Engineering Candida boidinii formate dehydrogenase for activity with the non-canonical cofactor 3'-NADP(H)

Oxidoreductases catalyze essential redox reactions, and many require a diffusible cofactor for electron transport, such as NAD(H). Non-canonical cofactor analogs have been explored as a means to create enzymatic reactions that operate orthogonally to existing metabolism. Here, we aimed to engineer the formate dehydrogenase from Candid boidinii (CbFDH) for activity with the non-canonical cofactor nicotinamide adenine dinucleotide 3'-phosphate (3'-NADP(H)). We used PyRosetta, the Cofactor Specificity Reversal Structural Analysis and Library Design (CSR-SALAD), and structure-guided saturation mutagenesis to identify mutations that enable CbFDH to use 3'-NADP+. Two single mutants, D195A and D195G, had the highest activities with 3'-NADP+, while the double mutant D195G/Y196S exhibited the highest cofactor selectivity reversal behavior. Steady state kinetic analyses were performed; the D195A mutant exhibited the highest KTS value with 3'-NADP+. This work compares the utility of computational approaches for cofactor specificity engineering while demonstrating the engineering of an important enzyme for novel non-canonical cofactor selectivity.