Identification of metal binding residues for the binuclear zinc phosphodiesterase reveals identical coordination as glyoxalase II.

Zinc phosphodiesterase (ZiPD) is a member of the metallo-beta-lactamase family with a binuclear zinc binding site. As an experimental attempt to identify the metal ligands of Escherichia coli ZiPD and to investigate their function in catalysis, we mutationally exchanged candidate metal coordinating residues and performed kinetic and X-ray absorption spectroscopic analysis of the mutant proteins. All mutants (H66E, H69A, H141A, D212A, D212C, H231A, H248A, and H270A) show significantly lower catalytic rates toward the substrate bis(p-nitrophenyl)phosphate. Substrate binding, represented by the kinetic value K', remains unchanged for six mutants, whereas it is increased 3-4-fold for H231A and H270A. Accordingly, these two residues are supposed to be involved in substrate binding, whereas the others are more important for catalytic turnover and thus are assumed to be involved in zinc ligation. Structural insight into the metal binding of D212 was gained by zinc K-edge extended X-ray absorption fine structure (EXAFS). The sulfur coordination number of the cysteine mutant was found to be 1, demonstrating binding to both zinc metals in a bridging mode. Taken together with two residues from a strictly conserved sequence region within the metallo-beta-lactamase family, the metal site of ZiPD is proposed with H64, H66, and H141 coordinating ZnA, D68, H69, and H248 coordinating ZnB, and D212 bridging both metals. Surprisingly, the same coordination sphere is found in glyoxalase II. This is further substantiated by comparable EXAFS spectra of both native enzymes. This is the first example of the same metal site in two members of the metallo-beta-lactamase domain proteins catalyzing different reactions. The kinetic analysis of mutants provides unexpected insights into the reaction mechanism of ZiPD.