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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  K  m  values for both NADH and Orange I were 170 and 8.6 μM, respectively. The  K  m  of NADPH for the enzyme is 1.0 μM. 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°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 (G 7 xxG 10 xxG 13 ) 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.

Identification and molecular characterization of a novel flavin-free NADPH preferred azoreductase encoded by azoB in Pigmentiphaga kullae K24