A Novel Fluorescent Probe-Based High-Throughput Screening Approach for Nicotinamide Cofactor Biomimetics-Dependent Oxidoreductases

This study demonstrates the successful development of a high-throughput screening platform for nicotinamide cofactor biomimetics (NCBs)-dependent oxidoreductases using a fluorescent probe ((E)-1-methyl-3-(2-(1,3,3-trimethyl-3H-indol-1-ium-2-yl)vinyl)-4,4a-dihydroquinolin-1-ium). By adapting the probe originally designed for NAD(P)H detection, we enabled real-time monitoring of enzymatic reactions involving NCBs like 1-benzyl-3-carbamoylpyridin-1-ium (BNA⁺). The assay exhibited high sensitivity, showing more than a 10-fold improvement in the molar extinction coefficient (72300 M^-1·cm^-1) compared with conventional UV-based activity assays that rely on the characteristic absorption of reduced cofactors (e.g., 6220 M^-1·cm^-1 for NAD(P)H at 340 nm and 7400 M^-1·cm^-1 for BNAH at 360 nm). This enhanced optical response makes the method particularly suitable for low-concentration detection and high-throughput screening applications. Using this fluorescent probe-based screening assay, SpALDH2, an aldehyde dehydrogenase from Sphingobium sp., was engineered for improved activity toward BNA⁺ and its analogs, yielding several mutants with enhanced catalytic efficiency. The optimal mutant A244D/M341S exhibited a 17.6-fold increase in catalytic efficiency compared with the wild type. The method was further validated by HPLC, showing a strong correlation between fluorescence intensity and product yield. Overall, this fluorescent probe-based assay provides a versatile and reliable analytical platform for NCB-related research and applications, including enzyme screening, kinetic analysis, and enzyme engineering.