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

ABSTRACT: 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.

SUBMITTER: Chen H

PROVIDER: S-EPMC2858024 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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

Chen Huizhong H Feng Jinhui J Kweon Ohgew O Xu Haiyan H Cerniglia Carl E CE

BMC biochemistry 20100316

<h4>Background</h4>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.<h4>Results</h4>The deduced amino acid sequence of azoB from P. kullae K24 showed 61% identity to a previously studied azoreductase (AzoA) from the same st ...[more]

PMID: 20233432

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Project description:Metazoan core promoters, which direct the initiation of transcription by RNA polymerase II (Pol II), may contain short sequence motifs termed core promoter elements/motifs (e.g. the TATA box, initiator (Inr) and downstream core promoter element (DPE)), which recruit Pol II via the general transcription machinery. The DPE was discovered and extensively characterized in Drosophila, where it is strictly dependent on both the presence of an Inr and the precise spacing from it. Since the Drosophila DPE is recognized by the human transcription machinery, it is most likely that some human promoters contain a downstream element that is similar, though not necessarily identical, to the Drosophila DPE. However, only a couple of human promoters were shown to contain a functional DPE, and attempts to computationally detect human DPE-containing promoters have mostly been unsuccessful. Using a newly-designed motif discovery strategy based on Expectation-Maximization probabilistic partitioning algorithms, we discovered preferred downstream positions (PDP) in human promoters that resemble the Drosophila DPE. Available chromatin accessibility footprints revealed that Drosophila and human Inr+DPE promoter classes are not only highly structured, but also similar to each other, particularly in the proximal downstream region. Clustering of the corresponding sequence motifs using a neighbor-joining algorithm strongly suggests that canonical Inr+DPE promoters could be common to metazoan species. Using reporter assays we demonstrate the contribution of the identified downstream positions to the function of multiple human promoters. Furthermore, we show that alteration of the spacing between the Inr and PDP by two nucleotides results in reduced promoter activity, suggesting a spacing dependency of the newly discovered human PDP on the Inr. Taken together, our strategy identified novel functional downstream positions within human core promoters, supporting the existence of DPE-like motifs in human promoters.

Dataset Information

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

Publications

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

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