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Oligopeptide Competition Assay for Phosphorylation Site Determination
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Oligopeptide Competition Assay for Phosphorylation Site Determination

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A single evolutionarily divergent mutation determines the different FAD-binding affinities of human and rat NQO1 due

Juan Luis Pacheco-Garcia1, Dmitry Loginov2, Bruno Rizzuti3,4

  • 1Departamento de Química Física, Facultad de Ciencias, Universidad de Granada, Spain.

FEBS Letters
|November 24, 2021
PubMed

Insights

Neutral mutations near a cancer-related enzyme

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • The cancer-associated enzyme human NADP(H):quinone oxidoreductase 1 (hNQO1) is FAD-dependent.
  • A phosphomimetic mutation (S82D) in hNQO1 reduces flavin-adenine dinucleotide (FAD)-binding affinity and intracellular stability.

Purpose of the Study:

  • To investigate if the neutral mutation R80H, located near S82, influences the functional impact of S82 phosphorylation via electrostatic interactions.
  • To understand how evolutionary divergence in mutations affects mammalian orthologue responses to phosphorylation.

Main Methods:

  • Biophysical analyses
  • Bioinformatic analyses
  • Site-directed mutagenesis (S82D, R80H, H80R)

Main Results:

  • The reverse mutation H80R counteracted the effects of S82D phosphorylation on hNQO1 by altering local stability.
  • Rat NQO1 (rNQO1), possessing R80, exhibited milder phosphorylation effects compared to humanized rNQO1 (R80H).

Conclusions:

  • Evolutionarily divergent, seemingly neutral mutations can significantly modulate the functional consequences of phosphorylation in mammalian enzyme orthologues.
  • Electrostatic interactions and local stability play crucial roles in mediating the effects of mutations on enzyme function.
Keywords:
epistasisflavoproteinmolecular evolutionprotein phosphorylation

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