Cloning, Overexpression, and Characterization of Glutaredoxin 2, An Atypical Glutaredoxin from Escherichia coli

• Published in: The Journal of biological chemistry 1997-04, Vol.272 (17), p.11236-11243
• Main Authors: Vlamis-Gardikas, A, Aslund, F, Spyrou, G, Bergman, T, Holmgren, A
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Bacterial Proteins - genetics; Base Sequence; Cloning, Molecular; Escherichia coli; Escherichia coli - genetics; Genes, Bacterial; Glutaredoxins; Glutathione - metabolism; Insulin - metabolism; Membrane Proteins - genetics; Models, Molecular; Molecular Sequence Data; Mutation; Oxidation-Reduction; Oxidoreductases; Protein Biosynthesis; Protein Structure, Secondary; Proteins - chemistry; Proteins - genetics; Recombinant Proteins - biosynthesis; Restriction Mapping; Sequence Analysis, DNA; Sequence Deletion; Sequence Homology, Amino Acid; Species Specificity
• ISSN: 0021-9258; 1083-351X; 1083-351X
• DOI: 10.1074/jbc.272.17.11236

Glutaredoxin 2 (Grx2) from Escherichia coli catalyzes GSH-disulfide oxidoreductions via two redox-active cysteine residues, but in contrast to glutaredoxin 1 (Grx1) and glutaredoxin 3 (Grx3), is not a hydrogen donor for ribonucleotide reductase. To characterize Grx2, a chromosomal fragment containing the E. coli Grx2 gene ( grxB ) was cloned and sequenced. grxB (645 base pairs) is located between the rimJ and pyrC genes while an open reading frame immediately upstream grxB encodes a novel transmembrane protein of 402 amino acids potentially belonging to class II of substrate export transporters. The deduced amino acid sequence for Grx2 comprises 215 residues with a molecular mass of 24.3 kDa. There is almost no similarity between the amino acid sequence of Grx2 and Grx1 or Grx3 (both 9-kDa proteins) with the exception of the active site which is identical in all three glutaredoxins (C 9 PYC 12 for Grx2). Only limited similarities were noted to glutathione S -transferases (Grx2 amino acids 16-72), and protein disulfide isomerases from different organisms (Grx2 amino acids 70-180). Grx2 was overexpressed and purified to homogeneity and its activity was compared with those of Grx1 and Grx3 using GSH, NADPH, and glutathione reductase in the reduction of 0.7 m M β-hydroxyethyl disulfide. The three glutaredoxins had similar apparent K m values for GSH (2-3 m M ) but Grx2 had the highest apparent k cat (554 s −1 ). Expression of two truncated forms of Grx2 (1-114 and 1-133) which have predicted secondary structures similar to Grx1 (βαβαββα) gave rise to inclusion bodies. The mutant proteins were resolubilized and purified but lacked GSH-disulfide oxidoreductase activity. The latter should therefore require the participation of amino acid residues from the COOH-terminal half of the molecule and is probably not confined to a Grx1-like NH 2 -terminal subdomain. Grx2 being radically different from the presently known glutaredoxins in terms of molecular weight, amino acid sequence, catalytic activity, and lack of a consensus GSH-binding site is the first member of a novel class of glutaredoxins.