5, containing 150 mM NaCl and the recombinant proteins were then purified using a one-step affinity chromatography. The diluted crude extract (5 mL) was applied to a 5 mL Strep-Tactin Superflow cartridge (IBA GmbH, Göttingen, Germany). The purification was performed according to the manufacturer’s protocol. The MT I enzyme assay was performed in anaerobic quartz cuvettes with N2 as the gas phase. The total volume FK228 manufacturer was 100 μL. The activity was determined by the formation of methylcobalamin (ɛ528nm=7.9 mM−1 cm−1; Friedrich, 1975). The enzyme assay contained 50 mM Tris-HCl, pH 7.5,
2 mM ATP, 10 mM MgCl2, 5 mM substrate, 50 mM dithiothreitol, 0.5 mM titanium(III) citrate, 20 μM CP and crude extract with recombinant
AE; AE in the enzyme assay was estimated to be about 1 μg. MT Ivan activity was determined with vanillate (4-hydroxy-3-methoxybenzoic acid) and MT Iver activity with veratrol (1,2-dimethoxybenzene) as a substrate. The assay was started by adding MT I. All enzyme activities were the result of at least duplicate DAPT solubility dmso determinations. The SDs were ≤10%. The protein determination was performed according to the method of Bradford (1976) with bovine serum albumin as a standard. The zinc content was determined photometrically using the method described by Zhou et al. (1999). To remove unspecifically bound zinc, the proteins were incubated with 2.5 mM EDTA in 25 mM Tris-HCl, pH 7.5, for 15 min at room temperature and were then applied onto a gel
filtration column Superdex 75 (16/60) equilibrated with 50 mM Tris-HCl pH 7.5. The same buffer was used as an eluent at a flow rate of 1 mL min−1 to separate the proteins from EDTA. Enzyme-containing fractions were pooled and subsequently concentrated using Vivaspin 50 centrifugation units (Vivascience, Hannover, Germany). The O-methylated flavonoid protein and the zinc contents of the mutated enzymes were determined as described above. Structure predictions of the methyltransferases were performed using the quickphyre program (Bennett-Lovsey et al., 2008). A crystal structure of MT I is not yet available. Attempts to crystallize the enzymes have resulted in nondiffracting crystals so far. In addition, the enzyme appeared to be rather unstable under the experimental conditions applied. Therefore, we attempted to identify the zinc-binding amino acids using site-directed mutagenesis. Potential zinc-binding partners are histidine, glutamate, aspartate and cysteine. Plenty of these amino acids are present in both MT I. The alignment of the amino acid sequences with other zinc-containing enzymes (Vallee and Auld, 1990b) did not provide a clue about the amino acids involved, indicating an unusual type of binding motif. Therefore, we exchanged several amino acids to alanine and tested the resulting recombinant enzymes for activity and zinc content.