Crystal structures of the substrate free-enzyme, and reaction intermediate of the HAD superfamily member, haloacid dehalogenase DehIVa from Burkholderia cepacia MBA4. (2NO4)

DehIVa is a haloacid dehalogenase (EC 3.8.1.2) from the soil and water borne bacterium Burkholderia cepacia MBA4, which belongs to the functionally variable haloacid dehalogenase (HAD) superfamily of enzymes. The haloacid dehalogenases catalyse the removal of halides from haloacids resulting in a hydroxlated product. These enzymes are of interest for their potential to degrade recalcitrant halogenated environmental pollutants and their use in the synthesis of industrial chemicals. The haloacid dehalogenases utilise a nucleophilic attack on the substrate by an aspartic acid residue to form an enzyme-substrate ester bond and concomitantly cleaving of the carbon-halide bond and release of a hydroxylated product following ester hydrolysis. We present the crystal structures of both the substrate-free DehIVa refined to 1.93 A resolution and DehIVa covalently bound to l-2-monochloropropanoate trapped as a reaction intermediate, refined to 2.7 A resolution. Electron density consistent with a previously unidentified yet anticipated water molecule in the active site poised to donate its hydroxyl group to the product and its proton to the catalytic Asp11 is evident. It has been unclear how substrate enters the active site of this and related enzymes. The results of normal mode analysis (NMA) are presented and suggest a means whereby the predicted global dynamics of the enzyme allow for entry of the substrate into the active site. In the context of these results, the possible role of Arg42 and Asn178 in a `lock down` mechanism affecting active site access is discussed. In silico substrate docking of enantiomeric substrates has been examined in order to evaluate the enzymes enantioselectivity.

DehIVa是一种卤酸脱卤酶（haloacid dehalogenase，EC 3.8.1.2），来源于土壤和水生细菌洋葱伯克霍尔德菌（Burkholderia cepacia）MBA4菌株，属于功能多样的卤酸脱卤酶（HAD）超家族酶类。卤酸脱卤酶可催化从卤酸中去除卤离子，生成羟基化产物。这类酶因具有降解难降解卤代环境污染物的潜力，以及在工业化学品合成中的应用价值而备受关注。卤酸脱卤酶通过天冬氨酸残基对底物发起亲核攻击，形成酶-底物酯键；随后酯水解伴随碳-卤键断裂，释放羟基化产物。本文报道了两种DehIVa的晶体结构：无底物DehIVa（分辨率优化至1.93 Å），以及与L-2-一氯丙酸共价结合并作为反应中间体捕获的DehIVa（分辨率优化至2.7 Å）。活性位点中存在一个此前未被鉴定但预期存在的水分子，其电子密度特征表明该水分子可向产物提供羟基，并向催化残基Asp11提供质子。底物如何进入该酶及相关酶的活性位点一直不明确。本文报道的正态模式分析（normal mode analysis，NMA）结果揭示了一种机制：酶的预测全局动力学特性允许底物进入活性位点。基于这些结果，本文讨论了Arg42和Asn178在影响活性位点可及性的“锁定”机制中可能发挥的作用。此外，通过对映体底物的计算机模拟对接（in silico substrate docking）研究，评估了该酶的对映选择性。