Department of Chemical Engineering, Columbia University, New York, NY 10027, USA.
Protein Eng Des Sel. 2023 Jan 21;36. doi: 10.1093/protein/gzad009.
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.
氧化还原酶催化重要的氧化还原反应，许多酶需要可扩散的辅酶来进行电子传递，例如 NAD(H)。非经典辅酶类似物已被探索用于创造与现有代谢正交运作的酶反应。在这里，我们旨在对 Candida boidinii（CbFDH）的甲酸脱氢酶进行工程改造，使其能够与非经典辅酶烟酰胺腺嘌呤二核苷酸 3'-磷酸（3'-NADP(H)）发生作用。我们使用 PyRosetta、辅酶特异性反转结构分析和文库设计（CSR-SALAD）以及结构指导饱和突变技术来鉴定使 CbFDH 能够使用 3'-NADP+的突变。两个单突变体 D195A 和 D195G 对 3'-NADP+的活性最高，而双突变体 D195G/Y196S 则表现出最高的辅酶选择性反转行为。进行了稳态动力学分析；D195A 突变体对 3'-NADP+的 KTS 值最高。这项工作比较了计算方法在辅酶特异性工程中的应用，同时展示了对新型非经典辅酶选择性的重要酶的工程改造。
