Molecular cloning, overexpression, purification, and characterization of an aerobic FMN-dependent azoreductase from Enterococcus faecalis

Abstract

Azo dyes represent a major class of synthetic colorants that are ubiquitous in foods and consumer products. Enterococcus faecalis is a predominant member of the human gastrointestinal microflora. Strain ATCC 19433 grew in the presence of azo dyes and metabolized them to colorless products. A gene encoding a putative FMN-dependent aerobic azoreductase that shares 34% identity with azoreductase (AcpD) of Escherichia coli has been identified in this strain. The gene in E. faecalis, designated as azoA, encoded a protein of 208 amino acids with a calculated isoelectric point of 4.8. AzoA was heterologously overexpressed in E. coli with a strong band of 23 kDa on SDS-PAGE. The purified recombinant enzyme was a homodimer with a molecular weight of 43 kDa, probably containing one molecule of FMN per dimer. AzoA required FMN and NADH, but not NADPH, as a preferred electron donor for its activity. The apparent Km values for both NADH and 2-[4-(dimethylamino)phenylazo]benzoic acid (Methyl red) substrates were 0.14 and 0.024 mM, respectively. The apparent Vmax was 86.2 microM/min/mg protein. The enzyme was not only able to decolorize Methyl red, but was also able to convert sulfonated azo dyes Orange II, Amaranth, Ponceau BS, and Ponceau S. AzoA is the first aerobic azoreductase to be identified and characterized from human intestinal gram-positive bacteria.

Fig. 1

Comparison of the deduced amino acid sequences of E. faecalis AzoA (top) and E. coli acpD (bottom). Amino acid residues with different degrees of conservation (+) are indicated.

Fig. 2

SDS–PAGE of the purified AzoA of E. faecalis expressed in E. coli. Lane 1, protein molecular mass standards; lane 2, crude cell extract (40 μg); and lane 3, the purified AzoA (3 μg).

Fig. 3

Comparison between spectra of E. faecalis AzoA and free FMN. After the final step purification by gel filtration, the absorption spectrum of the purified AzoA was measured. Solid and dotted lines, respectively, show spectra of AzoA at 29.5 μM and 32.5 μM free FMN in 50mM potassium phosphate buffer (pH 7.0) containing 100mM NaCl.

Fig. 4

Structures of azo dyes used for reduction assays and cultures.