Cloning And Analysis Of Genes Encoding 2-naphthoate Monooxygenase And NADH: Flavin Reductase

Azo aromatic compounds exist widely in the world and millions of tons of azo compounds are produced each year. Lots of Azo aromatic compounds are very important in industry, while serious pollution may appear when they are used and released. 2-naphthoate(2-NAT) is a kind of aromatic compounds that are similar with the basic chemical structure of azo aromatic compounds. 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 fragment 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 Rastonia 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 SA(only harboring orfA) showed very low oxygenase oxidation activity as detected by NADH decreasing, while the cell extracts of recombinant SB(only harboring orfB) did not show any oxidation activity at all. But when the cell extracts of SB and SA were mixed, the mixture 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 SB 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 O2 oxidizing 2-naphthoate. NmoB is NmoA's coupling protein. The discovery of 2-naphthoate monooxygenase is a new member of the family of two component nonheme flavin-diffusible monooxygenase(TC-FDM).