Structural and Functional Analysis of a Novel Haloalkane Dehalogenase with Two Halide-Binding Sites
Chaloupkova, R., Prudnikova, T., Rezacova, P., Prokop, Z., Koudelakova, T., Daniel, L., Brezovsky, J., Ikeda-Ohtsubo, W., Sato, Y., Kuty, M., Nagata, Y., Kuta Smatanova, I., Damborsky, J.
ACTA CRYSTALLOGRAPHICA D70: 1884-1897 (2014)
Crystal structure of novel haloalkane dehalogenase DbeA revealed the presence of two chloride ions buried in the protein interior. The first halide-binding site is involved in substrate binding and is present in all structurally characterized haloalkane dehalogenases. The second halide-binding site is unique to DbeA. To elucidate the role of the second halide-binding site in enzyme functionality, a two-point mutant lacking this site was constructed and characterized. These substitutions resulted in a shift of substrate-specificity class and were accompanied by decrease of enzyme activity, stability and elimination of the substrate inhibition. The changes in enzyme catalytic activity were attributed to deceleration of the rate-limiting hydrolytic step, mediated by lower basicity of the catalytic histidine.