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Related Experiment Videos

Towards a new interaction enzyme:coenzyme.

Marta Martínez-Júlvez1, Jesús Tejero, José R Peregrina

  • 1Departamento de Bioquímica y Biología Molecular y Celular and Institute of Biocomputation and Physics of Complex Systems (BiFi), Facultad de Ciencias, Universidad de Zaragoza, 50009 Zaragoza, Spain.

Biophysical Chemistry
|March 9, 2005
PubMed
Summary

Mutating Anabaena ferredoxin-NADP(+) reductase (FNR) altered its enzyme specificity. The modified FNR enzyme can no longer distinguish between nicotinamide adenine dinucleotide phosphate (NADP(+))/H and nicotinamide adenine dinucleotide (NAD(+))/H.

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Area of Science:

  • Biochemistry
  • Enzymology
  • Protein Engineering

Background:

  • Ferredoxin-NADP(+) reductase (FNR) is a key enzyme in photosynthesis, catalyzing NADP(+) reduction.
  • Understanding coenzyme specificity is crucial for enzyme function and engineering.

Purpose of the Study:

  • To investigate coenzyme specificity determinants in Anabaena FNR.
  • To engineer FNR for altered or reversed coenzyme specificity.

Main Methods:

  • Site-directed mutagenesis was employed to introduce mutations in the NADP(+)/H pyrophosphate binding and C-terminal regions of Anabaena FNR.
  • Characterization of the mutated enzyme's kinetic parameters (kcat, Km) for different coenzymes (NADPH, NADH).

Main Results:

  • A quadruple mutant (T155G/A160T/L263P/Y303S) showed significantly altered coenzyme specificity.

Related Experiment Videos

  • The mutated enzyme exhibited similar catalytic efficiency (kcat) for both NADPH and NADH, unlike the wild-type (WT).
  • The Michaelis constant (Km) for NADH decreased 20-fold, while Km for NADPH remained similar, drastically reducing specificity for NADPH over NADH.

    • Conclusions:

    • Simultaneous mutations in key regions can effectively reverse the coenzyme specificity of Anabaena FNR.
    • The study provides insights into the structural basis of coenzyme recognition and discrimination in FNR.
    • Further analysis is needed to fully elucidate the role of C-terminal residues in FAD binding energetics.