Crystal structure of thioredoxin from Escherichia coli at 1.68 Å resolution

Katti, Suresh; LeMaster, David M.; Eklund, Hans

doi:10.1016/0022-2836(90)90313-b

Cited by 569 publications

(641 citation statements)

References 66 publications

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“…This could result from interaction of the indole with Asp 26 directly, or from an interaction involving Asp 26 and another neighboring protein group that titrates with pK, in the 4-8 range. Specifically, His 6 is close to van der Waals contact with the indole of Trp 28 (Katti et al, 1990;Chandrasekhar et al, 1994), as can be seen in Figure 5. Involvement of His 6 with an indole is also supported by tryptophan fluorescence measurements conducted on the thioredoxin mutants H6A and D26A (Hanson, 1995).…”

Section: Structural Effects Of the D26a Mutationmentioning

confidence: 66%

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Conformation, stability, and active‐site cysteine titrations of Escherichia coli D26A thioredoxin probed by Raman spectroscopy

et al. 1998

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The active-site cysteines (Cys 32 and Cys 35) of Escherichia coli thioredoxin are oxidized to a disulfide bridge when the protein mediates substrate reduction. In reduced thioredoxin, Cys 32 and Cys 35 are characterized by abnormally low pK, values. A conserved side chain, Asp 26, which is sterically accessible to the active site, is also essential to oxidoreductase activity. pK, values governing cysteine thiol-thiolate equilibria in the mutant thioredoxin, D26A, have been determined by direct Raman spectrophotometric measurement of sulfhydryl ionizations. The results indicate that, in D26A thioredoxin, both sulfhydryls titrate with apparent pK, values of 7.5 f 0.2, close to values measured previously for wild-type thioredoxin. Sulfhydryl Raman markers of D26A and wild-type thioredoxin also exhibit similar band shapes, consistent with minimal differences in respective cysteine side-chain conformations and sulfhydryl interactions. The results imply that neither the Cys 32 nor Cys 35 SH donor is hydrogen bonded directly to Asp 26 in the wild-type protein. Additionally, the thioredoxin main-chain conformation is largely conserved with D26A mutation. Conversely, the mutation perturbs Raman bands diagnostic of tryptophan (Trp 28 and Trp 31) orientations and leads to differences in their pH dependencies, implying local conformational differences near the active site. We conclude that, although the carboxyl side chain of Asp 26 neither interacts directly with active-site cysteines nor is responsible for their abnormally low pK, values, the aspartate side chain may play a role in determining the conformation of the enzyme active site.Keywords: cysteine; pK,; Raman spectra; structure; thiol; thiolate; thioredoxin The thioredoxin fold is an extensively studied structural motif found in a variety of proteins that interact with cysteine-containing substrates (Edman et al., 1985;Martin, 1995). Both the thioredoxin fold and the local sequence at the active site (-W-C-G-P-C-) are highly conserved among thioredoxins of different species (Holmgren, 1985;Martin, 1995). In Escherichia coli, thioredoxin functions not only as an oxidoreductase, but also as a host regulator of prokaryotic viral assembly (reviewed by Holmgren, 1985). The active-site cysteines 32 and 35 in E. coli thioredoxin are oxidized to form a cystine bridge when the protein mediates the reduction of a suitable substrate. The reduced form of thioredoxin is subsequently regenerated by the enzyme thioredoxin reductase.

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Section: Structural Effects Of the D26a Mutationmentioning

confidence: 66%

“…The X-ray crystal structure of oxidized, wild-type thioredoxin has been solved at 1.68 8, resolution (Katti et al, 1990) and a solution structure has been determined by multidimensional NMR methods (Dyson et al, 1990;Jeng et al, 1994). The protein fold comprises a five-stranded &sheet core and four a-helical segments.…”

mentioning

confidence: 99%

Conformation, stability, and active‐site cysteine titrations of Escherichia coli D26A thioredoxin probed by Raman spectroscopy

et al. 1998

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“…Numbers in parentheses represent residues excised from the alignment. Predicted (Rost & Sander, 1993) secondary structure of Prxs (E/e represents a P-strand, whereas H/h represents an cy-helix; predicted accuracy 72% [lower case]/82% [upper case]) is given below the Prx alignment and above the known secondary structure of E. coli thioredoxin (Katti et al, 1990). Sequence analysis of Prx homologues used the PSI-BLAST algorithm (Altschul et al, 1997).…”

Section: Discussionmentioning

confidence: 99%

“…Alignment was guided by: ( I ) CLUSTAL-W-derived alignments (Thompson et al, 1994), (2) the predicted secondary structures of previously known Prx homologues (Rost & Sander, 1993), and (3) a multiple alignment derived from an automatically-derived structural alignment of known Trx-like structures (Epp et al, 1983; Katti et al, 1990Katti et al, , 1995Sodano et al, 1991;Martin et al, 1993;Saarinen et al, 1995) using Dali (Holm & Sander, 1998). The final alignment (Fig.…”

Section: Peroxiredoxins Are Thioredoxin Homologuesmentioning

confidence: 99%

Evidence that peroxiredoxins are novel members of the thioredoxin fold superfamily

Schröder

Ponting

1998

Protein Science

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Abstract:Peroxiredoxins catalyze reduction of hydrogen peroxide or alkyl peroxide, to water or the corresponding alcohol. Detailed analysis of their sequences indicates that these enzymes possess a thioredoxin (Trx)-like fold and consequently are homologues of both thioredoxin and glutathione peroxidase (GPx). Sequence-and structure-based multiple sequence alignments indicate that the peroxiredoxin active site cysteine and GPx active site selenocysteine are structurally equivalent. Homologous peroxiredoxin and GPx enzymes are predicted to catalyze equivalent reactions via similar reaction intermediates.

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“…For four members, both the crystal structure and the NMR structure are available. These are Escherichia coli thioredoxin (Katti et al 1990;Jeng and Dyson 1994), human thioredoxin (Qin et al 1994;Weichsel et al 1996), thioredoxin h from Chlamydomonas reinhardtii (Mittard et al 1997;Menchise et al 2001) and thioredoxin m (Trx-m) from spinach chloroplasts (Capitani et al 2000;Neira et al 2001). In the case of Trx-m, two crystal structures of the protein, in the oxidized and in the reduced state (PDB codes 1FB6 and 1FB0, respectively), were published first (Capitani et al 2000) and were followed one year later by the NMR structure of the oxidized protein (PDB code 1GL8) (Neira et al 2001).…”

Section: Introductionmentioning

confidence: 99%