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

Gamal Nasser Abdel-Hady; Takeshi Ikeda; Takenori Ishida; Hisakage Funabashi; Akio Kuroda; Ryuichi Hirota

doi:10.3389/fbioe.2021.647176

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

Front Bioeng Biotechnol. 2021 Apr 1:9:647176. doi: 10.3389/fbioe.2021.647176. eCollection 2021.

Authors

Gamal Nasser Abdel-Hady^{1

2}, Takeshi Ikeda^{1

3}, Takenori Ishida^{1

3}, Hisakage Funabashi^{1

3}, Akio Kuroda^{1

3}, Ryuichi Hirota^{1

3}

Affiliations

¹ Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Hiroshima, Japan.
² Department of Genetics, Faculty of Agriculture, Minia University, Minia, Egypt.
³ Unit of Biotechnology, Division of Biological and Life Sciences, Graduate School of Integrated Sciences for Life, Hiroshima University, Hiroshima, Japan.

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.

Keywords: cofactor regeneration; nicotinamide adenine dinucleotide phosphate; organic solvent tolerance; phosphite dehydrogenase; thermotolerance.