Metal specificity and catalytic mechanism of quercetinase QueD

Basic data for this project

Description

Quercetinases are metal-dependent, CO-forming flavonol 2,4-dioxygenases with a cupin fold. Whereas the reactivity of quercetinases of Aspergillus spp. depends on a mononuclear Cu(II) center, the enzymes from Bacillus subtilis and Streptomyces sp. strain FLA can use different divalent metal ions to catalyze flavonol ring cleavage. Interestingly, the Streptomyces enzyme is most active with Ni2+, which is a poor cofactor for the Bacillus enzyme. The mechanistic implications of such "metal promiscuity" are not well understood. The proposed project will address this question, using different metal forms of Streptomyces quercetinase (QueD) as model enzyme. The effect of metal ions on catalytic function, active-site geometry, and structure of QueD will be analyzed: Using different flavonol substrates, kinetic and electron paramagnetic resonance spectroscopic studies will be performed on different metal forms of QueD and on QueD variants generated by site-directed mutagenesis, to address (i) the mode of flavonol and/or O2 binding to the metal(s), (ii) the possible role of active-site residues in catalysis, e.g., as a general base, (iii) possible electron transfer (from flavonolate to dioxygen?). In collaboration with Prof. Dr. H. Dobbek, the crystal structure of QueD forms will be analyzed. The overall goal of this project is to mechanistically compare metal-dependent and cofactor-independent ring-cleavage dioxygenases, to contribute to an understanding of how dioxygenases work.