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


CONCLUSIONS
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.
Division of Microbiology, National Center for Toxicological Research, US Food and Drug Administration, Jefferson, AR 72079-9502, USA. huizhong.chen@fda.hhs.gov
Huizhong
H
Chen
Jinhui
J
Feng
Ohgew
O
Kweon
Haiyan
H
Xu
Carl E
CE
Cerniglia
D042401:Alcaligenaceae / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001391:Azo Compounds / Q000378:metabolism; D003001:Cloning, Molecular; D004396:Coloring Agents / Q000378:metabolism; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics*; D009249:NADP / Q000378:metabolism; D009601:Nitroreductases
D001391:Azo Compounds; D004396:Coloring Agents; D009249:NADP; D009247:NADH, NADPH Oxidoreductases; D009601:Nitroreductases; C021106:azoreductase