Characterization of a Novel Rieske-Type Alkane Monooxygenase System in Pusillimonas sp Strain T7-7
Characterization of a Novel Rieske-Type Alkane Monooxygenase System in Pusillimonas sp Strain T7-7
复制标题
微单胞菌菌株 T7-7 中新型 Rieske 型烷烃单加氧酶系统的表征
DOI：
10.1128/jb.02107-12
复制
发表时间：
2013-05-01
影响因子：
3.2
通讯作者：
Feng, Lu
中科院分区：
文献类型：
--
作者：
Li, Ping;Wang, Lei;Feng, Lu
耐冷细菌Pusillimonas sp.菌株T7-7能够利用柴油（C-5至C-30烷烃）作为唯一的碳和能量来源。在本研究中，生物信息学、蛋白质组学和实时逆转录酶PCR方法被用于鉴定该细菌中存在的烷烃羟基化系统。该系统由rieske型单加氧酶、铁氧还蛋白和nadh依赖性还原酶组成。该单加氧酶由一个大亚基（46.711 kDa）和一个小亚基（15.355 kDa）组成，通过体外生化分析和体内异源功能互补试验进一步研究了其功能。纯化的单加氧酶的大亚基能够以NADH作为辅助因子氧化从戊烷（C-5）到四烷（C-24）的烷烃，在C-15底物上具有最大的活性。大亚基对几种烷烃衍生物，包括硝基甲烷和甲烷磺酸也有活性，但对芳香烃没有作用。大亚基的最佳反应条件为30℃pH 7.5， Fe2+能明显提高酶的活性。这是首次在细菌中发现属于Rieske非血红素铁加氧酶家族的烷烃单加氧酶系统。
The cold-tolerant bacterium Pusillimonas sp. strain T7-7 is able to utilize diesel oils (C-5 to C-30 alkanes) as a sole carbon and energy source. In the present study, bioinformatics, proteomics, and real-time reverse transcriptase PCR approaches were used to identify the alkane hydroxylation system present in this bacterium. This system is composed of a Rieske-type monooxygenase, a ferredoxin, and an NADH-dependent reductase. The function of the monooxygenase, which consists of one large (46.711 kDa) and one small (15.355 kDa) subunit, was further studied using in vitro biochemical analysis and in vivo heterologous functional complementation tests. The purified large subunit of the monooxygenase was able to oxidize alkanes ranging from pentane (C-5) to tetracosane (C-24) using NADH as a cofactor, with greatest activity on the C-15 substrate. The large subunit also showed activity on several alkane derivatives, including nitromethane and methane sulfonic acid, but it did not act on any aromatic hydrocarbons. The optimal reaction condition of the large subunit is pH 7.5 at 30 degrees C. Fe2+ can enhance the activity of the enzyme evidently. This is the first time that an alkane monooxygenase system belonging to the Rieske non-heme iron oxygenase family has been identified in a bacterium.