Oakley Aaron J, Prokop Zbynek, Bohác Michal, Kmunícek Jan, Jedlicka Tomás, Monincová Marta, Kutá-Smatanová Ivana, Nagata Yuji, Damborský Jirí, Wilce Matthew C J
Department of Pharmacology/Crystallography Centre, University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia.
Biochemistry. 2002 Apr 16;41(15):4847-55. doi: 10.1021/bi015734i.
The hydrolysis of haloalkanes to their corresponding alcohols and inorganic halides is catalyzed by alpha/beta-hydrolases called haloalkane dehalogenases. The study of haloalkane dehalogenases is vital for the development of these enzymes if they are to be utilized for bioremediation of organohalide-contaminated industrial waste. We report the kinetic and structural analysis of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26 (LinB) in complex with each of 1,2-dichloroethane and 1,2-dichloropropane and the reaction product of 1-chlorobutane turnover. Activity studies showed very weak but detectable activity of LinB with 1,2-dichloroethane [0.012 nmol s(-1) (mg of enzyme)(-1)] and 1,2-dichloropropane [0.027 nmol s(-1) (mg of enzyme)(-1)]. These activities are much weaker compared, for example, to the activity of LinB with 1-chlorobutane [68.2 nmol s(-1) (mg of enzyme)(-1)]. Inhibition analysis reveals that both 1,2-dichloroethane and 1,2-dichloropropane act as simple competitive inhibitors of the substrate 1-chlorobutane and that 1,2-dichloroethane binds to LinB with lower affinity than 1,2-dichloropropane. Docking calculations on the enzyme in the absence of active site water molecules and halide ions confirm that these compounds could bind productively. However, when these moieties were included in the calculations, they bound in a manner similar to that observed in the crystal structure. These data provide an explanation for the low activity of LinB with small, chlorinated alkanes and show the importance of active site water molecules and reaction products in molecular docking.
卤代烷烃水解生成相应的醇类和无机卤化物，这一过程由名为卤代烷脱卤酶的α/β水解酶催化。如果要利用卤代烷脱卤酶对受有机卤化物污染的工业废料进行生物修复，那么对其进行研究至关重要。我们报告了少动鞘氨醇单胞菌UT26（LinB）的卤代烷脱卤酶与1,2 - 二氯乙烷、1,2 - 二氯丙烷以及1 - 氯丁烷周转反应产物形成复合物的动力学和结构分析。活性研究表明，LinB对1,2 - 二氯乙烷[0.012 nmol s(-1) (mg酶)(-1)]和1,2 - 二氯丙烷[0.027 nmol s(-1) (mg酶)(-1)]的活性非常微弱但可检测到。例如，与LinB对1 - 氯丁烷的活性[68.2 nmol s(-1) (mg酶)(-1)]相比，这些活性要弱得多。抑制分析表明，1,2 - 二氯乙烷和1,2 - 二氯丙烷均作为底物1 - 氯丁烷的简单竞争性抑制剂，且1,2 - 二氯乙烷与LinB的结合亲和力低于1,2 - 二氯丙烷。在没有活性位点水分子和卤离子的情况下对该酶进行对接计算，证实这些化合物能够有效结合。然而，当在计算中包含这些部分时，它们的结合方式与晶体结构中观察到的相似。这些数据解释了LinB对小分子氯化烷烃活性较低的原因，并表明活性位点水分子和反应产物在分子对接中的重要性。
