Insights into the role of the metal-binding sites of quercetin 2,3-dioxygenase from Bacillus subtilis

The metalloenzyme quercetin 2,3-dioxygenase (QueD) catalyzes the oxidative decomposition of the aromatic compound, quercetin. The most recently characterized example is a product of the bacterium Bacillus subtilis (BsQueD); all previous examples were fungal enzymes from the genus Aspergillus (AQueD). AQueD contains a single atom of Cu(II) per monomer. However, BsQueD, over expressed in Escherichia coli, contains Mn(II) and has two metal-binding sites, and therefore two possible active sites per monomer. To understand the contribution of each site to BsQueD's activity, the N-terminal and C-terminal metal-binding sites have been mutated individually in an effort to disrupt metal binding. In wild type BsQueD, each Mn(II) is ligated by three histidines (His) and one glutamate (Glu). All efforts to mutate His residues to non-ligating residues resulted in insoluble protein or completely inactive enzyme. A soluble mutant was expressed that replaced the Glu residue with a fourth His at the N-terminal domain. This mutant (E69H) has a specific activity of 0.00572 &mumol;/min/mg, which is nearly 3000-fold lower than the rate of wild type BsQueD (15.9 &mumol;/min/mg). Further analysis of E69H by inductively couple plasma mass spectrometry revealed that this mutant contains only 0.062 mol of Mn(II) per mol of enzyme. This is evidence that disabling metal-ligation at one domain influences metal-incorporation at the other. During the course of the mutagenic study, a second, faster purification method was developed. A hexahistidine tag and an enterokinase cleavage site were fused to the N-terminus of BsQueD (6xHis-BsQueD). Active enzyme was successfully expressed and purified with a nickel column in 3 hours. This is much faster than the previous multi-column purification, which took two full days to complete. However, the concentration of soluble, purified enzyme (1.8 mg/mL) was much lower than concentrations achieved with the traditional method (30 mg/mL). While the concentration of 6xHis-BsQueD is sufficient for some analyses, there are several characterization techniques that must be conducted at higher concentrations. Therefore, it will be advantageous to continue using both purification methods in the future.