A novel mechanism for azoreduction.
Azoreductases are important due to their ability to activate anti-inflammatory azo pro-drugs and to detoxify azo dyes. Three genes encoding azoreductases have been identified in Pseudomonas aeruginosa. We describe here a comparison of the three enzymes. The pure recombinant proteins each have a distinct substrate specificity profile against a range of azo substrates. Using the structure of P. aeruginosa azoreductase (paAzoR) 1 and the homology models of paAzoR2 and paAzoR3, we have identified residues important for substrate specificity. We have defined a novel flavin mononucleotide binding cradle, which is a recurrent motif in many flavodoxin-like proteins. A novel structure of paAzoR1 with the azo pro-drug balsalazide bound within the active site was determined by X-ray crystallography and demonstrates that the substrate is present in a hydrazone tautomer conformation. We propose that the structure with balsalazide bound represents an enzyme intermediate and, together with the flavin mononucleotide binding cradle, we propose a novel catalytic mechanism.
Ryan
Ali
A
Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, UK.
Laurieri
Nicola
N
Westwood
Isaac
I
Wang
Chan-Ju
CJ
Lowe
Edward
E
Sim
Edith
E
D000595:Amino Acid Sequence; D001391:Azo Compounds / Q000737:chemistry*; D001665:Binding Sites; D018360:Crystallography, X-Ray; D005486:Flavin Mononucleotide / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009601:Nitroreductases; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000201:enzymology; D016415:Sequence Alignment; D013379:Substrate Specificity
D001391:Azo Compounds; D005486:Flavin Mononucleotide; D009247:NADH, NADPH Oxidoreductases; D009601:Nitroreductases; C021106:azoreductase