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.
Chen
Huizhong
H
Division of Microbiology, National Center for Toxicological Research, US Food and Drug Administration, Jefferson, AR 72079-9502, USA. huizhong.chen@fda.hhs.gov
Feng
Jinhui
J
Kweon
Ohgew
O
Xu
Haiyan
H
Cerniglia
Carl E
CE
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