Structural basis and kinetics of inter- and intramolecular disulfide exchange in the redox catalyst DsbD

Rozhkova, Anna; Stirnimann, Christian U.; Frei, Patrick; Grauschopf, Ulla; Brunisholz, René A.; Grütter, Markus G.; Capitani, Guido; Glockshuber, Rudi

doi:10.1038/sj.emboj.7600178

Cited by 127 publications

(174 citation statements)

References 59 publications

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“…The equivalent residues in DsbC, H102, S180, T182, and P194, are also exposed in DsbC (Fig. 3B) and cluster in two regions that interact with the partner protein DsbD (19). These regions are postulated to be involved in DsbC's interaction with misoxidized substrate proteins.…”

Section: Structures Of Dsbg Mutants Conferring Coppermentioning

confidence: 99%

Laboratory evolution of one disulfide isomerase to resemble another

Hiniker

Ren

Heras

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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It is often difficult to determine which of the sequence and structural differences between divergent members of multigene families are functionally important. Here we use a laboratory evolution approach to determine functionally important structural differences between two distantly related disulfide isomerases, DsbC and DsbG from Escherichia coli. Surprisingly, we found single amino acid substitutions in DsbG that were able to complement dsbC in vivo and have more DsbC-like isomerase activity in vitro. Crystal structures of the three strongest point mutants, DsbG K113E, DsbG V216M, and DsbG T200M, reveal changes in highly surface-exposed regions that cause DsbG to more closely resemble the distantly related DsbC. In this case, laboratory evolution appears to have taken a direct route to allow one protein family member to complement another, with single substitutions apparently bypassing much of the need for multiple changes that took place over Ϸ0.5 billion years of evolution. Our findings suggest that, for these two proteins at least, regions important in determining functional differences may represent only a tiny fraction of the overall protein structure.chaperone ͉ protein folding ͉ directed evolution A number of models of the origin of life postulate that the primordial living cell contained only a small number of enzymes; years of gene duplication, divergence, and natural selection led to the current situation in which each organism possesses thousands of proteins with different functions (1). Identifying the mechanisms by which proteins can acquire new functions is important both in understanding this fundamental question of natural diversity and in elucidating the particular structural differences that allow related proteins to have distinct functions. Here we use a combination of directed evolution and structural biology to examine the functionally important structural differences of two distantly related Escherichia coli disulfide isomerases, DsbC and DsbG.Directed evolution is an elegant method that is often used to alter enzymatic function (usually by broadening enzymatic specificity), but it also has been used to change the properties of one enzyme so that it has some of the functional properties of another related enzyme (2). One way to do this requires the presence of a unique and selectable phenotype for one family member, allowing the selection of gain-of-function mutations in a related protein. Analysis of those gain-of-function mutants can provide information about functional differences between the two family members. This technique has been used to alter the enzymatic activity of a number of proteins so that they now have common properties, including the E. coli paralogs aspartate aminotransferase (AATase) and tyrosine aminotransferase (3), the structurally similar HisA and TrpF (4), and the human class 1-1 and rat class 2-2 glutathione transferases (5). Directed evolution has also been used to explore the differences between various E. coli folding catalysts, such as DsbC and DsbA (6), Gr...

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Section: Structures Of Dsbg Mutants Conferring Coppermentioning

confidence: 99%

Laboratory evolution of one disulfide isomerase to resemble another

Hiniker

Ren

Heras

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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“…Redox Potential Measurement-Proteins with redox potentials that were to be measured were incubated with the ␥-domain of DsbD, a protein with a known redox potential of Ϫ235 mV (27). At equilibrium, different protein species were separated by reverse-phase HPLC, 2 and the equilibrium constant (K eq ) of the reaction and the standard redox potential (EЈ 0 ) of the protein were determined as described (28).…”

Section: Methodsmentioning

confidence: 99%

The CXXC Motif Is More than a Redox Rheostat

Qin

Schneider

Pan

et al. 2007

Journal of Biological Chemistry

124

122

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The CXXC active-site motif of thiol-disulfide oxidoreductases is thought to act as a redox rheostat, the sequence of which determines its reduction potential and functional properties. We tested this idea by selecting for mutants of the CXXC motif in a reducing oxidoreductase (thioredoxin) that complement null mutants of a very oxidizing oxidoreductase, DsbA. We found that altering the CXXC motif affected not only the reduction potential of the protein, but also its ability to function as a disulfide isomerase and also impacted its interaction with folding protein substrates and reoxidants. It is surprising that nearly all of our thioredoxin mutants had increased activity in disulfide isomerization in vitro and in vivo. Our results indicate that the CXXC motif has the remarkable ability to confer a large number of very specific properties on thioredoxin-related proteins.

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“…Kinetic factors prevent the reduction of DsbA by DsbC or by DsbD (25). TrxA however is prone to reduction in the periplasm.…”

Section: A Screen For Periplasmic Protein Oxidation Using M13mentioning

confidence: 99%

Laboratory Evolution of Escherichia coli Thioredoxin for Enhanced Catalysis of Protein Oxidation in the Periplasm Reveals a Phylogenetically Conserved Substrate Specificity Determinant

Masip

Klein‐Marcuschamer

Qin

et al. 2008

Journal of Biological Chemistry

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Thioredoxin exported into the Escherichia coli periplasm catalyzes the oxidation of protein thiols in a DsbB-dependent function. However, the oxidative activity of periplasmic thioredoxin is insufficient to render dsbA ؊ cells susceptible to infection by M13, a phenotype that is critically dependent on disulfide bond formation in the cell envelope. We sought to examine the molecular determinants that are required in order to convert thioredoxin from a reductant into an efficient periplasmic oxidant. A genetic screen for mutations in thioredoxin that render dsbA ؊ cells sensitive to infection by M13 led to the isolation of a single amino acid substitution, G74S. In vivo the TrxA(G74S) mutant exhibited enhanced catalytic activity in the oxidation of alkaline phosphatase but was unable to oxidize FlgI and restore cell motility. In vitro studies revealed that the G74S substitution does not affect the redox potential of the thioredoxin-active site or its kinetics of oxidation by DsbB. Thus, the gain of function afforded by G74S stems in part from its altered substrate specificity, which also rendered the protein more resistant to reduction by DsbD/DsbC in the periplasm.Cells have evolved elaborate mechanisms for the spatial and temporal control of disulfide bond formation in proteins. Normally, protein oxidation occurs in exocytoplasmic compartments (the endoplasmic reticulum or the bacterial periplasm), whereas in the cytoplasm disulfide bond formation is strongly disfavored (1, 2). Thiol oxidation, disulfide reduction, and isomerization are all accomplished by the action of multiple thiol:disulfide oxidoreductases. In the course of these reactions, the enzyme transfers electrons to or from substrate proteins and becomes oxidized or reduced in the process. To complete the catalytic cycle, the proper redox state of the thiol:disulfide oxidoreductase has to be restored by specialized enzymes that transfer electrons from non-thiol redox couples (such as NAD: NADH) or to the respiratory chain. For example, in the bacterial periplasmic space, disulfide bond formation is catalyzed by the highly efficient protein thiol oxidase DsbA (3). Upon transferring its disulfide to a substrate protein, DsbA becomes reduced and has to be recycled by the action of the membrane enzyme DsbB, which then transfers the electrons to the quinones. Similarly, in the cytoplasm, disulfide bond reduction is catalyzed by thioredoxin that is recycled by thioredoxin reductase (the product of the trxB gene), which in turn uses NADPH as electron donor (1). Interestingly, thiol:disulfide oxidoreductases with broad substrate specificity, such as DsbA, DsbC, thioredoxin, and glutaredoxin, all employ catalytic domains that belong to the thioredoxin superfamily. In contrast, the enzymes that recycle the general thiol:disulfide oxidoreductases (respectively DsbB, DsbD, thioredoxin reductase, and glutaredoxin reductase) are more structurally diverse.Although DsbA and thioredoxin (TrxA) display a high degree of structural homology, in the cell they catalyze o...

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Structural basis and kinetics of inter- and intramolecular disulfide exchange in the redox catalyst DsbD

Cited by 127 publications

References 59 publications

Laboratory evolution of one disulfide isomerase to resemble another

Laboratory evolution of one disulfide isomerase to resemble another

The CXXC Motif Is More than a Redox Rheostat

Laboratory Evolution of Escherichia coli Thioredoxin for Enhanced Catalysis of Protein Oxidation in the Periplasm Reveals a Phylogenetically Conserved Substrate Specificity Determinant

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