Cloning, expression and characterization of a gene encoding nitroalkane-oxidizing enzyme from Streptomyces ansochromogenes.
A nitroalkane-oxidizing enzyme gene (naoA) was cloned from a genomic DNA library of Streptomyces ansochromogenes 7100. The deduced protein (NaoA) of this gene contains 363 amino acids and has high similarity to several nitroalkane-oxidizing enzymes from various micro-organisms. The naoA gene was subcloned into an expression vector pET23b and overexpressed in Escherichia coli BL21(DE3). The protein was then purified, and its characteristics were studied. Experimental results showed that NaoA can convert 1-nitropropane, 2-nitropropane and nitroethane into the corresponding carbonyl compounds. The optimal pH and temperature for NaoA was found to be pH 7-8 and 48-56 degrees C, respectively. The Km of NaoA for nitroethane is approximately 26.8 mm. NADH and nitro blue tetrazolium are strong inhibitors of NaoA, and thiol compounds and superoxide dismutase partially inhibit the enzyme activity. Therefore, superoxide may be an essential intermediate in the oxidation of nitroalkane by NaoA.
Zhang
Jihui
J
Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
Tan
Huarong
H
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D049308:Dioxygenases; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010105:Oxygenases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics*; D013696:Temperature
D004247:DNA; D010105:Oxygenases; D049308:Dioxygenases; C021876:2-nitropropane dioxygenase