Escherichia coli glutaredoxin: cloning and overexpression, thermodynamic stability of the oxidized and reduced forms, and report of an N-terminal extended species.


Abstract

Escherichia coli glutaredoxin (MW 9700) catalyzes intracellular redox reactions utilizing a disulfide/dithiol enzymatic mechanism involving the active-site residues -Cys-Pro-Tyr-Cys-. It is functionally related to the thioredoxin family and is expected to share similar three-dimensional structure [Eklund, H., Cambillau, C., Sjöberg, B.-M., Holmgren, A., Jörnvall, H., Höög, J.-O., & Brändén, C.-I. (1984) EMBO J. 3, 1443-1449]. We constructed an overexpression system in which production of glutaredoxin is controlled by temperature-sensitive expression of the phage T7 promoter. In addition to glutaredoxin, a second gene product is observed; this species, which we call glutaredoxin N, is glutaredoxin extended by the sequence Met-Arg-Arg-Glu-Ile- at the N terminus. We have begun characterization of the structure and stability of the oxidized and reduced forms of glutaredoxin (grx-S2 and grx-(SH)2, respectively). Secondary structure calculated from CD data agrees with that predicted from the three-dimensional model of Eklund et al. The cooperative denaturation reactions of oxidized and reduced glutaredoxin were measured in temperature-induced and guanidine hydrochloride induced unfolding experiments. Surprisingly, oxidized and reduced glutaredoxins are very similar in stability. In heat-induced denaturation, monitored by CD, Tm is 55 and 57 degrees C for oxidized and reduced, respectively. In GuHCl denaturation, monitored by fluorescence, the midpoint denaturant concentrations are 2 M for both oxidized and reduced. It follows that the redox potentials of the disulfide bond are similar in unfolded and folded glutaredoxin. This is unexpected because in E. coli thioredoxin the oxidized form is far more stable than the reduced [Kelley, R.F., Shalongo, W., Jagannadham, M.V., & Stellwagen, E. (1987) Biochemistry 26, 1406-1411] and the redox potential of folded thioredoxin is significantly more negative than that of unfolded thioredoxin [Lin, T.-Y., & Kim, P. (1989) Biochemistry 28, 5282-5287]. Study holds ProTherm entries: 4372, 4373, 4374, 4375, 4376, 4377, 4378, 4379 Extra Details: additive : EDTA(1 mM), active-site residues; temperature-sensitive; secondary structure;,cooperative denaturation;

Submission Details

ID: ZXcVDSAs

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:25 p.m.

Version: 1

Publication Details
Sandberg VA;Kren B;Fuchs JA;Woodward C,Biochemistry (1991) Escherichia coli glutaredoxin: cloning and overexpression, thermodynamic stability of the oxidized and reduced forms, and report of an N-terminal extended species. PMID:2036416
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1EGR 1993-10-31 SEQUENCE-SPECIFIC 1H N.M.R. ASSIGNMENTS AND DETERMINATION OF THE THREE-DIMENSIONAL STRUCTURE OF REDUCED ESCHERICHIA COLI GLUTAREDOXIN
1EGO 1993-10-31 NMR STRUCTURE OF OXIDIZED ESCHERICHIA COLI GLUTAREDOXIN: COMPARISON WITH REDUCED E. COLI GLUTAREDOXIN AND FUNCTIONALLY RELATED PROTEINS
1GRX 1994-01-31 STRUCTURE OF E. COLI GLUTAREDOXIN
1QFN 2000-01-01 GLUTAREDOXIN-1-RIBONUCLEOTIDE REDUCTASE B1 MIXED DISULFIDE BOND

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Glutaredoxin 1 P68689 GLRX1_SHIFL
100.0 Glutaredoxin 1 P68688 GLRX1_ECOLI