Expression and characteristics of the gene encoding azoreductase from Rhodobacter sphaeroides AS1.1737

Abstract

A gene that encodes a protein with azoreductase activity was obtained by PCR amplification from Rhodobacter sphaeroides AS1.1737. The enzyme, with a molecular weight of 18.7 kD, was heterologously expressed in Escherichia coli and its azoreductase activity was characterized. Furthermore, the reduction mechanism of azo dyes catalyzed by the azoreductase was studied in detail. The presence of a hydrazo-intermediate was identified, which provided a convincing evidence for the assumption that azo dyes were degraded via an incomplete reduction stage.

Functional Annotation of a Presumed Nitronate Monoxygenase Reveals a New Class of NADH:Quinone Reductases

“…PA1024 displayed a marked preference for NADH over NADPH, as indicated by the fact that flavin reduction with 500 M NADPH is ϳ3,500 times slower than with an equivalent concentration of NADH. Preference for NADH as a substrate was previously reported for some flavin-dependent monooxygenases (16), FMN-dependent quinone reductases in bacteria, such as tryptophan (W) repressor-binding protein (WrbA) (17), and the FMN-dependent azoreductases AzoA (18), AcpD (19), and AzoR (20). In the case of PA1024, the structural determinants for NADH specificity are currently unknown, and future crystallographic studies of the enzyme in complex with the product NAD ϩ will be required for their elucidation.…”

Reaction mechanism of azoreductases suggests convergent evolution with quinone oxidoreductases

“…The sequences for paAzoR1, 2 and 3 are azoreductases from P. aeruginosa. ecAzoR, rsAzoR, stAzoR and efAzoR are azoreductases from E. coli (Nakanishi et al, 2001), R. sphaeroides (Bin et al, 2004), S. typhimurium (Zhang et al, 2004) and E. faecalis (Chen et al, 2004). paWrbA is the sequence for WrbA from P. aeruginosa (Gorman and Shapiro, 2005), KefF is an enzyme from E. coli (Roosild et al, 2009), ecMdaB and paMdaB are the mediator of drug activity B from E. coli (Adams and Jia, 2006) and P. aeruginosa respectively, YhdA is an enzyme from B. subtilis (Binter et al, 2009), EmoB is an enzyme fromMesorhizobiums p. BNC1 (Nissen et al, 2008), ArsH is an enzyme from S. meliloti (Ye et al, 2007), Lot6p is an enzyme from S. cerevisiae (Liger et al, 2004), ppChrR is the chromate reductase from P. putida (Ackerley et al, 2004).…”

Expression and Characterization of the Genes Encoding Azoreductases fromBacillus subtilisandGeobacillus stearothermophilus

“…11) Moreover, a novel azoreductase gene of Xenophilus azovorans was cloned and characterized as carrying a -NAD(P)H dependent reductase motif by Blumel et al, 12) followed by reports on cloning and expression of azoreductase genes from Pigmentiphaga kullae, 13) Enterococcus faecalis, 14) Rhodobacter sphaeroides, 15) and Staphylococcus aureus 16) with similar characteristics. Additionally, crystal structure analysis of B. subtilis hypothetical protein YhdA, predicted as an azobenzen reductase has been performed and the results were presented as 1NNI 23) in the Protein Data Bank of Research Collaboratory for Structural Bioinformatics (RCSB).…”

“…Pigmentiphaga kullae, 13) Enterococcus faecalis, 14) Rhodobacter sphaeroides, 15) and Staphylococcus aureus. 16) Although these enzymes reduced some kinds of azo dyes, some dyes were not degraded efficiently.…”