A high-sensitivity strategy to screen NAD(P)H-dependent reductase activity by coupled enzyme cascade

Enzymes play a pivotal role in "green chemistry" as tools for biocatalysis. Oxidoreductase enzymes are especially useful for carrying out key electron transfer (redox) steps toward a wide range of chemical transformations (e.g., asymmetric hydrogenation, oxygenation, hydroxylation, epoxidation, or Baeyer-Villiger oxidation) that might not otherwise be available to chemists through conventional (nonbiological) synthetic approaches. The ability to screen oxidoreductase activity is important in identifying useful biocatalysts from nature, and also toward engineering novel ones through directed evolution. Many valuable redox enzymes are dependent upon NAD(P)H as an electron-donating co-substrate (or conversely, upon NAD(P)⁺ as an electron acceptor), and the common method to detect their activity is to monitor the change in absorbance at 340 nm as NAD(P)H is converted to NAD(P)⁺ (or vice versa). The limited sensitivity of this method presents a challenge in detecting very low levels of oxidoreductase activity, and this can prove very difficult to begin engineering enzymes as improved biocatalysts when the rates of natural enzymes may be slow for a desired redox reaction. Herein, we report a fluorescence-based, enzyme cascade-coupled system that we have developed to detect NAD(P)H-dependent enzyme-catalyzed reduction with orders of magnitude more sensitivity than conventional absorbance-based assays. While recycling NAD(P)H from NAD(P)⁺, the coupled enzyme cascade triggers cleavage of a fluorogenically labeled probe, releasing a strong fluorescent signal. This allows detection of very low levels of a specific oxidoreductase activity that 1 may wish to magnify by directed evolution using our assay in high-throughput screening.