Fortin Pascal D, Horsman Geoff P, Yang Hao M, Eltis Lindsay D
Department of Microbiology and Immunology, University of British Columbia, 1365-2350 Health Sciences Mall, Vancouver, BC V6T 1Z3, Canada.
J Bacteriol. 2006 Jun;188(12):4424-30. doi: 10.1128/JB.01849-05.
BphK is a glutathione S-transferase of unclear physiological function that occurs in some bacterial biphenyl catabolic (bph) pathways. We demonstrated that BphK of Burkholderia xenovorans strain LB400 catalyzes the dehalogenation of 3-chloro 2-hydroxy-6-oxo-6-phenyl-2,4-dienoates (HOPDAs), compounds that are produced by the cometabolism of polychlorinated biphenyls (PCBs) by the bph pathway and that inhibit the pathway's hydrolase. A one-column protocol was developed to purify heterologously produced BphK. The purified enzyme had the greatest specificity for 3-Cl HOPDA (kcat/Km, approximately 10(4) M(-1) s(-1)), which it dechlorinated approximately 3 orders of magnitude more efficiently than 4-chlorobenzoate, a previously proposed substrate of BphK. The enzyme also catalyzed the dechlorination of 5-Cl HOPDA and 3,9,11-triCl HOPDA. By contrast, BphK did not detectably transform HOPDA, 4-Cl HOPDA, or chlorinated 2,3-dihydroxybiphenyls. The BphK-catalyzed dehalogenation proceeded via a ternary-complex mechanism and consumed 2 equivalents of glutathione (GSH) (Km for GSH in the presence of 3-Cl HOPDA, approximately 0.1 mM). A reaction mechanism consistent with the enzyme's specificity is proposed. The ability of BphK to dehalogenate inhibitory PCB metabolites supports the hypothesis that this enzyme was recruited to facilitate PCB degradation by the bph pathway.
BphK是一种生理功能不明的谷胱甘肽S-转移酶，存在于一些细菌的联苯分解代谢（bph）途径中。我们证明，嗜麦芽窄食单胞菌LB400菌株的BphK催化3-氯-2-羟基-6-氧代-6-苯基-2,4-二烯酸酯（HOPDAs）的脱卤反应，这些化合物是由bph途径对多氯联苯（PCBs）进行共代谢产生的，并且会抑制该途径的水解酶。我们开发了一种单柱方案来纯化异源产生的BphK。纯化后的酶对3-氯-HOPDA具有最高的特异性（kcat/Km，约为10⁴ M⁻¹ s⁻¹），其脱氯效率比先前提出的BphK底物4-氯苯甲酸高约3个数量级。该酶还催化5-氯-HOPDA和3,9,11-三氯-HOPDA的脱氯反应。相比之下，BphK未检测到对HOPDA、4-氯-HOPDA或氯化2,3-二羟基联苯的转化。BphK催化的脱卤反应通过三元复合物机制进行，消耗2当量的谷胱甘肽（GSH）（在存在3-氯-HOPDA的情况下，GSH的Km约为0.1 mM）。我们提出了一种与该酶特异性一致的反应机制。BphK对抑制性PCB代谢物进行脱卤的能力支持了这样一种假设，即该酶被招募来促进bph途径对PCB的降解。
