Characterisation of the flavin-free oxygen-tolerant azoreductase from Xenophilus azovorans KF46F in comparison to flavin-containing azoreductases.
The flavin-free azoreductase from Xenophilus azovorans KF46F (AzoB), which has been the very first characterized oxygen-tolerant azoreductase, was analyzed in comparison to various recently described flavin-containing azoreductases from different bacterial sources. Sequence comparisons demonstrated that the azoreductase from X. azovorans KF46F is a member of the NmrA family of proteins and demonstrates 30% sequence identity with a NADPH-dependent quinone oxidoreductase from Escherichia coli (encoded by ytfG). In contrast, it was found that the flavin-containing azoreductases from E. coli OY1-2 (AZR), Bacillus sp. OY1-2 (AZR) and related azoreductases all belong to the FMN_red superfamily of enzymes. The substrate specificity of AzoB was reanalyzed in respect to the recently characterized flavin-containing azoreductases, and it was found that purified AzoB converted in addition to different ortho-hydroxy azo compounds [such as Orange II = 1-(4'-sulfophenylazo)-2-naphthol] also the simple non-hydroxylated non-sulfonated azo dye Methyl Red (4'-dimethylaminoazobenzene-2-carboxylic acid), but no indications for the conversion of quinones were obtained. Significant differences were observed in the substrate specificities between AzoB and the flavin-containing azoreductases. The kinetic analysis of the turn-over of Orange II by AzoB suggested an ordered bireactant reaction mechanism which was different from the ping-pong mechanism suggested for the flavin-containing azoreductases.
Bürger
Sibylle
S
Institut für Mikrobiologie, Universität Stuttgart, Germany.
Stolz
Andreas
A
D000595:Amino Acid Sequence; D001419:Bacteria / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D042621:Comamonadaceae / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D005415:Flavins / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009601:Nitroreductases; D010100:Oxygen / Q000378:metabolism*; D016415:Sequence Alignment; D013379:Substrate Specificity
D001426:Bacterial Proteins; D005415:Flavins; D009247:NADH, NADPH Oxidoreductases; D009601:Nitroreductases; C021106:azoreductase; D010100:Oxygen