State Key Joint Laboratory on Environment Simulation and Pollution Control, School of Environment, Tsinghua University, Beijing, People's Republic of China.
PLoS One. 2012;7(9):e45887. doi: 10.1371/journal.pone.0045887. Epub 2012 Sep 20.
The occurrence of cytochrome P450 and P450-mediated pentachlorophenol oxidation in a white rot fungus Phanerochaete chrysosporium was demonstrated in this study. The carbon monoxide difference spectra indicated induction of P450 (103±13 pmol P450 per mg protein in the microsomal fraction) by pentachlorophenol. The pentachlorophenol oxidation by the microsomal P450 was NADPH-dependent at a rate of 19.0±1.2 pmol min(-1) (mg protein)(-1), which led to formation of tetrachlorohydroquinone and was significantly inhibited by piperonyl butoxide (a P450 inhibitor). Tetrachlorohydroquinone was also found in the cultures, while the extracellular ligninases which were reported to be involved in tetrachlorohydroquinone formation were undetectable. The formation of tetrachlorohydroquinone was not detectable in the cultures added with either piperonyl butoxide or cycloheximide (an inhibitor of de novo protein synthesis). These results revealed the pentachlorophenol oxidation by induced P450 in the fungus, and it should be the first time that P450-mediated pentachlorophenol oxidation was demonstrated in a microorganism. Furthermore, the addition of the P450 inhibitor to the cultures led to obvious increase of pentachlorophenol, suggesting that the relationship between P450 and pentachlorophenol methylation is worthy of further research.
本研究证明了白腐真菌糙皮侧耳中细胞色素 P450 和 P450 介导的五氯苯酚氧化作用的发生。一氧化碳差示光谱表明五氯苯酚诱导了 P450（在微粒体部分每毫克蛋白中为 103±13 pmol P450）的产生。在 NADPH 依赖性条件下，微粒体 P450 以 19.0±1.2 pmol min(-1)（mg 蛋白）(-1)的速度氧化五氯苯酚，导致四氯对苯二酚和醌的形成，该过程被胡椒基丁醚（一种 P450 抑制剂）显著抑制。在培养物中也发现了四氯对苯二酚，而据报道参与四氯对苯二酚形成的细胞外木质素酶则无法检测到。在添加胡椒基丁醚或环己亚胺（一种新蛋白质合成抑制剂）的培养物中，无法检测到四氯对苯二酚的形成。这些结果揭示了真菌中诱导的 P450 对五氯苯酚的氧化作用，这应该是首次在微生物中证明 P450 介导的五氯苯酚氧化作用。此外，向培养物中添加 P450 抑制剂会导致五氯苯酚明显增加，这表明 P450 与五氯苯酚甲基化之间的关系值得进一步研究。
