2-萘酸加单氧酶基因及NADH:黄素还原酶基因的克隆与分析

伯克霍尔德氏菌(Burkholderiasp.)JT1500对2-萘酸(2-naphthoate)生物降解的关键步骤之一是通过2-萘酸加单氧酶羟化2-萘酸生成1-羟基-2-萘酸(1-hydroxy-2-naphthoate)。在已确定2-萘酸加单氧酶基因及其功能的基础上对含有该基因的一个4.8kb长度的基因簇进行了克隆测序。该序列上含有4个可能的阅读框orfB、orfC、orfD、orfA。序列比对发现,orfA序列与JaponicumUSDA110和RalstoniaeutrophaHF39中的加单氧酶基因同源性较高,orfB序列与BordetllapertussisTohamaI、RalstoniasolanacearumGMI1000和BordetellabronchisepticaRB50等菌中的黄素还原酶基因有一定的同源性。酶活分析发现只含基因orfA的重组大肠杆菌SA细胞提取液有很低的加氧活性,含基因orfB的重组子SB细胞提取液没有加氧活性,但在反应体系中同时加入SA和SB的细胞提取液后,其加氧活性显著增强,包含片段orfB orfA的重组子SB A在黄素(FMN、FAD)存在的情况下也表现出很强的加氧活性;在厌氧条件下,能检测出SB细胞提取液的黄素还原活性。基于以上信息,认为2-萘酸加单氧酶基因簇含有两个重要的组分黄素还原酶基因(nmoB)和加单氧酶基因(nmoA)。2-萘酸加单氧酶Nmo羟化2-萘酸的过程为先由黄素还原酶(NmoB)在NADH存在的条件下将黄素(FMN、FAD)还原为还原型黄素(FMNH2、FADH2),然后加单氧酶(NmoA)利用还原型黄素和O2羟化底物2-萘酸,生成1-羟基-2-萘酸。NmoB是NmoA的偶联蛋白。

Cloning and analysis of genes encoding 2-naphthoate monooxygenase and NADH: flavin oxidoreductase

In Burkholderia sp.JT1500, a key step of 2-naphthoate biodegradation pathway is carried out by 2-naphthoate monooxygenase(Nmo) in which 2-naphthoate is oxidized to 1-hydroxy-2-naphthoate. A gene cluster of 4.8kb from Burkholderia sp. JT1500 was cloned and sequenced, four open reading frames named orfB, orfC, orfD and orfA were identified in this region. Sequence alignment showed that orfA had a high homology of nucleotide acid composition to monooxygenase genes from both Japonicum USDA 110 and Ralstonia eutropha HF 39, orfB had some homology to the component of flavin reductase genes from Bordetlla pertussis Tohama I, Ralstonia solanacearum GMI1000 and Bordetella bronchiseptica RB50.Enzyme activity analysis showed that the cell extracts of recombinant E.coli S- A (only harboring orfA) showed very low oxygenase oxidation activity as detected by NADH decreasing, while the cell extracts of recombinant S- B (only harboring orfB) did not show any oxidation activity at all. But when the cell extracts of S- B and S- A were mixed, which showed very strong oxidation activity when flavin (FMN or FAD) provided; the recombinant S- B A cells harboring both orfB and orfA genes also showed strong oxidation activity when flavin provided; weak flavin deoxidization activity could be detected from the cell extracts of E.coli S- B under anaerobic conditions. Based on above message, a conclusion was drawn that Nmo is consisted of two components: a flavin oxidoreductase (NmoB) and a monooxygenase (NmoA). First NmoB uses NADH to reduce flavin and supplies reduced flavin to NmoA to catalyze O- 2 oxidizing 2-NAT. NmoB is NmoA's coupling protein.