一种对多环芳烃二氢二醇具有广泛底物特异性的芳烃顺式二氢二醇脱氢酶的纯化和性质
Purification and Characterization of an Arene cis-Dihydrodiol Dehydrogenase Endowed with Broad Substrate Specificity toward Polycyclic Aromatic Hydrocarbon Dihydrodiols
摘要：
多环芳烃（PAH）的细菌降解的初始反应包括由双加氧酶催化的环二羟基化和随后由脱氢酶氧化二氢二醇产物。在这项研究中，二氢二醇脱氢酶从多环芳烃降解鞘氨醇单胞菌株CHY-1的特点。将编码PAH二氢二醇脱氢酶（PDDH）的bphB基因克隆并作为His标记的蛋白质过表达。重组蛋白纯化为表观Mr为110，000的同源四聚体。PDDH氧化联苯和八种多环烃（包括苯并蒽、苯并[a]蒽和苯并[a]芘）衍生的顺式二氢二醇为相应的儿茶酚。值得注意的是，该酶氧化芘4，5-二氢二醇，而芘不被菌株CHY-1代谢。由PDDH产生的PAH儿茶酚在空气中快速自氧化，但在与NADH反应形成的邻醌后再生。在缺氧条件下进行的动力学分析表明，该酶有效地利用两个到四个环的二氢二醇，与Km值的范围为1.4至7.1 $μ$ M，并表现出高得多的米氏常数NAD+（160 $μ$ M的Km）。在pH 7.0时，特异性常数范围为（1.3 $\pm$ 0.1）× 106 M？1秒？1与苯并[a]蒽1，2-二氢二醇反应至（20.0 $\pm$ 0.8）x 106 M？1秒？1与萘1，2-二氢二醇反应。该酶的催化活性在pH 9.5时高13倍。PDDH受NADH和3，4-二羟基菲的抑制，其抑制模式表明反应机理为Bi Bi。PDDH活性的调节似乎是防止PAH儿茶酚在细菌细胞中积累的一种手段。
英文摘要：
Initial reactions involved in the bacterial degradation of polycyclic aromatic hydrocarbons (PAHs) include a ring-dihydroxylation catalyzed by a dioxygenase and a subsequent oxidation of the dihydrodiol products by a dehydrogenase. In this study, the dihydrodiol dehydrogenase from the PAH-degrading Sphingomonas strain CHY-1 has been characterized. The bphB gene encoding PAH dihydrodiol dehydrogenase (PDDH) was cloned and overexpressed as a His-tagged protein. The recombinant protein was purified as a homotetramer with an apparent Mr of 110,000. PDDH oxidized the cis-dihydrodiols derived from biphenyl and eight polycyclic hydrocarbons, including chrysene, benz[a]anthracene, and benzo[a]pyene, to corresponding catechols. Remarkably, the enzyme oxidized pyrene 4,5-dihydrodiol, whereas pyrene is not metabolized by strain CHY-1. The PAH catechols produced by PDDH rapidly auto-oxidized in air but were regenerated upon reaction of the o-quinones formed with NADH. Kinetic analyses performed under anoxic conditions revealed that the enzyme efficiently utilized two- to four-ring dihydrodiols, with Km values in the range of 1.4 to 7.1 $μ$M, and exhibited a much higher Michaelis constant for NAD+ (Km of 160 $μ$M). At pH 7.0, the specificity constant ranged from (1.3 $\pm$ 0.1) x 106 M?1 s?1 with benz[a]anthracene 1,2-dihydrodiol to (20.0 $\pm$ 0.8) x 106 M?1 s?1 with naphthalene 1,2-dihydrodiol. The catalytic activity of the enzyme was 13-fold higher at pH 9.5. PDDH was subjected to inhibition by NADH and by 3,4-dihydroxyphenanthrene, and the inhibition patterns suggested that the mechanism of the reaction was ordered Bi Bi. The regulation of PDDH activity appears as a means to prevent the accumulation of PAH catechols in bacterial cells.
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