CYP153A6, a soluble P450 oxygenase catalyzing terminal-alkane hydroxylation
CYP153A6, a soluble P450 oxygenase catalyzing terminal-alkane hydroxylation
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DOI：
10.1128/jb.00286-06
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发表时间：
2006-07-01
影响因子：
3.2
通讯作者：
van Beilen, Jan B.
中科院分区：
文献类型：
--
作者：
Funhoff, Enrico G.;Bauer, Ulrich;van Beilen, Jan B.
烷烃代谢的第一步也是关键步骤是烷烃末端羟基化为1-烷醇，该反应由恶臭假单胞菌GPo1 AMB相关的整膜双铁酶家族、多种甲烷、丙烷和丁烷单加氧酶以及一些膜结合的细胞色素P450催化。最近，在原核生物中发现了一个细胞质P450酶家族，使它们的宿主能够生长在脂肪烷烃上。该家族的一个成员，来自分枝杆菌属的CYP153A6。HXN-1500将中链烷烃(C-6至C-11)羟基化为1-烷醇，最大周转次数为70min(-1)，末端碳原子位置的区域专一性为~gt；=95%。光谱结合研究表明，C-6-到-C-11脂肪族烷烃结合在活性中心，K-d值在20~3.7mM之间变化，较长的烷烃比较短的烷烃结合更强，而空间位阻基团的引入降低了亲和力。这表明底物结合口袋的形状使得直链烷烃是首选的。在底物存在的情况下，电子顺磁共振波谱显示形成了酶-底物络合物，这证实了光学滴定中观察到的底物的结合。为了在分子水平上合理地进行实验观察，使用了CYP153A6的同源建模和底物对接来提供对末端烷烃羟化所需的结构特征的第一次洞察。
The first and key step in alkane metabolism is the terminal hydroxylation of alkanes to 1-alkanols, a reaction catalyzed by a family of integral-membrane diiron enzymes related to Pseudomonas putida GPo1 AMB, by a diverse group of methane, propane, and butane monooxygenases and by some membrane-bound cytochrome P450s. Recently, a family of cytoplasmic P450 enzymes was identified in prokaryotes that allow their host to grow on aliphatic alkanes. One member of this family, CYP153A6 from Mycobacterium sp. HXN-1500, hydroxylates medium-chain-length alkanes (C-6 to C-11) to 1-alkanols with a maximal turnover number of 70 min(-1) and has a regiospecificity of >= 95% for the terminal carbon atom position. Spectroscopic binding studies showed that C-6-to-C-11 aliphatic alkanes bind in the active site with K-d values varying from similar to 20 nM to 3.7 mu M. Longer alkanes bind more strongly than shorter alkanes, while the introduction of sterically hindering groups reduces the affinity. This suggests that the substrate-binding pocket is shaped such that linear alkanes are preferred. Electron paramagnetic resonance spectroscopy in the presence of the substrate showed the formation of an enzyme-substrate complex, which confirmed the binding of substrates observed in optical titrations. To rationalize the experimental observations on a molecular scale, homology modeling of CYP153A6 and docking of substrates were used to provide the first insight into structural features required for terminal alkane hydroxylation.