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The energy released from the breakdown of the chemical bonds within nutrients can be stored either through the reduction of electron carriers or in the bonds of adenosine triphosphate (ATP). In living systems, a small class of compounds functions as mobile electron carriers, molecules that bind to and shuttle high-energy electrons between compounds in pathways. The principal electron carriers that will be considered originate from the B vitamin group and are derivatives of nucleotides; they are...
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Updated: Jul 23, 2025

Protein Film Infrared Electrochemistry Demonstrated for Study of H2 Oxidation by a [NiFe] Hydrogenase
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Engineering a Formate Dehydrogenase for NADPH Regeneration.

Wei Ma1, Qiang Geng1, Cheng Chen1

  • 1State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Centre for Biomanufacturing, East China University of Science and Technology, Meilong Road 130, Shanghai, 200237, China.

Chembiochem : a European Journal of Chemical Biology
|July 16, 2023
PubMed
Summary

Engineered formate dehydrogenase (FDH) from Candida dubliniensis efficiently utilizes nicotinamide adenine dinucleotide phosphate (NADPH). This enhanced enzyme shows significant catalytic improvements, enabling robust NADPH regeneration for biocatalysis.

Keywords:
cofactor preferencecofactor regenerationformate dehydrogenaseprotein engineering

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Area of Science:

  • Biocatalysis and Enzyme Engineering
  • Synthetic Biology
  • Green Chemistry

Background:

  • Nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH) are key hydrogen donors in biological redox reactions.
  • Efficient regeneration of NAD(P)H is crucial for many enzymatic biotransformations.
  • Existing formate dehydrogenases (FDHs) often exhibit strict NAD+ dependence and limited reactivity, hindering NADPH regeneration.

Purpose of the Study:

  • To engineer a novel formate dehydrogenase (FDH) capable of efficiently utilizing nicotinamide adenine dinucleotide phosphate (NADP+) for robust NADPH regeneration.
  • To improve the catalytic efficiency and stability of FDHs for application in biocatalytic processes.

Main Methods:

  • Structure-guided rational and semi-rational design approaches were employed to engineer the FDH from Candida dubliniensis (CdFDH).
  • Combinatorial mutagenesis was used to generate variants with improved properties.
  • Characterization of enzyme kinetics and performance in asymmetric oxidative/reductive transformations.

Main Results:

  • A combinatorial mutant, CdFDH-M4, demonstrated a 75-fold increase in catalytic efficiency (kcat/Km) compared to the wild-type enzyme.
  • CdFDH-M4 exhibited no strict NAD+ preference, effectively utilizing NADP+.
  • The engineered enzyme was successfully applied in various asymmetric biocatalytic processes, achieving high cofactor total turnover numbers (TTNs) from 135 to 986.

Conclusions:

  • The engineered CdFDH-M4 represents a significant advancement in NADP+-dependent FDHs.
  • This enzyme provides a robust and efficient system for NADPH regeneration, essential for various NADPH-dependent biocatalytic applications.
  • The findings pave the way for more sustainable and efficient enzymatic synthesis pathways.