Rational Engineering of Formate Dehydrogenase Substrate/Cofactor Affinity for Better Performance in NADPH Regeneration
- PDF / 1,004,585 Bytes
- 14 Pages / 439.37 x 666.142 pts Page_size
- 84 Downloads / 167 Views
Rational Engineering of Formate Dehydrogenase Substrate/Cofactor Affinity for Better Performance in NADPH Regeneration He-Wen Jiang 1 & Qi Chen 1 & Jiang Pan 1 & Gao-Wei Zheng 1 & Jian-He Xu 1 Received: 7 February 2020 / Accepted: 23 April 2020/ # Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
Formate dehydrogenases are critical tools for nicotinamide cofactor regeneration, but their limited catalytic efficiency (kcat/Km) is a major drawback. A formate dehydrogenase from Burkholderia stabilis 15516 (BstFDH) was the first native NADP+-dependent formate dehydrogenase reported and has the highest kcat/Km toward NADP+ (kcat/ KmNADP+) compared with other FDHs that can utilize NADP+ as a hydrogen acceptor. However, the substrate and cofactor affinities of BstFDH are inferior to those of other FDHs, making its practical application difficult. Herein, we engineered recombinant BstFDH to enhance its HCOO− and NADP+ affinities. Based on sequence information analysis and homologous modeling results, I124, G146, S262, and A287 were found to affect the binding affinity for HCOO− and NADP+. By combining these mutations, we identified a BstFDH variant (G146M/A287G) that reduced KmNADP+ to 0.09 mM, with a concomitant decrease in KmHCOO−, and gave 1.6-fold higher kcat/KmNADP+ than the wild type (WT). Furthermore, BstFDH I124V/G146H/A287G, with the lowest KmHCOO− of 8.51 mM, showed a catalytic efficiency that was 2.3-fold higher than that of the wild type and a decreased KmNADP+ of 0.11 mM. These results are beneficial for improving the performance of NADP+-dependent formate dehydrogenase in the NADPH regeneration of various bioreductive reactions and provide a useful guide for engineering of the substrate and cofactor affinity of other enzymes. Keywords Binding affinity . Burkholderia stabilis . Formate dehydrogenase . NADPH regeneration . Rational engineering
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12010-02003317-7) contains supplementary material, which is available to authorized users.
* Gao-Wei Zheng [email protected] * Jian-He Xu [email protected] Extended author information available on the last page of the article
Applied Biochemistry and Biotechnology
Introduction Oxidoreductases that use NAD(P)H as cofactor play an indispensable role in industrial biocatalysis and biotransformation [1–4]. Owing to the consumption of expensive cofactors, the in situ regeneration method used is vital for industrial applications [5, 6]. One general method involves coupling with a second oxidoreductase to recycle the cofactor at the expense of a cheap cosubstrate. Several enzymes, such as glucose dehydrogenase (GDH), formate dehydrogenase (FDH), phosphite dehydrogenase (PTDH), alcohol dehydrogenase (ADH), and glucose-6-phosphate dehydrogenase (G6PDH) can be used as auxiliary tools to regenerate NAD(P)H in oxidoreductase-catalyzed reactions [7, 8]. Currently, GDH is the most widely used tool enzyme owing to its high activity and low glucose pri
Data Loading...
