The group II azoreductase BTI1 utilizes NADPH to directly cleave azo bonds in water-soluble azo dyes, including quenchers of fluorescence. Unexpectedly, optimal reduction was dye specific, ranging from a pH of <5.5 for Janus green B, to pH 6.0 for methyl red, methyl orange, and BHQ-10, to pH >8.3 for flame orange.Azo dyes are vivid colorants that consist of aromatic rings connected by one or more azo bonds. Thanks to the lack of native fluorescence, the DAB(C/S)YL and Black Hole Quencher (BHQ) azo dyes can be used as true dark quenchers of fluorescence in genetic assays, such as real-time quantitative PCR (5). Three groups of azoreductases (EC 1.7.1.6) cleave azo dyes into their colorless aromatic amines. Group II flavodoxin-related enzymes have been described from Bacillus sp. strain OY1-2, Bacillus subtilis, and others. They exist in a dimer-tetramer equilibrium, utilize flavin mononucleotide (FMN) as a noncovalently bound cofactor, and spend NADPH for direct reduction (1, 2, 4, 10, 11, 13). To date, azoreductase substrate specificities have been assessed mostly around physiological pH, which possibly underestimates optimal reactivity. Data on the pH-dependent reduction by the group II BTI1 azoreductase (Biosearch Technologies Inc.) of water-soluble azo dye quenchers are here presented.Enzyme. The cloning of BTI1, based on the Bacillus OY1-2 azoreductase (11), as well as expression and verification of the functionalized BTI10 variant used throughout this study as a tetrameric FMN-containing azoreductase is described in the supplemental material.Azoreductase activity measurements. Potential substrates (Fig. 1) in fresh double-distilled water (ddH 2 O) at 1.0 or 0.10 mM were kept at ϩ4°C until used at 20 to 40 M for substrate susceptibility testing and at 1.0 to 20 M for kinetics measurements. Stock solutions of ␤-NADH and ␤-NADPH (5 mM) in ddH 2 O were prepared fresh weekly, stored at Ϫ20°C (12), and used at 200 M. Reduction was carried out in 200-l reaction mixtures in 96-well plates, with 0.5 to 2 g of enzyme for substrate susceptibility testing and 10.0 ng to 1.0 g of enzyme for kinetics measurements. Reaction rates were calculated by fitting the initial part of the absorbance decay by linear regression to find the slope. Extinction coefficients and absorbance maxima ( max ) are in Table 1. All experiments were repeated at least three times, with similar results. The data were processed and plotted and kinetic parameters calculated by using Origin 6.0 (OriginLab, Corp., Northampton, MA).Reaction conditions. The initial reduction by BTI10 of the test dye methyl orange (MeO) in 20 mM sodium phosphate (pH 7.0) buffer, 250 M ␤-NADPH at room temperature, and a 20 M concentration of the substrate (11) was slow (0.80 mol ⅐ min Ϫ1 ⅐ mg Ϫ1 protein) and failed to reach completion. Addition of more NADPH, but not NADH, extended the reaction but did not affect the rate, verifying hydride donor depletion (4, 7). Upon cessation of reduction, the absorbance increased, suggesting azo bond reformation from a short-lived hydr...

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Reliable duplex and multiplex assays remain a challenge for current fluorescence reporter enzyme technology. To this end we have developed a system that takes advantage of pro‐fluorescent probes and a novel azoreductase.The BTI1 azoreductase gene was cloned from a soil sample, optimized and used to bacterially express the protein. Biochemical characterization of the purified enzyme in solution revealed a tetramer carrying a non‐covalently bound flavin mononucleotide cofactor. BTI1 readily reduced an array of carboxylated and sulfonated azo dyes including methyl orange (DABSYL) and the Black Hole Quencher (BHQ(tm)) dyes. Enzyme kinetic parameters of the four‐hydride transfer reaction were consistent with the bi‐bi ping‐pong mechanism of related oxidoreductases. In our optimized colorimetric assay, the detection limit of the BTI1 azoreductase was >10 fold lower than that of beta‐galactosidase.Reduction of the azo‐quenchers in pro‐fluorescent substrates containing BHQs and a range of fluorophores readily released fluorescence in an NADPH‐dependent manner with robust signal to noise ratios.In short, by matching BTI azoreductase with pro‐fluorescent substrates we have created a simple, yet adaptable, reporter system that generates diverse fluorescence signals.This research was supported in part by a grant from NIH to HEJ (R43/R44 GM076843).