The BTI1 Azoreductase Colorimetric and Fluorometric Reporter System

Abstract

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).