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

Azo dyes are a predominant class of colourants used in tattooing, cosmetics, foods and consumer products. A gene encoding NADPH-flavin azoreductase (Azol) from the skin bacterium Staphylococcus aureus ATCC 25923 was identified and overexpressed in Escherichia coli. RT-PCR results demonstrated that the azol gene was constitutively expressed at the mRNA level in S. aureus. Azol was found to be a tetramer with a native molecular mass of 85 kDa containing four non-covalently bound FMN. Azol 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 N,N-dimethyl-p-phenylenediamine and 2-aminobenzoic acid. The apparent K-m values for NADPH and Methyl Red substrates were 0-074 and 0(.)057 mM, respectively. The apparent V-max was 0(.)4 mu M min(-1) (mg protein)(-1). Azol was also able to metabolize Orange 11, Amaranth, Ponceau BS and Ponceau S azo dyes. Azol represents the first azoreductase to be identified and characterized from human skin microflora.