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http://purl.uniprot.org/citations/20233432http://www.w3.org/1999/02/22-rdf-syntax-ns#typehttp://purl.uniprot.org/core/Journal_Citation
http://purl.uniprot.org/citations/20233432http://www.w3.org/1999/02/22-rdf-syntax-ns#typehttp://purl.uniprot.org/core/Journal_Citation
http://purl.uniprot.org/citations/20233432http://www.w3.org/2000/01/rdf-schema#comment"

Background

Microbial degradation of azo dyes is commonly initiated by the reduction of the azo bond(s) by a group of NADH or NADPH dependant azoreductases with many requiring flavin as a cofactor. In this study, we report the identification of a novel flavin-free NADPH preferred azoreductase encoded by azoB in Pigmentiphaga kullae K24.

Results

The deduced amino acid sequence of azoB from P. kullae K24 showed 61% identity to a previously studied azoreductase (AzoA) from the same strain. azoB encoded a protein of 203 amino acids and heterologously expressed in Escherichia coli. The purified recombinant enzyme was a monomer with a molecular mass of 22 kDa. Both NADH and NADPH can be used as an electron donor for its activity with 4-(4-hydroxy-1-naphthylazo) benzenesulfonic acid (Orange I) as substrate. The apparent Km values for both NADH and Orange I were 170 and 8.6 microM, respectively. The Km of NADPH for the enzyme is 1.0 microM. When NADPH served as the electron donor, the activity of the enzyme is 63% higher than that when NADH was used. The pH and temperature optima for activity of the enzyme with Orange I as the substrate were at pH 6.0 and between 37 and 45 degrees C. Phylogenetic analysis shows that AzoB belongs to the flavin-free azoreductase group which has a key fingerprint motif GXXGXXG for NAD(P)H binding at the N-terminus of the amino acid sequences. The 3D structure of AzoB was generated by comparative modeling approach. The structural combination of three conserved glycine residues (G7xxG10xxG13) in the pyrophosphate-binding loop with the Arg-32 explains the preference for NADPH of AzoB.

Conclusion

The biochemical and structural properties of AzoB from P. kullae K24 revealed its preference for NADPH over NADH and it is a member of the monomeric flavin-free azoreductase group. Our studies show the substrate specificity of AzoB based on structure and cofactor requirement and the phylogenetic relationship among azoreductase groups."xsd:string
http://purl.uniprot.org/citations/20233432http://purl.org/dc/terms/identifier"doi:10.1186/1471-2091-11-13"xsd:string
http://purl.uniprot.org/citations/20233432http://purl.org/dc/terms/identifier"doi:10.1186/1471-2091-11-13"xsd:string
http://purl.uniprot.org/citations/20233432http://purl.uniprot.org/core/author"Chen H."xsd:string
http://purl.uniprot.org/citations/20233432http://purl.uniprot.org/core/author"Chen H."xsd:string
http://purl.uniprot.org/citations/20233432http://purl.uniprot.org/core/author"Xu H."xsd:string
http://purl.uniprot.org/citations/20233432http://purl.uniprot.org/core/author"Xu H."xsd:string
http://purl.uniprot.org/citations/20233432http://purl.uniprot.org/core/author"Feng J."xsd:string
http://purl.uniprot.org/citations/20233432http://purl.uniprot.org/core/author"Feng J."xsd:string
http://purl.uniprot.org/citations/20233432http://purl.uniprot.org/core/author"Cerniglia C.E."xsd:string
http://purl.uniprot.org/citations/20233432http://purl.uniprot.org/core/author"Cerniglia C.E."xsd:string
http://purl.uniprot.org/citations/20233432http://purl.uniprot.org/core/author"Kweon O."xsd:string
http://purl.uniprot.org/citations/20233432http://purl.uniprot.org/core/author"Kweon O."xsd:string
http://purl.uniprot.org/citations/20233432http://purl.uniprot.org/core/date"2010"xsd:gYear
http://purl.uniprot.org/citations/20233432http://purl.uniprot.org/core/date"2010"xsd:gYear
http://purl.uniprot.org/citations/20233432http://purl.uniprot.org/core/name"BMC Biochem."xsd:string
http://purl.uniprot.org/citations/20233432http://purl.uniprot.org/core/name"BMC Biochem"xsd:string
http://purl.uniprot.org/citations/20233432http://purl.uniprot.org/core/pages"13"xsd:string
http://purl.uniprot.org/citations/20233432http://purl.uniprot.org/core/pages"13"xsd:string
http://purl.uniprot.org/citations/20233432http://purl.uniprot.org/core/title"Identification and molecular characterization of a novel flavin-free NADPH preferred azoreductase encoded by azoB in Pigmentiphaga kullae K24."xsd:string
http://purl.uniprot.org/citations/20233432http://purl.uniprot.org/core/title"Identification and molecular characterization of a novel flavin-free NADPH preferred azoreductase encoded by azoB in Pigmentiphaga kullae K24."xsd:string
http://purl.uniprot.org/citations/20233432http://purl.uniprot.org/core/volume"11"xsd:string
http://purl.uniprot.org/citations/20233432http://purl.uniprot.org/core/volume"11"xsd:string
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