Exploring the cupin-type metal-coordinating signature of acetylacetone dioxygenase Dke1 with site-directed mutagenesis: catalytic reaction profile and Fe2+ binding stability of Glu-69->Gln mutant.

Glu-69 belongs to a proposed active-site consensus motif His62-X-His64-X4-Glu69 (where X is any amino acid) that acetylacetone dioxygenase Dke1 from Acinetobacter johnsonii shares with structurally related non-heme metal enzymes of the cupin protein superfamily.We report functional consequences of the site-directed replacement Glu-69¨Gln based on a detailed biochemical and kinetic characterization of the purified Dke1 mutant. Perturbations of the free energy profile of the wild-type caused by the mutation were surprisingly small, with key points of the reaction pathway such as _-diketone substrate binding, the rate-limiting reduction of dioxygen, and C C bond cleavage essentially left unaltered. Release of Fe2+ from the mutant active site occurred at twice the wild-type rate, and the thermal stability of _-sheet secondary structure in Fe2+-depleted apo-proteins was lower in the mutant. The substitution Glu-69¨Gln is thus remarkably silent regarding Dke1 function. These results do not support a unified catalytic or metal-coordinating role of Glu-69 (and its positional homologues) in O2-dependent cupin-fold enzymes.