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Volume 151, Issue 5

Biochemical and molecular characterization of an azoreductase from , a tetrameric NADPH-dependent flavoprotein Free

Abstract

Azo dyes are a predominant class of colourants used in tattooing, cosmetics, foods and consumer products. A gene encoding NADPH-flavin azoreductase (Azo1) from the skin bacterium ATCC 25923 was identified and overexpressed in . RT-PCR results demonstrated that the gene was constitutively expressed at the mRNA level in . Azo1 was found to be a tetramer with a native molecular mass of 85 kDa containing four non-covalently bound FMN. Azo1 requires NADPH, but not NADH, as an electron donor for its activity. The enzyme was resolved to dimeric apoprotein by removing the flavin prosthetic groups using hydrophobic-interaction chromatography. The dimeric apoprotein was reconstituted on-column and in free stage with FMN, resulting in the formation of a fully functional native-like tetrameric enzyme. The enzyme cleaved the model azo dye 2-[4-(dimethylamino)phenylazo]benzoic acid (Methyl Red) into ,-dimethyl--phenylenediamine and 2-aminobenzoic acid. The apparent values for NADPH and Methyl Red substrates were 0·074 and 0·057 mM, respectively. The apparent was 0·4 μM min (mg protein). Azo1 was also able to metabolize Orange II, Amaranth, Ponceau BS and Ponceau S azo dyes. Azo1 represents the first azoreductase to be identified and characterized from human skin microflora.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Keyword(s): Azo1, NADPH-flavin azoreductase
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2005-05-01
2024-04-19
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References

  1. Aliverti A., Curti B., Vanoni M. A. 1999; Identifying and quantitating FAD and FMN in simple and in iron-sulfur-containing flavoproteins. Methods Mol Biol 131:9–23
     [Google Scholar]
  2. Baumler W., Eibler E. T., Hohenleutner U., Sens B., Sauer J., Landthaler M. 2000; Q-switched laser and tattoo pigment: first results of the chemical and photophysical analysis of 41 compounds. Lasers Surg Med 26:13–21 [CrossRef]
     [Google Scholar]
  3. Ben-Bassat A., Bauer K., Chang S. Y., Myambo K., Boosman A., Chang S. 1987; Processing of the initiation methionine from proteins: properties of the Escherichia coli methionine aminopeptidase and its gene structure. J Bacteriol 169:751–757
     [Google Scholar]
  4. Blumel S., Stolz A. 2003; Cloning and characterization of the gene coding for the aerobic azoreductase from Pigmentiphaga kullae K24. Appl Microbiol Biotechnol 62:186–190 [CrossRef]
     [Google Scholar]
  5. Blumel S., Knackmuss H.-J., Stolz A. 2002; Molecular cloning and characterization of the gene coding for the aerobic azoreductase from Xenophilus azovorans KF46F. Appl Environ Microbiol 68:3948–3955 [CrossRef]
     [Google Scholar]
  6. Chapes S. K., Baharka A. A., Hart M. E., Smeltzer M. S., Iandolo J. J. 1994; Differential RNA regulation by staphylococcal enterotoxins A and B in murine macrophages. J Leukocyte Biol 55:523–529
     [Google Scholar]
  7. Chen H., Wang R. F., Cerniglia C. E. 2004; Molecular cloning, overexpression, purification, and characterization of an aerobic FMN-dependent azoreductase from Enterococcus faecalis. Protein Expr Purif 34:302–310 [CrossRef]
     [Google Scholar]
  8. Chung K.-T. 1983; The significance of azo-reduction in the mutagenesis and carcinogenesis of azo dyes. Mutat Res 114:269–281 [CrossRef]
     [Google Scholar]
  9. Chung K.-T., Stevens S. E., Cerniglia C. E. Jr 1992; The production of azo dyes by the intestinal microflora. Crit Rev Microbiol 18:175–190 [CrossRef]
     [Google Scholar]
  10. Combes R. D., Haveland-Smith R. B. 1982; A review of the genotoxicity of food, drug and cosmetic colours and other azo, triphenylmethane and xanthene dyes. Mutat Res 98:101–248 [CrossRef]
     [Google Scholar]
  11. Hefti M. H., Vervoort J., van Berkel W. J. H. 2003; Deflavination and reconstitution of flavoproteins: tacking fold and function. Eur J Biochem 270:4227–4242 [CrossRef]
     [Google Scholar]
  12. Kloos W. E., Schleifer K. H. 1975; Isolation and characterization of staphylococci from human skin: II. Description of four new species; Staphylococcus warneri, Staphylococcus capitis, Staphylococcus hominis, and Staphylococcus simulans . Int J Syst Bacteriol 25:62–79 [CrossRef]
     [Google Scholar]
  13. Kuroda M., Ohta T., Uchiyama I. & 34 other authors; 2001; Whole genome sequencing of meticillin-resistant Staphylococcus aureus. Lancet 357:1225–1240 [CrossRef]
     [Google Scholar]
  14. Lake B. D., Goodwin H. J. 1976; Lipids. In Chromatographic and Electrophoretic Techniques 1 pp 345–366 Edited by Smith I., Seakin J. M. T. Bath, UK: Pitman Press;
     [Google Scholar]
  15. Levine W. G. 1991; Metabolism of azo dyes: implication for detoxification and activation. Drug Metab Rev 23:253–309 [CrossRef]
     [Google Scholar]
  16. Liger D., Graille M., Zhou C. Z., Leulliot N., Quevillon-Cheruel S., Blondeau K., Janin J, van Tilbeurgh H. 2004; Crystal structure and functional characterization of yeast YLR011wp, an enzyme with NAD(P)H-FMN and ferric iron reductase activities. J Biol Chem 279:34890–34897 [CrossRef]
     [Google Scholar]
  17. Mortl M., Diederichs K., Welte W., Molla G., Motteran L., Andriolo G., Pilone M. S., Pollegioni L. 2004; Structure-function correlation in glycine oxidase from Bacillus subtilis. J Biol Chem 279:29718–29727 [CrossRef]
     [Google Scholar]
  18. Moutaouakkil A., Zeroual Y., Zzayri F. Z., Talbi M., Lee K., Blaghen M. 2003; Purification and partial characterization of azoreductase from Enterobacter agglomerans . Arch Biochem Biophys 413:139–146 [CrossRef]
     [Google Scholar]
  19. Nagase N., Sasaki A., Yamashita K., Shimizu A., Wakita Y., Kitai S., Kawano J. 2002; Isolation and species distribution of Staphyloccocci from animal and human skin. J Vet Med Sci 64:245–250 [CrossRef]
     [Google Scholar]
  20. Nakanishi M., Yatome C., Ishida N., Kitade Y. 2001; Putative ACP phosphodiesterase gene (acpD) encodes an azoreductase. J Biol Chem 276:46394–46399 [CrossRef]
     [Google Scholar]
  21. Nakayama T., Kimura T., Kodama M., Nagata C. 1983; Generation of hydrogen peroxide and superoxide anion from active metabolites of naphthylamines and aminoazo dyes: its possible role in carcinogenesis. Carcinogenesis 4:765–769 [CrossRef]
     [Google Scholar]
  22. Platzek T., Lang C., Grohmann G., Gi U.-S., Baltes W. 1999; Formation of a carcinogenic aromatic amine from an azo dye by human skin bacteria. Hum Exp Toxicol 18:552–559 [CrossRef]
     [Google Scholar]
  23. Suzuki Y., Yoda T., Ruhul A., Sugiura W. 2001; Molecular cloning and characterization of the gene coding for azoreductase from Bacillus sp. OY1-1 isolated from soil. J Biol Chem 276:9059–9065 [CrossRef]
     [Google Scholar]
  24. Van Berkel W. J., van den Berg W. A., Muller F. 1988; Large-scale preparation and reconstitution of apo-flavoproteins with special reference to butyryl-CoA dehydrogenase from Megasphaera elsdenii . Hydrophobic-interaction chromatography. Eur J Biochem 178:197–207 [CrossRef]
     [Google Scholar]
  25. Von Lehmann G., Pierchalla P. 1988; Tatowierungsfarbstoffe. Derm Beruf Umwelt 36:152–156
     [Google Scholar]
  26. Wang R. F., Kim S.-J., Robertson L. H., Cerniglia C. E. 2002; Development of a membrane-array method for the detection of human intestinal bacteria in fecal samples. Mol Cell Probes 16:341–350 [CrossRef]
     [Google Scholar]
  27. Wong P. K., Yuen P. Y. 1998; Decolourization and biodegradation of N,N′-dimethyl-p-phenylenediamine by Klebsiella pneumoniae RS-13 and Acetobacter liquefaciens S-1. J Appl Microbiol 85:79–87 [CrossRef]
     [Google Scholar]
  28. Yamashita S., Hemmi H., Ikeda Y., Nakayama T., Nishino T. 2004; Type 2 isopentenyl diphosphate isomerase from a thermoacidophilic archaeon Sulfolobus shibatae. Eur J Biochem 271:1087–1093 [CrossRef]
     [Google Scholar]
  29. Yan B., Zhou J., Wang J., Du C., Hou H., Song Z., Bao Y. 2004; Expression and characteristics of the gene encoding azoreductase from Rhodobacter sphaeroides AS1.1737. FEMS Microbiol Lett 236:129–136 [CrossRef]
     [Google Scholar]
  30. Zimmermann T., Kulla H. G., Leisinger T. 1982; Properties of purified Orange II azoreductase, the enzyme initiating azo dye degradation by Pseudomonas KF46. Eur J Biochem 129:197–203 [CrossRef]
     [Google Scholar]
  31. Zimmermann T., Gasser F., Kulla H. G., Leisinger T. 1984; Comparison of two bacterial azoreductases acquired during adaptation to growth on azo dyes. Arch Microbiol 138:37–43 [CrossRef]
     [Google Scholar]
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Biochemical and molecular characterization of an azoreductase from Staphylococcus aureus, a tetrameric NADPH-dependent flavoprotein
Microbiology 151, 1433 (2005); https://doi.org/10.1099/mic.0.27805-0
/content/journal/micro/10.1099/mic.0.27805-0
/content/journal/micro/10.1099/mic.0.27805-0
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