Characterization of Escherichia coli thioredoxins with altered active site residues

Abstract

Escherichia coli thioredoxin is a small disulfide-containing redox protein with the active site sequence Cys-Gly-Pro-Cys-Lys. Mutations were made in this region of the thioredoxin gene and the mutant proteins expressed in E. coli strains lacking thioredoxin. Mutant proteins with a 17-membered or 11-membered disulfide ring were inactive in vivo. However, purified thioredoxin with the active site sequence Cys-Gly-Arg-Pro-Cys-Lys is still able to serve as a substrate for thioredoxin reductase and a reducing agent in the ribonucleotide reductase reaction, although with greatly reduced catalytic efficiency. A smaller disulfide ring, with the active site sequence Cys-Ala-Cys, does not turn over at a sufficient rate to be an effective reducing agent. Strain in the small ring favors the formation of intermolecular disulfide bonds. Alteration of the invariant proline to a serine has little effect on redox activity. The function of this residue may be in maintaining the stability of the active site region rather than participation in redox activity or protein-protein interactions. Mutation of the positively charged lysine in the active site to a glutamate residue raises the Km values with interacting enzymes. Although it has been proposed that the positive residue at position 36 is conserved to maintain the thiolate anion on Cys-32 (Kallis & Holmgren, 1985), the presence of the negative charge at this position does not alter the pH dependence of activity or fluorescence behavior. The lysine is most likely conserved to facilitate thioredoxin-protein interactions.

Facile sulfitolysis of the disulfide bonds in oxidized thioredoxin and glutaredoxin

“…Thioredoxin from E. coli was prepared by the published procedure [ 161. Mutant thioredoxins D26A [ 171, K36E, R insert [18] and the protein of Anabaena 7120 [19] were obtained from Drs F. K. Gleason and J. A. Fuchs, Uni-versity of Minnesota, St. Paul, USA; E. coli glutaredoxin and thioredoxin P34H [20] were kindly provided by Dr A. Holmgren, Karolinska Institutet, Stockholm, Sweden; spinach chloroplast thioredoxins [9] were gifts of Dr P. Schiirmann, University of Neuchatel, Switzerland.…”

“…We have made use of the availability of proteins variants produced by genetic engineering [18,20,271 to probe the dependence of E. coli thioredoxin sulfitolysis on charge and other structure elements near the active site. The thioredoxin of a cyanobacterium, Anabaena 7120, and glutaredoxin of E. coli were of interest because they lack negatively charged amino acids in that region altogether.…”

The CXC Motif: A Functional Mimic of Protein Disulfide Isomerase

“…Most attempts at tuning the physico-chemical properties of CXXC motifs have relied on swapping one natural -XX-sequence for another (14,19) or creating random libraries of -XXsequences (7,22,52). Only one attempt has been made to examine the functional consequences of altering the number of intervening amino acids in a CXXC motif (38).…”

The high-resolution three-dimensional solution structures of the oxidized and reduced states of human thioredoxin

“…Subtle structural and dynamic differences between the reduced and oxidized forms have been invoked to explain these functional changes [20]. Mutagenesis studies indicate that the conserved active site region plays an important role in interacting with other proteins for both redox and non-redox related functions of thioredoxin [18,19,[21][22][23][24]. In contrast to our extensive knowledge about E. coli thioredoxin, little is known about the functional activities of human thioredoxin beyond its general redox properties.…”