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Related Experiment Videos

Putidaredoxin reductase, a new function for an old protein.

Irina F Sevrioukova1, Thomas L Poulos

  • 1Department of Molecular Biology and the Program in Macromolecular Structure, University of California, Irvine 92612-3900, USA. sevrioui@uci.edu

The Journal of Biological Chemistry
|May 16, 2002
PubMed
Summary
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Recombinant putidaredoxin reductase (Pdr) exhibits novel NAD(H)-dependent dithiol/disulfide oxidoreductase activity, distinct from its electron transfer role. This function is influenced by C-terminal modifications and thiol groups.

Area of Science:

  • Biochemistry
  • Enzymology
  • Protein Chemistry

Background:

  • Putidaredoxin reductase (Pdr) is a FAD-containing enzyme crucial for the Pseudomonas putida cytochrome P450cam monooxygenase system.
  • Recombinant wild-type (WT) and six-histidine tag-fused (His(6)) Pdr were investigated to understand their properties and functions.
  • Both Pdr forms exhibit monomer-dimer equilibrium and can bind NAD(+).

Purpose of the Study:

  • To characterize the properties of recombinant WT and His(6) Pdr.
  • To investigate the effect of the His(6) tag on Pdr's interaction with NAD(+) and its enzymatic activity.
  • To explore the potential oxidoreductase function of Pdr beyond its role in electron transfer.

Main Methods:

  • Expression and purification of recombinant WT and His(6) Pdr.

Related Experiment Videos

  • Spectroscopic analysis (UV-Vis) to monitor spectral changes and complex formation.
  • Enzyme assays to determine electron transfer and oxidoreductase activities.
  • Investigation of reaction kinetics under anaerobic conditions with various additives.

    • Main Results:

    • His(6) Pdr showed altered interaction with NAD(+) compared to WT Pdr, forming a charge-transfer complex and undergoing flavin reduction.
    • NAD(+) induced flavin reduction in WT Pdr in the presence of free histidine or thiol-reducing agents (dithiothreitol, reduced glutathione).
    • Free thiol groups within Pdr act as internal electron sources, enabling dithiol/disulfide oxidoreductase activity, independent of electron transfer to putidaredoxin.

    Conclusions:

    • Recombinant Pdr, both WT and His(6), functions as an NAD(H)-dependent dithiol/disulfide oxidoreductase.
    • This oxidoreductase activity can be dissociated from the enzyme's role in the cytochrome P450cam system's electron transfer chain.
    • Pdr shares functional similarities with enzymes from the glutathione reductase family.