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

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

doi:10.3389/fbioe.2021.647176

Gamal Nasser Abdel-Hady, Hisakage Funabashi + Show 4 more

Open Access

https://doi.org/10.3389/fbioe.2021.647176

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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 RsPtxDHARRA for NADP (Kcat/KM)NADP was 44.1 μM–1 min–1, which was the highest among the previously reported phosphite dehydrogenases. Moreover, the RsPtxDHARRA 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 RsPtxDHARRA 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 RsPtxDHARRA mutant might be a promising alternative NADPH regeneration system for practical applications.

Highlights

Biocatalysts play a crucial role in the chemical, pharmaceutical, and energy industries (Bornscheuer et al, 2012; Straathof, 2014; Wang et al, 2017)
The specificity of a nicotinamide cofactor to dehydrogenase is influenced by the amino acids after the second β-strand because this position engages with the 2 -hydroxyl of adenine ribose where the phosphate group is present in Nicotinamide adenine dinucleotide phosphate (NADP) (Carugo and Argos, 1997)
The replacements of the amino acids at this position with basic amino acids in several NAD-dependent dehydrogenases resulted in increased affinity toward NADP, whereas replacing with acidic amino acids resulted in increased affinity for NAD (Chánique and Parra, 2018)

Summary

Introduction

Biocatalysts play a crucial role in the chemical, pharmaceutical, and energy industries (Bornscheuer et al, 2012; Straathof, 2014; Wang et al, 2017). Over the last few decades, the use of enzymes in various biotechnology-related fields has drastically increased and is growing continuously (Wandrey et al, 2000; Schmid et al, 2001). Their catalytic performance in benign reactions along. Dehydrogenases, a family of enzymes catalyzing a wide range of industrially useful reactions such as chiral conversions of pharmaceutical building blocks, require nicotinamide cofactors, NAD(H) or NADP(H), which precludes their large-scale use in stoichiometric amounts (Hummel and Kula, 1989; Krix et al, 1997)

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