A mutant thioredoxin from Escherichia coli tsnC 7007 that is nonfunctional as subunit of phage T7 DNA polymerase.

Holmgren, Arne; Kallis, G.B.; Nordström, B

doi:10.1016/s0021-9258(19)69732-2

Cited by 43 publications

(9 citation statements)

References 34 publications

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“…Reaction of the hybrid thioredoxins to these antibodies indicates that the antigenic determinants for thioredoxin are located in the C-terminal two-thirds of the molecule. Similar findings had previously been reported for E. coli thioredoxin (Holmgren et al, 1981). Since antibodies apparently recognize unique sequences in the native, folded protein (Benjamin et al, 1984), our data indicate that the hybrid thioredoxins maintain the conformational integrity of their parent proteins at antigenic sites.…”

Section: Discussionsupporting

confidence: 89%

Characterization of Escherichia coli-Anabaena sp. hybrid thioredoxins

Lim¹,

Gleason²,

Jacobson³

et al. 1988

Biochemistry

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Thioredoxin is a small redox protein with an active-site disulfide/dithiol. The protein from Escherichia coli has been well characterized. The genes encoding thioredoxin in E. coli and in the filamentous cyanobacterium Anabaena PCC 7119 have been cloned and sequenced. Anabaena thioredoxin exhibits 50% amino acid identity with the E. coli protein and interacts with E. coli enzymes. The genes encoding Anabaena and E. coli thioredoxin were fused via a common restriction site in the nucleotide sequence coding for the active site of the proteins to generate hybrid genes, coding for two chimeric thioredoxins. These proteins are designated Anabaena-E. coli (A-E) thioredoxin for the construct with the Anabaena sequence from the N-terminus to the middle of the active site and the E. coli sequence to the C-terminus, and E. coli-Anabaena (E-A) for the opposite construct. The gene encoding the A-E thioredoxin complements all phenotypes of an E. coli thioredoxin-deficient strain, whereas the gene encoding E-A thioredoxin is only partially effective. Purified E-A thioredoxin exhibits a much lower catalytic efficiency with E. coli thioredoxin reductase and ribonucleotide reductase than either E. coli or Anabaena thioredoxin. In contrast, the A-E thioredoxin has a higher catalytic efficiency in these reactions than either parental protein. Reaction with antibodies to E. coli and Anabaena thioredoxins shows that the antigenic determinants for thioredoxin are located in the C-terminal part of the molecule and retain the native conformation in the hybrid proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Discussionsupporting

confidence: 89%

Characterization of Escherichia coli-Anabaena sp. hybrid thioredoxins

Lim¹,

Gleason²,

Jacobson³

et al. 1988

Biochemistry

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“…This was confirmed by their ultraviolet spectra, their nonidentity with native thioredoxin in immuno double diffusion, and their amino acid compositions. The low yield of the complex suggested that either only a fraction of the peptides Mg of thioredoxin-S2 0) and 10 Mg each of thioredoxin-C' (2), thioredoxin-C-(38-l08) (3), and thioredoxin-C-(l-37) (4) were run in native gels at pH 8.9. For experimental details, see Slaby &…”

Section: Resultsmentioning

confidence: 99%

Thioredoxin-C': mechanism of noncovalent complementation and reactions of the refolded complex and the active site containing fragment with thioredoxin reductase

Holmgren¹,

Slaby²

1979

Biochemistry

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“…The fact that thioredoxin P34S can restore the wild-type phenotype in various E. coli mutants indicates that it functions as an effective reducing agent in vivo. Since the flat proline residue has been replaced by a short, hydrophilic hydroxyl group in P34S, it would appear that a strictly hydrophobic character at this site is not essential for protein-protein interactions, although similar mutations at the other proposed interaction sites, glycine-74 and glycine-92 (Holmgren et al, 1981), caused a marked decline in enzymatic activity. Proline-34 is conserved in all known thioredoxins but is replaced by a histidine in protein disulfide isomerase (Edman et al, 1985).…”

Section: Discussionmentioning

confidence: 99%

Characterization of Escherichia coli thioredoxins with altered active site residues

Gleason

Lim²,

Gerami-Nejad³

et al. 1990

Biochemistry

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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.

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A mutant thioredoxin from Escherichia coli tsnC 7007 that is nonfunctional as subunit of phage T7 DNA polymerase.

Cited by 43 publications

References 34 publications

Characterization of Escherichia coli-Anabaena sp. hybrid thioredoxins

Characterization of Escherichia coli-Anabaena sp. hybrid thioredoxins

Thioredoxin-C': mechanism of noncovalent complementation and reactions of the refolded complex and the active site containing fragment with thioredoxin reductase

Characterization of Escherichia coli thioredoxins with altered active site residues

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