Engineering Cofactor Specificity of a Thermostable Phosphite Dehydrogenase for a Highly Efficient and Robust NADPH Regeneration System.

ABSTRACT: Nicotinamide adenine dinucleotide phosphate (NADP)-dependent dehydrogenases catalyze a range of chemical reactions useful for practical applications. However, their dependence on the costly cofactor, NAD(P)H remains a challenge which must be addressed. Here, we engineered a thermotolerant phosphite dehydrogenase from Ralstonia sp. 4506 (RsPtxD) by relaxing the cofactor specificity for a highly efficient and robust NADPH regeneration system. The five amino acid residues, Cys174-Pro178, located at the C-terminus of β7-strand region in the Rossmann-fold domain of RsPtxD, were changed by site-directed mutagenesis, resulting in four mutants with a significantly increased preference for NADP. The catalytic efficiency of mutant RsPtxD_HARRA for NADP (K _cat/K _M)^NADP was 44.1 μM^-1 min^-1, which was the highest among the previously reported phosphite dehydrogenases. Moreover, the RsPtxD_HARRA mutant exhibited high thermostability at 45°C for up to 6 h and high tolerance to organic solvents, when bound with NADP. We also demonstrated the applicability of RsPtxD_HARRA as an NADPH regeneration system in the coupled reaction of chiral conversion of 3-dehydroshikimate to shikimic acid by the thermophilic shikimate dehydrogenase of Thermus thermophilus HB8 at 45°C, which could not be supported by the parent RsPtxD enzyme. Therefore, the RsPtxD_HARRA mutant might be a promising alternative NADPH regeneration system for practical applications.

SUBMITTER: Abdel-Hady GN 

PROVIDER: S-EPMC8047080 | biostudies-literature |

REPOSITORIES: biostudies-literature