Engineering Candida boidinii formate dehydrogenase for activity with the non-canonical cofactor 3′-NADP(H)
Abstract
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.
Document Details
- Document Type
- Pub Defense Publication
- Publication Date
- Jan 01, 2023
- Source ID
- 10.1093/protein/gzad009
Entities
People
- Salomon Vainstein
- Scott Banta
Organizations
- Army Research Office
- Columbia University
- United States Department of Defense