Department of Biochemistry and Microbiology, Rutgers University, New Brunswick, NJ, USA Shaw Environmental, Inc., Lawrenceville, NJ, USA.
Lett Appl Microbiol. 2012 Sep;55(3):175-81. doi: 10.1111/j.1472-765X.2012.03290.x. Epub 2012 Jul 31.
Mycobacterium sp. strain ENV421 has the ability to cometabolize a variety of chemicals following growth on propane as a sole source of carbon and energy. In this study, we used genetic and biochemical approaches to identify and characterize multiple propane-inducible oxygenase genes in ENV421.
Gene clusters encoding a CYP153-type cytochrome P450 oxygenase (P450), an AlkB-type alkane monooxygenase (AlkB) and a soluble diiron monooxygenase were identified and cloned using degenerate PCR primers. Reverse transcriptase PCR showed that all three gene clusters were induced by propane. Substrate specificity studies revealed that despite the fact that ENV421 does not grow on medium length alkanes, cloned versions of both the AlkB and P450 were capable of octane oxidation, forming n-octanol. Additionally, the P450 oxygenase had the ability to oxidize indole, medium-to-long-chain alkylbenzenes and a variety of para-substituted methylalkylbenzenes. Successful cloning and expression of the diiron monooxygenase was not achieved, so its substrate specificity could not be determined.
Three types of short-to-medium-chain alkane oxygenases were induced by propane in ENV421, even though the cloned AlkB and P450 oxygenases did not oxidize propane. Curiously, they both oxidized octane, which is not a growth substrate for ENV421. Furthermore, the P450, typically operating as terminal alkane hydroxylase, exhibited interesting regio- and stereoselectivity, catalysing linear alkanes, alkylbenzenes and indole.
This study describes the first example of a propane-inducible P450 with a broad substrate specificity, including linear alkanes, alkylbenzenes and a multiring compound. The induction of three distinct oxygenase classes by propane is also an interesting finding because it might explain why propane serves as an effective stimulant that promotes the biodegradation of a various environmental contaminants.
分枝杆菌株 ENV421 能够在以丙烷为唯一碳源和能源的条件下生长，同时代谢多种化学物质。在本研究中，我们使用遗传和生化方法鉴定和表征了 ENV421 中多个丙烷诱导的加氧酶基因。
使用简并 PCR 引物鉴定和克隆了编码 CYP153 型细胞色素 P450 加氧酶（P450）、AlkB 型烷烃单加氧酶（AlkB）和可溶性二铁单加氧酶的基因簇。逆转录 PCR 显示，所有三个基因簇均受丙烷诱导。底物特异性研究表明，尽管 ENV421 不能在中长链烷烃上生长，但克隆的 AlkB 和 P450 都能够氧化辛烷，形成正辛醇。此外，P450 加氧酶能够氧化吲哚、中长链烷基苯和各种对位取代的甲基烷基苯。未能成功克隆和表达二铁单加氧酶，因此无法确定其底物特异性。
即使克隆的 AlkB 和 P450 加氧酶不氧化丙烷，丙烷仍能诱导 ENV421 中三种类型的短至中链烷烃加氧酶。奇怪的是，它们都能氧化辛烷，而辛烷不是 ENV421 的生长底物。此外，通常作为末端烷烃羟化酶起作用的 P450 表现出有趣的区域和立体选择性，催化直链烷烃、烷基苯和吲哚。
本研究描述了第一个具有广泛底物特异性的丙烷诱导 P450 的实例，包括直链烷烃、烷基苯和多环化合物。丙烷诱导三种不同加氧酶类的现象也很有趣，因为它可能解释了为什么丙烷是一种有效的刺激物，能促进各种环境污染物的生物降解。
