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

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. To cite this data use the following DOI: 10.4225/52/557F9CB23E046

DehIVa是一种源自土壤与水环境洋葱伯克霍尔德菌（Burkholderia cepacia）MBA4的卤酸脱卤酶（haloacid dehalogenase，EC 3.8.1.2），隶属于功能多样的卤酸脱卤酶（haloacid dehalogenase, HAD）超家族。卤酸脱卤酶可催化卤酸脱除卤原子，生成羟基化产物。此类酶因具备降解难降解卤代环境污染物的潜力，且可用于工业化学品合成，因而受到广泛关注。该类酶通过天冬氨酸残基对底物进行亲核攻击，形成酶-底物酯键，同时断裂碳-卤键；随后经酯水解，释放羟基化产物。本研究解析了两种DehIVa的晶体结构：无底物结合的DehIVa精修至1.93埃分辨率，与作为反应中间体被捕获的L-2-一氯丙酸共价结合的DehIVa精修至2.7埃分辨率。活性位点中存在一个此前未被鉴定但预期存在的水分子，其电子密度清晰可辨；该水分子可将羟基供给产物，并将质子传递给催化性天冬氨酸残基Asp11。此前尚不明确底物如何进入该酶及其同源酶的活性位点。本研究呈现了简正模式分析（normal mode analysis, NMA）的结果，结果表明，该酶的预测全局动态特性可允许底物进入活性位点。结合上述结果，本文讨论了精氨酸残基Arg42与天冬酰胺残基Asn178在调控活性位点准入的"lock down"机制中的潜在作用。为评估该酶的对映选择性，本研究还开展了对映体底物的计算机模拟（in silico）对接分析。引用本数据集请使用以下DOI：10.4225/52/557F9CB23E046