The Role of the Thioredoxin and Glutaredoxin Pathways in Reducing Protein Disulfide Bonds in the Escherichia coliCytoplasm

Prinz, William A.; Åslund, Fredrik; Holmgren, Arne; Beckwith, Jon

doi:10.1074/jbc.272.25.15661

Cited by 583 publications

(464 citation statements)

References 48 publications

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“…This construct produces Dom1PI with an NH 2 -terminal Escherichia coli thioredoxin fusion (Trx tag, Novagen, Madison, WI) connected by a 43-amino acid residue linker containing six histidine residues and a factor Xa cleavage site. To ensure accurate disulfide formation, an E. coli trxB -/gor522 -double mutant [E. coli Origami (DE3), Novagen] with an oxidative cytoplasm was used (21,22). This strategy has been shown to result in the correct LEKTI domain 1 disulfide pattern (20).…”

Section: Methodsmentioning

confidence: 99%

The Solution Structure of a Chimeric LEKTI Domain Reveals a Chameleon Sequence

et al. 2004

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The conversion of an R-helical to a -strand conformation and the presence of chameleon sequences are fascinating from the perspective that such structural features are implicated in the induction of amyloid-related fatal diseases. In this study, we have determined the solution structure of a chimeric domain (Dom1PI) from the multidomain Kazal-type serine proteinase inhibitor LEKTI using multidimensional NMR spectroscopy. This chimeric protein was constructed to investigate the reasons for differences in the folds of the homologous LEKTI domains 1 and 6 [Lauber, T., et al. (2003) J. Mol. Biol. 328, 205-219]. In Dom1PI, two adjacent phenylalanine residues (F28 and F29) of domain 1 were substituted with proline and isoleucine, respectively, as found in the corresponding P4′ and P5′ positions of domain 6. The three-dimensional structure of Dom1PI is significantly different from the structure of domain 1 and closely resembles the structure of domain 6, despite the sequence being identical to that of domain 1 except for the two substituted phenylalanine residues and being only 31% identical to the sequence of domain 6. The mutation converted a short 3 10 -helix into an extended loop conformation and parts of the long COOH-terminal R-helix of domain 1 into a -hairpin structure. The latter conformational change occurs in a sequence stretch distinct from the region containing the substituted residues. Therefore, this switch from an R-helical structure to a -hairpin structure indicates a chameleon sequence of seven residues. We conclude that the secondary structure of Dom1PI is determined not only by the local protein sequence but also by nonlocal interactions.

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Section: Methodsmentioning

confidence: 99%

The Solution Structure of a Chimeric LEKTI Domain Reveals a Chameleon Sequence

et al. 2004

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“…The ScFv-fragment is fused with the NusA protein (N utilization substance protein A), which shows the highest solubility in E. coli (16). A stain bearing a defect in the reductase for thioredoxin and glutathione, which has been shown to accumulate active oxidizing enzymes in the cytoplasm (17), provides a potentially favorable environment for disulfide bond formation of single chain Fv. We show that the combination of NusA fusion and an oxidizing cytoplasmic environment facilitates the expression of a functional catalytic antibody fragment.…”

mentioning

confidence: 99%

Production of a Functional Catalytic Antibody ScFv-NusA Fusion Protein in Bacterial Cytoplasm

Zheng

Baumann

Reymond

2003

Journal of Biochemistry

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Functional expression of catalytic antibodies in the cytoplasm of E. coli is potentially of great interest in searching for new catalysts by genetic selection. Herein, a catalytic antibody single chain Fv (ScFv) 14D9, which catalyzes a highly enantioselective protonation, was expressed as a NusA fusion protein under the T7 promoter. A functional disulfide-containing ScFv fusion protein was obtained in the oxidizing environment of bacterial cytoplasm. The 14D9 ScFv could not be overexpressed alone without NusA fusion. The highly soluble NusA protein most likely retards aggregate formation of ScFv and indirectly supports correct folding and disulfide bridge formation in the fusion construct ScFv-NusA. The ScFv-NusA fusion product shows highly enantioselective, specific, hapten inhibited catalytic activity comparable to its parent monoclonal antibody, 14D9. The NusA fusion method might be generally helpful for functional antibody expression in vivo and for the new development of biocatalysts by genetic selection.

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“…Reduced Tr has been implicated in a variety of physiological functions, including the reduction of disulfide bonds in enzymes such as ribonucleotide reductase which require such a step for turnover (12,13). It has recently been recognized that TrR in many eukaryotic organisms is structurally quite distinct from bacterial TrR and is likely to catalyze Tr reduction through an additional redox center at the C-terminus of the protein (14)(15)(16).…”

mentioning

confidence: 99%

Attachment of the N-Terminal Domain of Salmonella typhimurium AhpF to Escherichia coli Thioredoxin Reductase Confers AhpC Reductase Activity but Does Not Affect Thioredoxin Reductase Activity

and

Poole

2000

Biochemistry

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AhpF of Salmonella typhimurium, the flavoprotein reductase required for catalytic turnover of AhpC with hydroperoxide substrates in the alkyl hydroperoxide reductase system, is a 57 kDa protein with homology to thioredoxin reductase (TrR) from Escherichia coli. Like TrR, AhpF employs tightly bound FAD and redox-active disulfide center(s) in catalyzing electron transfer from reduced pyridine nucleotides to the disulfide bond of its protein substrate. Homology of AhpF to the smaller (35 kDa) TrR protein occurs in the C-terminal part of AhpF; a stretch of about 200 amino acids at the N-terminus of AhpF contains an additional redox-active disulfide center and is required for catalysis of AhpC reduction. We have demonstrated that fusion of the N-terminal 207 amino acids of AhpF to full-length TrR results in a chimeric protein (Nt-TrR) with essentially the same catalytic efficiency (k cat /K m ) as AhpF in AhpC reductase assays; both k cat and the K m for AhpC are decreased about 3-4-fold for Nt-TrR compared with AhpF. In addition, Nt-TrR retains essentially full TrR activity. Based on results from two mutants of Nt-TrR (C129,132S and C342,345S), AhpC reductase activity requires both centers while TrR activity requires only the C-terminal-most disulfide center in Nt-TrR. The high catalytic efficiency with which Nt-TrR can reduce thioredoxin implies that the attached N-terminal domain does not block access of thioredoxin to the TrR-derived Cys342-Cys345 center of Nt-TrR nor does it impede the putative conformational changes that this part of Nt-TrR is proposed to undergo during catalysis. These studies indicate that the C-terminal part of AhpF and bacterial TrR have very similar mechanistic properties. These findings also confirm that the N-terminal domain of AhpF plays a direct role in AhpC reduction.

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The Role of the Thioredoxin and Glutaredoxin Pathways in Reducing Protein Disulfide Bonds in the Escherichia coliCytoplasm

Cited by 583 publications

References 48 publications

The Solution Structure of a Chimeric LEKTI Domain Reveals a Chameleon Sequence

The Solution Structure of a Chimeric LEKTI Domain Reveals a Chameleon Sequence

Production of a Functional Catalytic Antibody ScFv-NusA Fusion Protein in Bacterial Cytoplasm

Attachment of the N-Terminal Domain of Salmonella typhimurium AhpF to Escherichia coli Thioredoxin Reductase Confers AhpC Reductase Activity but Does Not Affect Thioredoxin Reductase Activity

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