Wang Susan C, Person Maria D, Johnson William H, Whitman Christian P
Division of Medicinal Chemistry, College of Pharmacy, The University of Texas, Austin, Texas 78712-1074, USA.
Biochemistry. 2003 Jul 29;42(29):8762-73. doi: 10.1021/bi034598+.
Various soil bacteria use 1,3-dichloropropene, a component of the commercially available fumigants Shell D-D and Telone II, as a sole source of carbon and energy. One enzyme involved in the catabolism of 1,3-dichloropropene is trans-3-chloroacrylic acid dehalogenase (CaaD), which converts the trans-isomers of 3-bromo- and 3-chloroacrylate to malonate semialdehyde. Sequence analysis suggested a relationship between the heterohexameric CaaD and the bacterial isomerase, 4-oxalocrotonate tautomerase (4-OT), thereby distinguishing CaaD from a number of dehalogenases whose mechanisms proceed through an alkyl- or aryl-enzyme intermediate. In this study, the genes for the alpha- and beta-subunits of CaaD have been synthesized using a polymerase chain reaction-based strategy, cloned into separate plasmids, and the proteins expressed and purified as the functional heterohexamer. Subsequently, the product of the reaction was confirmed to be malonate semialdehyde by (1)H and (13)C NMR spectroscopy, and kinetic constants were determined using a UV spectrophotometric assay. In view of the proposed hydrolytic nature of the CaaD-catalyzed reaction, three acetylene compounds were investigated as substrates for the enzyme. One compound, 2-oxo-3-pentynoate, a potent active site-directed irreversible inhibitor of 4-OT, is a substrate for CaaD, and was processed to acetopyruvate with kinetic constants similar to those determined for the trans-isomers of 3-bromo- and 3-chloroacrylate. The remaining two compounds, 3-bromo- and 3-chloropropiolic acid, were transformed into potent irreversible inhibitors of CaaD. The inactivation observed for 3-bromopropiolic acid is due to the covalent modification of Pro-1 of the beta-subunit. The results provide evidence for a hydratase activity and set the stage to use the 3-halopropiolic acids as ligands in inactivated CaaD complexes that can be studied by X-ray crystallography.
多种土壤细菌利用1,3 - 二氯丙烯（市售熏蒸剂壳牌D - D和泰隆二号的一种成分）作为唯一的碳源和能源。参与1,3 - 二氯丙烯分解代谢的一种酶是反式 - 3 - 氯丙烯酸脱卤酶（CaaD），它将3 - 溴丙烯酸和3 - 氯丙烯酸的反式异构体转化为丙二酸半醛。序列分析表明异源六聚体CaaD与细菌异构酶4 - 草酰巴豆酸互变异构酶（4 - OT）之间存在关联，从而将CaaD与许多通过烷基或芳基酶中间体起作用的脱卤酶区分开来。在本研究中，使用基于聚合酶链反应的策略合成了CaaD的α和β亚基的基因，将其克隆到单独的质粒中，并表达和纯化出具有功能的异源六聚体蛋白。随后，通过¹H和¹³C核磁共振光谱确认反应产物为丙二酸半醛，并使用紫外分光光度法测定动力学常数。鉴于CaaD催化反应的水解性质，研究了三种乙炔化合物作为该酶的底物。一种化合物2 - 氧代 - 3 - 戊炔酸是4 - OT的一种有效的活性位点定向不可逆抑制剂，它是CaaD的底物，并被加工成乙酰丙酮酸，其动力学常数与为3 - 溴丙烯酸和3 - 氯丙烯酸的反式异构体测定的相似。其余两种化合物3 - 溴丙炔酸和3 - 氯丙炔酸被转化为CaaD的有效不可逆抑制剂。观察到的3 - 溴丙炔酸的失活是由于β亚基的Pro - 1的共价修饰。这些结果为水合酶活性提供了证据，并为将3 - 卤代丙炔酸用作可通过X射线晶体学研究的失活CaaD复合物中的配体奠定了基础。
