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Properties of the thioredoxin fold superfamily are modulated by a single amino acid residue.

Ren G, Stephan D, Xu Z, Zheng Y, Tang D, Harrison RS, Kurz M, Jarrott R, Shouldice SR, Hiniker A, Martin JL, Heras B, Bardwell JC - J. Biol. Chem. (2009)

Bottom Line: The ubiquitous thioredoxin fold proteins catalyze oxidation, reduction, or disulfide exchange reactions depending on their redox properties.It is isoleucine 75 in the case of thioredoxin.Our findings support the conclusion that a very small percentage of the amino acid residues of thioredoxin-related proteins are capable of dictating the functions of these proteins.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Departments of Molecular, Cellular, and Developmental Biology and Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.

ABSTRACT
The ubiquitous thioredoxin fold proteins catalyze oxidation, reduction, or disulfide exchange reactions depending on their redox properties. They also play vital roles in protein folding, redox control, and disease. Here, we have shown that a single residue strongly modifies both the redox properties of thioredoxin fold proteins and their ability to interact with substrates. This residue is adjacent in three-dimensional space to the characteristic CXXC active site motif of thioredoxin fold proteins but distant in sequence. This residue is just N-terminal to the conservative cis-proline. It is isoleucine 75 in the case of thioredoxin. Our findings support the conclusion that a very small percentage of the amino acid residues of thioredoxin-related proteins are capable of dictating the functions of these proteins.

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In vitro activity assays for DsbA, DsbG, and thioredoxin cisPro-1 variants. A, reductase activity was assayed using insulin and DTT as substrates for DsbA (black), DsbG (red), and thioredoxin (blue) (same in B and C). The orange line shows the DTT-only control. WT proteins are shown with solid lines and cisPro-1 mutants with dashed lines, sample names are as indicated in the panels. B, oxidase activity was assayed using reduced hirudin as a substrate. C, isomerase activity was assayed with scrambled hirudin as a substrate. The scrambled hirudin starting material is shown in Fig. S2C. Equimolar quantities of reduced proteins and scrambled hirudin were incubated for 30 min for the DsbA and thioredoxin samples and 18 h for the DsbG samples. The reactions were then acid-quenched, and samples were analyzed by reverse-phase HPLC on a Vydac™218TP54 C18 column. N and R indicate native and reduced hirudin, respectively.
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fig3: In vitro activity assays for DsbA, DsbG, and thioredoxin cisPro-1 variants. A, reductase activity was assayed using insulin and DTT as substrates for DsbA (black), DsbG (red), and thioredoxin (blue) (same in B and C). The orange line shows the DTT-only control. WT proteins are shown with solid lines and cisPro-1 mutants with dashed lines, sample names are as indicated in the panels. B, oxidase activity was assayed using reduced hirudin as a substrate. C, isomerase activity was assayed with scrambled hirudin as a substrate. The scrambled hirudin starting material is shown in Fig. S2C. Equimolar quantities of reduced proteins and scrambled hirudin were incubated for 30 min for the DsbA and thioredoxin samples and 18 h for the DsbG samples. The reactions were then acid-quenched, and samples were analyzed by reverse-phase HPLC on a Vydac™218TP54 C18 column. N and R indicate native and reduced hirudin, respectively.

Mentions: Perhaps the most widely used kinetic assay for thiol-disulfide oxidoreductases is measurement of the rate at which these proteins accelerate the reduction of disulfides in insulin (27). We initially used DTT as the electron donor in this assay, which allowed a direct comparison of the reductase activity of the cisPro-minus1 variants in DsbA, DsbG, DsbC, and thioredoxin. As shown in Figs. 3A and 2B and supplemental Fig. S2A, mutation of the cisPro-minus1 residue in DsbA, DsbC, DsbG, and thioredoxin significantly affects the reductase activity of these proteins, confirming the key role this residue plays in redox enzymatic activity. In the case of Dsb proteins, the cisPro-minus1 modification affected the reductase activity in ways that reflected changes in the redox potential (see Table 2). Thus, DsbA V150T has a substantially more oxidizing redox potential and, not surprisingly, is less efficient in reducing insulin. The cisPro-minus1 mutants of DsbC and DsbG are more efficient in reducing insulin, in keeping with their more reducing redox potentials.


Properties of the thioredoxin fold superfamily are modulated by a single amino acid residue.

Ren G, Stephan D, Xu Z, Zheng Y, Tang D, Harrison RS, Kurz M, Jarrott R, Shouldice SR, Hiniker A, Martin JL, Heras B, Bardwell JC - J. Biol. Chem. (2009)

In vitro activity assays for DsbA, DsbG, and thioredoxin cisPro-1 variants. A, reductase activity was assayed using insulin and DTT as substrates for DsbA (black), DsbG (red), and thioredoxin (blue) (same in B and C). The orange line shows the DTT-only control. WT proteins are shown with solid lines and cisPro-1 mutants with dashed lines, sample names are as indicated in the panels. B, oxidase activity was assayed using reduced hirudin as a substrate. C, isomerase activity was assayed with scrambled hirudin as a substrate. The scrambled hirudin starting material is shown in Fig. S2C. Equimolar quantities of reduced proteins and scrambled hirudin were incubated for 30 min for the DsbA and thioredoxin samples and 18 h for the DsbG samples. The reactions were then acid-quenched, and samples were analyzed by reverse-phase HPLC on a Vydac™218TP54 C18 column. N and R indicate native and reduced hirudin, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2665069&req=5

fig3: In vitro activity assays for DsbA, DsbG, and thioredoxin cisPro-1 variants. A, reductase activity was assayed using insulin and DTT as substrates for DsbA (black), DsbG (red), and thioredoxin (blue) (same in B and C). The orange line shows the DTT-only control. WT proteins are shown with solid lines and cisPro-1 mutants with dashed lines, sample names are as indicated in the panels. B, oxidase activity was assayed using reduced hirudin as a substrate. C, isomerase activity was assayed with scrambled hirudin as a substrate. The scrambled hirudin starting material is shown in Fig. S2C. Equimolar quantities of reduced proteins and scrambled hirudin were incubated for 30 min for the DsbA and thioredoxin samples and 18 h for the DsbG samples. The reactions were then acid-quenched, and samples were analyzed by reverse-phase HPLC on a Vydac™218TP54 C18 column. N and R indicate native and reduced hirudin, respectively.
Mentions: Perhaps the most widely used kinetic assay for thiol-disulfide oxidoreductases is measurement of the rate at which these proteins accelerate the reduction of disulfides in insulin (27). We initially used DTT as the electron donor in this assay, which allowed a direct comparison of the reductase activity of the cisPro-minus1 variants in DsbA, DsbG, DsbC, and thioredoxin. As shown in Figs. 3A and 2B and supplemental Fig. S2A, mutation of the cisPro-minus1 residue in DsbA, DsbC, DsbG, and thioredoxin significantly affects the reductase activity of these proteins, confirming the key role this residue plays in redox enzymatic activity. In the case of Dsb proteins, the cisPro-minus1 modification affected the reductase activity in ways that reflected changes in the redox potential (see Table 2). Thus, DsbA V150T has a substantially more oxidizing redox potential and, not surprisingly, is less efficient in reducing insulin. The cisPro-minus1 mutants of DsbC and DsbG are more efficient in reducing insulin, in keeping with their more reducing redox potentials.

Bottom Line: The ubiquitous thioredoxin fold proteins catalyze oxidation, reduction, or disulfide exchange reactions depending on their redox properties.It is isoleucine 75 in the case of thioredoxin.Our findings support the conclusion that a very small percentage of the amino acid residues of thioredoxin-related proteins are capable of dictating the functions of these proteins.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Departments of Molecular, Cellular, and Developmental Biology and Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.

ABSTRACT
The ubiquitous thioredoxin fold proteins catalyze oxidation, reduction, or disulfide exchange reactions depending on their redox properties. They also play vital roles in protein folding, redox control, and disease. Here, we have shown that a single residue strongly modifies both the redox properties of thioredoxin fold proteins and their ability to interact with substrates. This residue is adjacent in three-dimensional space to the characteristic CXXC active site motif of thioredoxin fold proteins but distant in sequence. This residue is just N-terminal to the conservative cis-proline. It is isoleucine 75 in the case of thioredoxin. Our findings support the conclusion that a very small percentage of the amino acid residues of thioredoxin-related proteins are capable of dictating the functions of these proteins.

Show MeSH
Related in: MedlinePlus