Characterization of two components of the 2-naphthoate monooxygenase system from Burkholderia sp. strain JT1500.
2-Naphthoate monooxygenase, a two-protein system, encoded by the nmoA and nmoB genes, was heterologously overexpressed in Escherichia coli. The proteins used for functional characterization were purified to over 90% homogeneity by affinity chromatography. The oxidative component EnmoA (47.9 kDa) lacked substrate catalysis capability on its own, and the reductive component EnmoB (33.4 kDa) and its truncated derivate EnmoB(T) (25 kDa) possessed nearly identical independent flavin reductase activities, c. 130 micromol min(-1) mg(-1) of protein. The inframe fusioned protein EnmoB(T)A (65.2 kDa), containing NmoB(T) and NmoA peptides showed a stable 2-naphthoate monooxygenase activity of 1.2 micromol min(-1) mg(-1) of protein. This is the first report on the purification of a fused form of a two-component flavoprotein monooxygenase. In the specificity experiment, FAD and NADH were shown to be preferred cosubstrates for EnmoB and EnmoB(T). All these data suggest that NmoB(T)A is a two-component flavoprotein monooxygenase, consisting of an oxygenase and a reductase component. NmoA is a member of the class D flavoprotein monooxygenase, and NmoB is an independent NADH:Flavin oxidoreductase.
Deng
Daiyong
D
Microbiology and Environmental Biotechnology, Guangdong Institute of Microbiology, Guangdong Province, China.
Li
Xiaobo
X
Fang
Xiangping
X
Sun
Guoping
G
D019117:Burkholderia / Q000201:enzymology*; D002264:Carboxylic Acids / Q000378:metabolism*; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005415:Flavins / Q000378:metabolism; D005420:Flavoproteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015870:Gene Expression; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D009281:Naphthalenes / Q000378:metabolism*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013379:Substrate Specificity
D002264:Carboxylic Acids; D005415:Flavins; D005420:Flavoproteins; D009281:Naphthalenes; D011993:Recombinant Fusion Proteins; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; C085312:1-naphthoic acid; D006899:Mixed Function Oxygenases; D010088:Oxidoreductases