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Related Experiment Video

Updated: Jan 29, 2026

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues
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Cofactor Specificity Switch on Peach Glucitol Dehydrogenase.

Matías D Hartman1, Romina I Minen1, Alberto A Iglesias1

  • 1Instituto de Agrobiotecnología del Litoral, UNL, CONICET, FBCB , 3000 Santa Fe , Argentina.

Biochemistry
|February 7, 2019
PubMed
Summary
This summary is machine-generated.

Researchers engineered peach glucitol dehydrogenase (GolDHase) to reverse cofactor specificity. The modified enzyme efficiently uses NADP+ over NAD+, showing potential for metabolic engineering and industrial applications.

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Area of Science:

  • Enzyme engineering
  • Biocatalysis
  • Protein engineering

Background:

  • Oxidoreductases typically exhibit strong cofactor preference for NAD+ or NADP+.
  • Peach glucitol dehydrogenase (GolDHase) shows a 1800-fold higher catalytic efficiency for NAD+ compared to NADP+.

Purpose of the Study:

  • To reverse the cofactor specificity of peach GolDHase.
  • To engineer an enzyme that preferentially utilizes NADP+.
  • To explore applications in metabolic engineering and industrial biocatalysis.

Main Methods:

  • Site-saturation mutagenesis was employed to generate the D216A mutant.
  • Site-directed mutagenesis was used to introduce further modifications, guided by fruit fly NADP-dependent GolDHase.
  • Kinetic data and structural information were combined to guide enzyme modification.

Main Results:

  • The D216A mutant demonstrated dual cofactor utilization (NAD+ and NADP+).
  • The D216A/V217R/D218S triple mutant exhibited a 2-fold higher catalytic efficiency with NADP+ than with NAD+.
  • The engineered enzyme successfully reversed the native cofactor preference.

Conclusions:

  • The engineered triple mutant GolDHase shows reversed cofactor specificity, favoring NADP+.
  • This modified enzyme holds potential for applications in metabolic engineering.
  • The enzyme could be valuable for cofactor recycling in industrial processes.