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Rational Engineering of Formate Dehydrogenase Substrate/Cofactor Affinity for Better Performance in NADPH Regeneration

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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.

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Acknowledgments

We thank Simon Partridge, Ph.D., from Liwen Bianji, Edanz Editing China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.

Funding

This work was financially supported by the National Natural Science Foundation of China (Nos. 21536004, 21871085, and 21878085), the National Key Research and Development Program of China (No. 2018YFC1706200), and the Fundamental Research Funds for the Central Universities (No. 22221818014).

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  1. State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing, East China University of Science and Technology, Shanghai, 200237, People’s Republic of China

    He-Wen Jiang, Qi Chen, Jiang Pan, Gao-Wei Zheng & Jian-He Xu

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  1. He-Wen Jiang
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  2. Qi Chen
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  3. Jiang Pan
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Correspondence to Gao-Wei Zheng or Jian-He Xu.

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Jiang, HW., Chen, Q., Pan, J. et al. Rational Engineering of Formate Dehydrogenase Substrate/Cofactor Affinity for Better Performance in NADPH Regeneration. Appl Biochem Biotechnol 192, 530–543 (2020). https://doi.org/10.1007/s12010-020-03317-7

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