理性设计真菌 P450 单加氧酶 CYP5136A3 以提高其对多环芳烃的氧化活性。
Rational engineering of the fungal P450 monooxygenase CYP5136A3 to improve its oxidizing activity toward polycyclic aromatic hydrocarbons.
机构信息
Department of Environmental Health, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0056, USA.
出版信息
Protein Eng Des Sel. 2013 Sep;26(9):553-7. doi: 10.1093/protein/gzt036. Epub 2013 Jul 31.
Abstract
A promising polycyclic aromatic hydrocarbon-oxidizing P450 CYP5136A3 from Phanerochaete chrysosporium was rationally engineered to enhance its catalytic activity. The residues W129 and L324 found to be critical in substrate recognition were transformed by single (L324F) and double (W129L/L324G, W129L/L324F, W129A/L324G, W129F/L324G and W129F/L324F) mutations, and the engineered enzyme forms were expressed in Pichia pastoris. L324F and W129F/L324F mutations enhanced the oxidation activity toward pyrene and phenanthrene. L324F also altered the regio-selectivity favoring C position 4 over 9 for hydroxylation of phenanthrene. This is the first instance of engineering a eukaryotic P450 for enhanced oxidation of these fused-ring hydrocarbons.
摘要
从黄孢原毛平革菌中鉴定出一种具有应用前景的多环芳烃氧化酶 CYP5136A3，通过理性设计提高了其催化活性。在底物识别中起关键作用的残基 W129 和 L324 经单点突变（L324F）和双点突变（W129L/L324G、W129L/L324F、W129A/L324G、W129F/L324G 和 W129F/L324F）转化，突变酶在毕赤酵母中进行了表达。L324F 和 W129F/L324F 突变提高了对芘和菲的氧化活性。L324F 还改变了对菲的羟基化区域选择性，有利于 C 位置 4 而非 9。这是首例为增强这些稠环烃的氧化而对真核 P450 进行的工程改造。
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