Determinants of activity in glutaredoxins: an in vitro evolved Grx1-like variant of Escherichia coli Grx3

Biochem J. 2010 Sep 15;430(3):487-95. doi: 10.1042/BJ20100289.


The Escherichia coli glutaredoxins 1 and 3 (Grx1 and Grx3) are structurally similar (37% sequence identity), yet have different activities in vivo. Unlike Grx3, Grx1 efficiently reduces protein disulfides in proteins such as RR (ribonucleotide reductase), whereas it is poor at reducing S-glutathionylated proteins. An E. coli strain lacking genes encoding thioredoxins 1 and 2 and Grx1 is not viable on either rich or minimal medium; however, a M43V mutation in Grx3 restores growth under these conditions and results in a Grx1-like protein [Ortenberg, Gon, Porat and Beckwith (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 7439-7944]. To uncover the structural basis of this change in activity, we have compared wild-type and mutant Grx3 using CD and NMR spectroscopy. Ligand-induced stability measurements demonstrate that the Grx3(M43V/C65Y) mutant has acquired affinity for RR. Far-UV CD spectra reveal no significant differences, but differences are observed in the near-UV region indicative of tertiary structural changes. NMR (1)H-(15)N HSQC (heteronuclear single quantum coherence) spectra show that approximately half of the 82 residues experience significant (Deltadelta>0.03 p.p.m.) chemical shift deviations in the mutant, including nine residues experiencing extensive (Deltadelta > or =0.15 p.p.m.) deviations. To test whether the M43V mutation alters dynamic properties of Grx3, H/D (hydrogen/deuterium) exchange experiments were performed demonstrating that the rate at which backbone amides exchange protons with the solvent is dramatically enhanced in the mutant, particularly in the core of the protein. These data suggest that the Grx1-like activity of the Grx3(M43V/C65Y) mutant may be explained by enhanced intrinsic motion allowing for increased specificity towards larger substrates such as RR.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Algorithms
  • Amino Acid Substitution
  • Circular Dichroism
  • Enzyme Stability
  • Escherichia coli / enzymology*
  • Escherichia coli / genetics
  • Escherichia coli / growth & development
  • Escherichia coli Proteins / chemistry
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism*
  • Glutaredoxins / chemistry
  • Glutaredoxins / genetics
  • Glutaredoxins / metabolism*
  • Glutathione Disulfide / metabolism
  • Isoenzymes / genetics
  • Isoenzymes / metabolism
  • Magnetic Resonance Spectroscopy
  • Models, Molecular
  • Mutant Proteins / chemistry
  • Mutant Proteins / genetics
  • Mutant Proteins / metabolism*
  • Mutation
  • Oxidation-Reduction
  • Protein Conformation
  • Protein Structure, Tertiary
  • Thermodynamics


  • Escherichia coli Proteins
  • Glutaredoxins
  • Isoenzymes
  • Mutant Proteins
  • Glutathione Disulfide