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

Oxidoreductases catalyze critical essential redox reactions, and many of these enzymes require a diffusible secondary substrate, or cofactor, for electron transport. Nicotinamide adenine dinucleotide NAD(H) and nicotinamide adenine dinucleotide phosphate (NADP(H)) differ by a 2'-phosphate group and their inability to cross-react allows these cofactor pools to be used by a number of cellular processes. Non-canonical cofactor analogs have been explored as a means to create enzymatic reactions that can operate orthogonally to existing metabolic networks. Here, we aim 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+ for the oxidation of formate. Two single mutants, D195A and D195G, had the highest activity with 3'-NADP(H), while the double mutant D195G/Y196S exhibited the highest cofactor selectivity reversal behavior. Steady state kinetic analyses were performed to explore the overall impact of the mutations on the apparent kinetic parameters for the different cofactors, and the D195A mutant exhibited the highest KTS value with 3'-NADP(H) as a cofactor. This work compares the utility of computational approaches available for cofactor specificity engineering while demonstrating the engineering of an important enzyme for novel non-canonical cofactor selectivity.© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Citation

Salomon Vainstein, Scott Banta. Engineering Candida boidinii formate dehydrogenase for activity with the non-canonical cofactor 3'-NADP(H). Protein engineering, design & selection : PEDS. 2023 Sep 02

PMID: 37658768

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