State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, People's Republic of China.
Appl Microbiol Biotechnol. 2010 Jan;85(3):615-24. doi: 10.1007/s00253-009-2118-1. Epub 2009 Jul 25.
In this study, a 3.7-kb DNA fragment was cloned from Rhodococcus sp. ECU0066, and the sequence was analyzed. It was revealed that the largest one (2,361 bp) of this gene fragment encodes a protein consisting of 787 amino acids, with 73% identity to P450RhF (accession number AF45924) from Rhodococcus sp. NCIMB 9784. The gene of this new P450 monooxygenase (named as P450SMO) was successfully expressed in Escherichia coli BL21 (DE3), and the enzyme was also purified and characterized. In the presence of reduced nicotinamide adenine dinucleotide phosphate, the enzyme showed significant sulfoxidation activity towards several sulfides, with (S)-sulfoxides as the predominant product. The p-chlorothioanisole, p-fluorothioanisole, p-tolyl methyl sulfide, and p-methoxythioanisole showed relatively higher activities than the other sulfides, but the stereoselectivity for p-methoxythioanisole was much lower. The optimal activity of the purified enzyme toward p-chlorothioanisole occurred at pH 7.0 and 30 degrees C. The current study is the first to report a recombinant cytochrome P450 enzyme of Rhodococcus sp. which is responsible for the asymmetric oxidation of sulfides. The new enzymatic activity of P450SMO on the above compounds makes it an attractive biocatalyst for asymmetric synthesis of enantiopure sulfoxides.
在这项研究中，从 Rhodococcus sp. ECU0066 中克隆了一个 3.7kb 的 DNA 片段，并对其序列进行了分析。结果表明，该基因片段中最大的一个（2361bp）编码一种蛋白，由 787 个氨基酸组成，与 Rhodococcus sp. NCIMB 9784 的 P450RhF（登录号 AF45924）具有 73%的同源性。这种新的 P450 单加氧酶（命名为 P450SMO）的基因成功地在大肠杆菌 BL21（DE3）中表达，并且该酶也被纯化和表征。在还原型烟酰胺腺嘌呤二核苷酸磷酸存在的情况下，该酶对几种硫醚表现出显著的氧化脱硫活性，以（S）-亚砜为主要产物。对氯苯甲硫醚、对氟苯甲硫醚、对甲苯甲基硫醚和对甲氧基苯甲硫醚的活性相对较高，但对甲氧基苯甲硫醚的立体选择性要低得多。该酶对 p-氯苯甲硫醚的最适活性出现在 pH7.0 和 30℃。本研究首次报道了一种负责不对称氧化硫醚的 Rhodococcus sp. 的重组细胞色素 P450 酶。P450SMO 对上述化合物的新酶活性使其成为手性纯亚砜不对称合成的有吸引力的生物催化剂。
