TY - JOUR
T1 - Structural and functional analysis of a novel haloalkane dehalogenase with two halide-binding sites
AU - Chaloupkova, Radka
AU - Prudnikova, Tatyana
AU - Rezacova, Pavlina
AU - Prokop, Zbynek
AU - Koudelakova, Tana
AU - Daniel, Lukas
AU - Brezovsky, Jan
AU - Ikeda-Ohtsubo, Wakako
AU - Sato, Yukari
AU - Kuty, Michal
AU - Nagata, Yuji
AU - Smatanova, Ivana Kuta
AU - Damborsky, Jiri
PY - 2014/7
Y1 - 2014/7
N2 - The crystal structure of the novel haloalkane dehalogenase DbeA from Bradyrhizobium elkanii USDA94 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 in the substrate-specificity class and were accompanied by a decrease in enzyme activity, stability and the elimination of substrate inhibition. The changes in enzyme catalytic activity were attributed to deceleration of the rate-limiting hydrolytic step mediated by the lower basicity of the catalytic histidine.
AB - The crystal structure of the novel haloalkane dehalogenase DbeA from Bradyrhizobium elkanii USDA94 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 in the substrate-specificity class and were accompanied by a decrease in enzyme activity, stability and the elimination of substrate inhibition. The changes in enzyme catalytic activity were attributed to deceleration of the rate-limiting hydrolytic step mediated by the lower basicity of the catalytic histidine.
KW - catalytic activity
KW - enzyme stability
KW - halide-binding site
KW - haloalkane dehalogenase
KW - substrate specificity
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U2 - 10.1107/S1399004714009018
DO - 10.1107/S1399004714009018
M3 - Article
C2 - 25004965
AN - SCOPUS:84903955828
SN - 0907-4449
VL - 70
SP - 1884
EP - 1897
JO - Acta Crystallographica Section D: Biological Crystallography
JF - Acta Crystallographica Section D: Biological Crystallography
IS - 7
ER -