forward primer with primer 2 (MT Ivan) or 4 (MT Iver) (Table 1).

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