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

摘要

Background: Microbial degradation of azo dyes is commonly initiated by the reduction of the azo bond(s) by agroup of NADH or NADPH dependant azoreductases with many requiring flavin as a cofactor. In this study, wereport the identification of a novel flavin-free NADPH preferred azoreductase encoded by azoB in Pigmentiphagakullae K24.Results: The deduced amino acid sequence of azoB from P. kullae K24 showed 61% identity to a previouslystudied azoreductase (AzoA) from the same strain. azoB encoded a protein of 203 amino acids and heterologouslyexpressed 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 and8.6 μM, respectively. The K_m of NADPH for the enzyme is 1.0 μM. When NADPH served as the electron donor, theactivity of the enzyme is 63% higher than that when NADH was used. The pH and temperature optima for activityof the enzyme with Orange I as the substrate were at pH 6.0 and between 37 and 45°C. Phylogenetic analysisshows that AzoB belongs to the flavin-free azoreductase group which has a key fingerprint motif GXXGXXG forNAD(P)H binding at the N-terminus of the amino acid sequences. The 3D structure of AzoB was generated bycomparative modeling approach. The structural combination of three conserved glycine residues (G_(7xx)G_(10xx)G_(13))inthe 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 forNADPH over NADH and it is a member of the monomeric flavin-free azoreductase group. Our studies show thesubstrate specificity of AzoB based on structure and cofactor requirement and the phylogenetic relationshipamong azoreductase groups.