Department of Molecular, Cellular and Developmental Biology, University of Colorado Boulder, Boulder, Colorado 80309, USA.
Biochemistry. 2012 May 8;51(18):3848-60. doi: 10.1021/bi300261p. Epub 2012 Apr 27.
Several strains of Sphingobium chlorophenolicum have been isolated from soil that was heavily contaminated with pentachlorophenol (PCP), a toxic pesticide introduced in the 1930s. S. chlorophenolicum appears to have assembled a poorly functioning pathway for degradation of PCP by patching enzymes recruited via two independent horizontal gene transfer events into an existing metabolic pathway. Flux through the pathway is limited by PCP hydroxylase. PCP hydroxylase is a dimeric protein that belongs to the family of flavin-dependent phenol hydroxylases. In the presence of NADPH, PCP hydroxylase converts PCP to tetrachlorobenzoquinone (TCBQ). The k(cat) for PCP (0.024 s(-1)) is very low, suggesting that the enzyme is not well evolved for turnover of this substrate. Structure-activity studies reveal that substrate binding and activity are enhanced by a low pK(a) for the phenolic proton, increased hydrophobicity, and the presence of a substituent ortho to the hydroxyl group of the phenol. PCP hydroxylase exhibits substantial uncoupling; the C4a-hydroxyflavin intermediate, instead of hydroxylating the substrate, can decompose to produce H(2)O(2) in a futile cycle that consumes NADPH. The extent of uncoupling varies from 0 to 100% with different substrates. The extent of uncoupling is increased by the presence of bulky substituents at position 3, 4, or 5 and decreased by the presence of a chlorine in the ortho position. The effectiveness of PCP hydroxylase is additionally hindered by its promiscuous activity with tetrachlorohydroquinone (TCHQ), a downstream metabolite in the degradation pathway. The conversion of TCHQ to TCBQ reverses flux through the pathway. Substantial uncoupling also occurs during the reaction with TCHQ.
已从受五氯苯酚（PCP）严重污染的土壤中分离出几株鞘氨醇单胞菌（Sphingobium chlorophenolicum），PCP 是一种 20 世纪 30 年代引入的有毒农药。鞘氨醇单胞菌似乎通过两种独立的水平基因转移事件将募集的酶修补到现有代谢途径中，从而组装了一条用于降解 PCP 的功能不佳的途径。该途径的通量受到 PCP 羟化酶的限制。PCP 羟化酶是一种二聚体蛋白，属于黄素依赖性酚羟基酶家族。在 NADPH 的存在下，PCP 羟化酶将 PCP 转化为四氯苯醌（TCBQ）。PCP 的 kcat（0.024 s-1）非常低，表明该酶在这种底物的周转率方面不是很好进化。结构-活性研究表明，酚质子的低 pKa、增加的疏水性以及酚羟基邻位取代基的存在增强了底物结合和活性。PCP 羟化酶表现出大量解偶联；C4a-羟黄素中间产物而不是羟基化底物，可以在无效循环中分解产生 H2O2，消耗 NADPH。不同的底物解偶联程度从 0 到 100%不等。在位置 3、4 或 5 处存在大取代基和邻位存在氯时，解偶联程度增加，而在邻位存在氯时，解偶联程度降低。PCP 羟化酶的活性还受到其与四氯对苯二酚（TCHQ）的混杂活性的阻碍，TCHQ 是降解途径中的下游代谢物。TCHQ 转化为 TCBQ 会逆转途径中的通量。在与 TCHQ 的反应过程中也会发生大量解偶联。
