Chaloupkova Radka, Prudnikova Tatyana, Rezacova Pavlina, Prokop Zbynek, Koudelakova Tana, Daniel Lukas, Brezovsky Jan, Ikeda-Ohtsubo Wakako, Sato Yukari, Kuty Michal, Nagata Yuji, Kuta Smatanova Ivana, Damborsky Jiri
Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment RECETOX, Masaryk University, 625 00 Brno, Czech Republic.
Faculty of Science, University of South Bohemia in Ceske Budejovice, Branisovska 31, 370 05 Ceske Budejovice, Czech Republic.
Acta Crystallogr D Biol Crystallogr. 2014 Jul;70(Pt 7):1884-97. doi: 10.1107/S1399004714009018. Epub 2014 Jun 29.
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.
来自慢生根瘤菌USDA94的新型卤代烷脱卤酶DbeA的晶体结构显示，在蛋白质内部存在两个氯离子。第一个卤化物结合位点参与底物结合，并且存在于所有结构特征明确的卤代烷脱卤酶中。第二个卤化物结合位点是DbeA所特有的。为了阐明第二个卤化物结合位点在酶功能中的作用，构建并表征了一个缺少该位点的双点突变体。这些取代导致底物特异性类别发生转变，并伴随着酶活性、稳定性的降低以及底物抑制作用的消除。酶催化活性的变化归因于催化组氨酸碱性较低介导的限速水解步骤的减速。
