Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “ISOMERASES”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 649 records · Page 36Linked to original sources
PubMed1989

Cyclophilin and peptidyl-prolyl cis-trans isomerase are probably identical proteins.

The enzyme peptidyl-prolyl cis-trans isomerase (PPIase) was recently discovered in mammalian tissues and purified from porcine kidney. It catalyses the slow cis-trans isomerization of proline peptide (Xaa-Pro) bonds in oligopeptides and accelerates slow, rate-limiting steps in the folding of several proteins. Here, we report the N-terminal sequence of PPIase together with further chemical and enzymatic properties. The results indicate that this enzyme is probably identical to cyclophilin, a recently discovered mammalian protein which binds tightly to cyclosporin A (CsA). Cyclophilin is thought to be linked to the immunosuppressive action of CsA. The first 38 amino-acid residues of porcine PPIase and of bovine cyclophilin are identical and the two proteins both have a relative molecular mass of about 17,000 (ref. 7). The catalysis of prolyl isomerization in oligopeptides and of protein folding by PPIase are strongly inhibited in the presence of low levels of CsA. The activities of both PPIase and cyclophilin depend on a single sulphydryl group. At present it is unknown whether the inhibition of prolyl isomerase activity is related with the immunosuppressive action of CsA.

Amino Acid Isomerases↗
PubMed1989

A receptor for the immunosuppressant FK506 is a cis-trans peptidyl-prolyl isomerase.

The structurally novel macrolide FK506 (refs 1,2) has recently been demonstrated to have potent immunosuppressive activity at concentrations several hundredfold lower than cyclosporin A (CsA). Cyclosporin A, a cyclic peptide, has found widespread clinical use in the prevention of graft rejection following bone marrow and organ transplantation. The mechanisms of immunosuppression mediated by FK506 and CsA appear to be remarkably similar, suggesting that these unrelated structures act on a common receptor or on similar molecular targets, perhaps the CsA receptor, cyclophilin, which has recently been shown by Fischer et al. and Takahashi et al. to have cis-trans peptidyl-prolyl isomerase activity. We have prepared an FK506 affinity matrix and purified a binding protein for FK506 from bovine thymus and from human spleen. This FK506-binding protein (FKBP) has a relative molecular mass (Mr) of approximately 14,000(14K), a pI of 8.8-8.9, and does not cross-react with antisera against cyclophilin. The first 40 N-terminal residues of the bovine and 16 residues of the human FKBP were determined; the 16-residue fragments are identical to each other and unrelated to any known sequences. This protein catalyses the cis-trans isomerization of the proline amide in a tetrapeptide substrate and FK506 inhibits the action of this new isomerase. The FKBP and cyclophilin appear to be members of an emerging class of novel proteins that regulate T cell activation and other metabolic processes, perhaps by the recognition (and possibly the isomerization) of proline-containing epitopes in target proteins.

Amino Acid Isomerases↗
PubMed2002

The prolyl isomerase Pin1 is a regulator of p53 in genotoxic response.

p53 is activated in response to various genotoxic stresses resulting in cell cycle arrest or apoptosis. It is well documented that DNA damage leads to phosphorylation and activation of p53 (refs 1-3), yet how p53 is activated is still not fully understood. Here we report that DNA damage specifically induces p53 phosphorylation on Ser/Thr-Pro motifs, which facilitates its interaction with Pin1, a member of peptidyl-prolyl isomerase. Furthermore, the interaction of Pin1 with p53 is dependent on the phosphorylation that is induced by DNA damage. Consequently, Pin1 stimulates the DNA-binding activity and transactivation function of p53. The Pin1-mediated p53 activation requires the WW domain, a phosphorylated Ser/Thr-Pro motif interaction module, and the isomerase activity of Pin1. Moreover, Pin1-deficient cells are defective in p53 activation and timely accumulation of p53 protein, and exhibit an impaired checkpoint control in response to DNA damage. Together, these data suggest a mechanism for p53 regulation in cellular response to genotoxic stress.

Amino Acid Motifs↗
PubMed2007

Peptidyl-prolyl cis/trans isomerases and transcription: is there a twist in the tail?

Eukaryotic transcription is regulated predominantly by the post-translational modification of the participating components. One such modification is the cis-trans isomerization of peptidyl-prolyl bonds, which results in a conformational change in the protein involved. Enzymes that carry out this reaction include the yeast peptidyl-prolyl cis/trans isomerase Ess1 and its human counterpart Pin1, both of which recognize phosphorylated target motifs exclusively. Consequently, they operate together with proline-directed serine-threonine kinases and phosphatases. High-profile client proteins involved in transcription include steroid hormone receptors, cell-cycle regulators and immune mediators. Other key targets are elements of the transcription machinery, including the multiply phosphorylated carboxy-terminal domain of RNA polymerase II. Changes in isomerase activity have been shown to alter the transactivation potential, protein stability or intracellular localization of these client proteins. The resulting disruption to developmental processes and cell proliferation has been linked, in some cases, to human cancers.

Cell Cycle Proteins↗
PubMed2003

Occurrence of a putative ancient-like isomerase involved in histidine and tryptophan biosynthesis.

We report the occurrence of an isomerase with a putative (betaalpha)(8)-barrel structure involved in both histidine and trypto-phan biosynthesis in Streptomyces coelicolor A3(2) and Mycobacterium tuberculosis HR37Rv. Deletion of a hisA homologue (SCO2050) putatively encoding N'-[(5'-phosphoribosyl)-formimino]-5 amino-imidazole-4-carboxamide ribonucleotide isomerase from the chromosome of S. coelicolor A3(2) generated a double auxotrophic mutant for histidine and tryptophan. The bifunctional gene SCO2050 and its orthologue Rv1603 from M. tuberculosis complemented both hisA and trpF mutants of Escherichia coli. Expression of the E. coli trpF gene in the S. coelicolor mutant only complemented the tryptophan auxo-trophy, and the hisA gene only complemented the histidine auxotrophy. The discovery of this enzyme, which has a broad-substrate specificity, has implications for the evolution of metabolic pathways and may prove to be important for understanding the evolution of the (betaalpha)(8)-barrels.

Aldose-Ketose Isomerases↗
PubMedDate not supplied

Proteasomal degradation of human peptidyl prolyl isomerase pin1-pointing phospho Bcl2 toward dephosphorylation.

Microtubule inhibitor-induced Bcl2 phosphorylation is detrimental to its antiapoptotic function. Phosphorylation of Bcl2 predominantly occurs on two serine residues (70 and 87) in cells arrested at G2-M phase by microtubule disarraying agents. Phospho Bcl2 can associate with a cis-trans peptidyl prolyl isomerase, Pin1. Pin1 and its homologues are known to target the proline residue carboxyl terminal to the phosphorylated threonine or serine residue of mitotic phosphoproteins, such as Bcl2. However, it was not clear how an extranuclear protein could associate with nuclear Pin1. The confocal images of the immunofluorescence studies employing phospho Bcl2-specific antibody developed in the laboratory demonstrated the translocation of phospho Bcl2 inside the nucleus. Interestingly, proteasomal degradation of Pin1 facilitates dephosphorylation of phospho Bcl2 due to longer exposure of Taxol. Here we show for the first time that proteasomal degradation of Pin1 is the key factor to determine the fate of phosphoforms of Bcl2. When Pin1 is degraded by proteasomes, phospho Bcl2 is converted to its native form. Thus, transient conformational change of Bcl2 due to association with peptidyl prolyl isomerase can contribute to irreversible apoptotic signaling.

Antineoplastic Agents↗
PubMed1977

The substrate specificity and stereochemistry, reversibility and inhibition of the 3-oxo steroid delta 4-delta 5-isomerase component of cholesterol oxidase.

1. 5-Cholesten-3-one was shown to be an intermediate in the conversion of cholesterol into 4-cholesten-3-one by Nocardia cholesterol oxidase. 2. The absence of a C-17 side chain from 5-androstene-3,17-dione slightly increased the Vmax. of the isomerase activity relative to 5-cholesten-3-one (1.7-fold), but greatly increased the Km. 3. Incubations of [4alpha-2H]-and [4beta-2H]-cholesterol with cholesterol oxidase showed that the 4beta-hydrogen atom can be transferred to the 6beta-position. However, incubations of cholesterol, 5-cholesten-3-one and 4-cholesten-3-one with the enzyme in 2H2O led to some incorporation of 2H into the 4-cholesten-3-one products, mostly at position 6beta. 4. Both the isomerase and the oxidase activities of cholesterol oxidase were inhibited by 5,10-seco-19-nor-5-cholestyne-3,10-dione.

3-Hydroxysteroid Dehydrogenases↗
PubMed1980

Mechanistic and stereochemical studies on 3-oxo steroid delta 4-delta 5-isomerase from human placenta.

The mechanism of isomerization of delta 5-3-ox steroids to delta 4-3-oxo steroids was examined by using the membrane-bound 3-oxo steroid delta 4-delta 5-isomerase (EC 5.3.3.1) and the 3 beta-hydroxy steroid dehydrogenase present in the microsomal fraction obtained from full-term human placenta. (1) Methods for the preparation of androst-5-ene-3 beta, 17 beta-diol specifically labelled at the 4 alpha-, 4 beta- or 6-positions are described. (2) Incubations with androst-5-ene-3 beta, 17 beta-diol stereospecifically 3H-labelled either in the 4 alpha- or 4 beta-position showed that the isomerization reaction occurs via a stereospecific elimination of the 4 beta hydrogen atom. In addition, the complete retention of 3H in the delta 4-3-oxo steroids obtained from [4 alpha-3H]androst-5-ene-3 beta, 17 beta-diol indicates that the non-enzymic contribution to these experiments was negligible. (3) To study the stereochemistry of the insertion of the incoming proton at C-6, the [6-3H]androst-4-ene-3, 17-dione obtained from the oxidation isomerization of [6-3H]androst-5-ene-3 beta, 17 beta-diol was enzymically hydroxylated in the 6 beta-position by the fungus Rhizopls stolonifer. Retention of 3H in the 6 alpha-position of the isolated 6 beta-hydroxyandrost-4-ene-3, 17-dione indicates that in the isomerase-catalysed migration of the C(5) = C(6) double bond, the incoming proton from the acidic group on the enzyme must enter C-6 from the beta-face, forcing the existing 3H into the 6 alpha-position.

Androstenediol↗
PubMed1980

Protein disulphide-isomerase activity in chick-embryo tissues. Correlation with the biosynthesis of procollagen.

Protein disulphide-isomerase activity was determined in homogenates of chick-embryo tissues. Activities were highest in tissues active in procollagen synthesis and were maximal at the developmental stage of maximal procollagen synthesis. These variations in protein disulphide-isomerase activity correlate closely with those previously observed for protocollagen hydroxylase activities.

Animals↗
PubMed1983

Structural properties of homogeneous protein disulphide-isomerase from bovine liver purified by a rapid high-yielding procedure.

Protein disulphide-isomerase from bovine liver was purified to homogeneity as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, two-dimensional electrophoresis and N-terminal amino acid analysis. The preparative procedure, a modification of that of Carmichael, Morin & Dixon [(1977) J. Biol. Chem. 252, 7163-7167], is much faster and higher-yielding than previous procedures, and the final purified material is of higher specific activity. The enzyme has Mr 57 000 as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, both in the presence and in the absence of thiol compounds. Gel-filtration studies on Sephadex G-200 indicate an Mr of 107 000, suggesting that the native enzyme is a homodimer with no interchain disulphide bonds. Ultracentrifugation studies give a sedimentation coefficient of 3.5S, implying that the enzyme sediments as the monomer. The isoelectric point, in the presence of 8 M-urea, is 4.2, and some microheterogeneity is detectable. The amino acid composition is comparable with previous analyses of this enzyme from bovine liver and of other preparations of thiol:protein disulphide oxidoreductases whose relation to protein disulphide-isomerase has been controversial. The enzyme contains a very high proportion of Glx + Asx residues (27%). The N-terminal residue is His. The pure enzyme has a very small carbohydrate content, determined as 0.5-1.0% by the phenol/H2SO4 assay. Unless specific steps are taken to remove it, the purified enzyme contains a small amount (5 mol/mol of enzyme) of Triton X-100 carried through the purification.

Amino Acids↗
PubMed1995

Time-dependent inhibition of peptidylprolyl cis-trans-isomerases by FK506 is probably due to cis-trans isomerization of the inhibitor's imide bond.

Free in solution, the immunosuppressive compounds cyclosporin A (CsA), FK506, ascomycin and rapamycin are present in many solvents in various slowly interconverting conformations. Together with their cellular receptor proteins, cyclophilin (CyP) and FK506-binding protein (FKBP), however, these inhibitors have been shown to have a homogeneous conformation. The existence of a slow cis-trans interconversion of an imidic bond in the inhibitor molecule during the course of the formation of the CsA-CyP18cy complex (where CyP18cy is human 18 kDa cytosolic CyP) prompted us to investigate the reaction of the peptidomacrolides FK506, ascomycin and rapamycin with two specific binding-proteins in more detail. Since formation of the FK506-FKBP complex results in the inhibition of the peptidylprolyl cis-trans-isomerase activity of the binding protein, we used the enzyme's decrease in enzymic activity to monitor binding of the inhibitors to their enzyme targets. For FK506, the kinetics of inhibition of human 12 kDa cytosolic FKBP (FKBP12cy) were clearly dependent on time. Subsequent to a rapid inactivation reaction, not resolved in its kinetics due to manual mixing, a slow dominant first-order inactivation process with a relaxation time of 1163 s at 10 degrees C was observed. Concomitantly the Ki value of the slow phase dropped 2.6-fold within the first 60 min of incubation. Using the FKBP12cy homologue 25 kDa membrane FKBP (FKBP25mem), a bacterial peptidylprolyl cis-trans-isomerase, the rate and amplitudes of the inhibition reactions were very similar to FKBP12cy. On the other hand, the kinetics and amplitudes of the inhibition of FKBP12cy varied significantly if rapamycin was used as an inhibitor instead of FK 506. Owing to reduced conformation transition in rapamycin upon binding to FKBP12cy, the slow phase during inhibition was significantly decreased in amplitude. A likely reason for this became apparent when the activation-enthalpy and the pH-dependence of the rate constants of the slow phase were determined. We conclude that the cis to trans interconversion of the pipecolinyl bond of the three peptidomacrolides may be responsible for the slow process. There was no indication of a suicide catalysis of this process by FKBPs.

Amino Acid Isomerases↗
PubMed2003

[Unexpected roles of the peptidyl-prolyl cis/trans isomerase Pin1].

Peptidyl-prolyl isomerases (PPIases) are chaperone enzymes which alter the peptide bond between a given amino acid and a proline, changing it from the cis to the trans conformation and vice versa. This modification can cause dramatic structural modifications which can affect the properties of targeted proteins. The ubiquitous PPIase Pin1, conserved from yeast to human, has been shown to be necessary for entry into mitosis. The yeast homologue, Ess1, is essential for cell survival. Pin1 possesses a WW domain which specifically recognizes pSer-Pro and pThr-Pro motifs in which the first amino acid is phosphorylated. Pin1 binds to many proteins implicated in cell cycle regulation (e.g. p53, Myt1, Wee1, and Cdc25C). Pin1 also targets tau, a protein forming part of hte neuronal cytoskeleton which is hyper-phosphorylated in patients suffering from Alzheimer's disease (AD). Pin1 could, therefore, be involved in the pathogenesis of Ad. Furthermore, Pin1 also binds two proteins involved in transcription: Rpb1, the largest subunit of RNA polymerase II and Spt5, a regulator of the elongation of transcription. Both theses proteins possess domains rich in S/T-P motifs which can be targeted by Pin1 when phosphorylated. Recent studies show that Pin1 modulates the dephosphorylation of some proteins by allowing trans-specific phosphatases to recognize their target after isomerization. This unexpected role might allow protein regulation via peptidyl-prolyl isomerase activity.

Alzheimer Disease↗
PubMed1991

Human cyclophilin B: a second cyclophilin gene encodes a peptidyl-prolyl isomerase with a signal sequence.

We report the cloning and characterization of a cDNA encoding a second human cyclosporin A-binding protein (hCyPB). Homology analyses reveal that hCyPB is a member of the cyclophilin B (CyPB) family, which includes yeast CyPB, Drosophila nina A, and rat cyclophilin-like protein. This family is distinguished from the cyclophilin A (CyPA) family by the presence of endoplasmic reticulum (ER)-directed signal sequences. hCyPB has a hydrophobic leader sequence not found in hCyPA, and its first 25 amino acids are removed upon expression in Escherichia coli. Moreover, we show that hCyPB is a peptidyl-prolyl cis-trans isomerase which can be inhibited by cyclosporin A. These observations suggest that other members of the CyPB family will have similar enzymatic properties. Sequence comparisons of the CyPB proteins show a central, 165-amino acid peptidyl-prolyl isomerase and cyclosporin A-binding domain, flanked by variable N-terminal and C-terminal domains. These two variable regions may impart compartmental specificity and regulation to this family of cyclophilin proteins containing the conserved core domain. Northern blot analyses show that hCyPB mRNA is expressed in the Jurkat T-cell line, consistent with its possible target role in cyclosporin A-mediated immunosuppression.

Amino Acid Isomerases↗
PubMed1992

Characterization of the human prolyl 4-hydroxylase tetramer and its multifunctional protein disulfide-isomerase subunit synthesized in a baculovirus expression system.

Prolyl 4-hydroxylase (EC 1.14.11.2), an alpha 2 beta 2 tetramer, catalyzes the posttranslational formation of 4-hydroxyproline in collagens. The enzyme can easily be dissociated into its subunits, but all attempts to associate a tetramer from the dissociated subunits in vitro have been unsuccessful. Molecular cloning of the catalytically important alpha subunit has identified two types of cDNA clone due to mutually exclusive alternative splicing. The beta subunit is a highly unusual multifunctional polypeptide, being identical to the enzyme protein disulfide-isomerase (EC 5.3.4.1). We report here on expression of the alpha and beta subunits of prolyl 4-hydroxylase and a fully active enzyme tetramer in Spodoptera frugiperda insect cells by baculovirus vectors. When the beta subunit was expressed alone, the polypeptide produced was found in a 0.1% Triton X-100 extract of the cell homogenate and was a fully active protein disulfide-isomerase. When either form of the alpha subunit was expressed alone, only traces of the alpha subunit could be extracted from the cell homogenate with 0.1% Triton X-100, and 1% SDS was required to obtain efficient solubilization. These alpha subunits had no prolyl 4-hydroxylase activity. When the cells were coinfected with both alpha- and beta-subunit-producing viruses, an enzyme tetramer was formed, but significant amounts of alpha and beta subunits remained unassociated. The recombinant tetramer was indistinguishable from that isolated from vertebrate tissue in terms of its specific activity and kinetic constants for cosubstrates and the peptide substrate. The two alternatively spliced forms of the alpha subunit gave enzyme tetramers with identical catalytic properties. Baculovirus expression seems to be an excellent system for mass production of the enzyme tetramer and for detailed investigation of the mechanisms involved in the association of the monomers.

Amino Acid Sequence↗
PubMed1995

Cloning, baculovirus expression, and characterization of a second mouse prolyl 4-hydroxylase alpha-subunit isoform: formation of an alpha 2 beta 2 tetramer with the protein disulfide-isomerase/beta subunit.

Prolyl 4-hydroxylase (EC 1.14.11.2) catalyzes the posttranslational formation of 4-hydroxyproline in collagens. The vertebrate enzyme is an alpha 2 beta 2 tetramer, the beta subunit of which is a highly unusual multifunctional polypeptide, being identical to protein disulfide-isomerase (EC 5.3.4.1). We report here the cloning of a second mouse alpha subunit isoform, termed the alpha (II) subunit. This polypeptide consists of 518 aa and a signal peptide of 19 aa. The processed polypeptide is one residue longer than the mouse alpha (I) subunit (the previously known type), the cloning of which is also reported here. The overall amino acid sequence identity between the mouse alpha (II) and alpha (I) subunits is 63%. The mRNA for the alpha (II) subunit was found to be expressed in a variety of mouse tissues. When the alpha (II) subunit was expressed together with the human protein disulfide-isomerase/beta subunit in insect cells by baculovirus vectors, an active prolyl 4-hydroxylase was formed, and this protein appeared to be an alpha (II) 2 beta 2 tetramer. The activity of this enzyme was very similar to that of the human alpha (I) 2 beta 2 tetramer, and most of its catalytic properties were also highly similar, but it differed distinctly from the latter in that it was inhibited by poly(L-proline) only at very high concentrations. This property may explain why the type II enzyme was not recognized earlier, as an early step in the standard purification procedure for prolyl 4-hydroxylase is affinity chromatography on a poly(L-proline) column.

Amino Acid Sequence↗
PubMed1997

Peptidyl prolyl cis-trans isomerase activity of cyclophilin A in functional homo-oligomeric receptor expression.

The functional expression of homo-oligomeric alpha7 neuronal nicotinic and type 3 serotonin receptors is dependent on the activity of a cyclophilin. In this paper we demonstrate that the mechanism of cyclophilin action during functional homo-oligomeric receptor expression in Xenopus oocytes is distinct from the calcineurin-dependent immunosuppressive mechanism by showing that a nonimmunosuppressive analog of cyclosporin A (CsA), SDZ 211-811, reduces functional receptor expression to the same extent as CsA. The cytoplasmic subtype of cyclophilin, cyclophilin A (CyPA), appears to be required for functional receptor expression. This is because overexpression of CyPA and a CyPA mutant that is deficient in CsA binding activity reverses CsA-induced reduction in functional receptor expression. The mechanism of action of CyPA is likely to involve its prolyl isomerase activity because a mutant CyPA with a single amino acid substitution (arginine 55 to alanine) that is predicted to produce a 1000-fold attenuation in isomerase activity fails to reverse the cyclosporin A effect. Our data also suggest that CyPA does not form a stable complex with receptor subunits.

Amino Acid Isomerases↗
PubMed1995

Double bond removal from odd-numbered carbons during peroxisomal beta-oxidation of arachidonic acid requires both 2,4-dienoyl-CoA reductase and delta 3,5,delta 2,4-dienoyl-CoA isomerase.

The pathway for the peroxisomal beta-oxidation of arachidonic acid (5,8,11,14-20:4) was elucidated by comparing its metabolism with 4,7,10-hexadecatrienoic acid (4,7,10-16:3) and 5,8-tetradecadienoic acid (5,8-14:2) which are formed, respectively, after two and three cycles of arachidonic acid degradation. When [1-14C]4,7,10-16:3 was incubated with peroxisomes in the presence of NAD+ and NADPH, it resulted in a time-dependent increase in the production of acid-soluble radioactivity which was accompanied by the synthesis of 2-trans-4,7,10-hexadecatetraenoic acid and two 3,5,7,10-hexadecatetraenoic acid isomers. The formation of conjugated trienoic acids suggests that peroxisomes contain delta 3,5,delta 2,4-dienoyl-CoA isomerase with the ability to convert 2-trans-4,7,10-hexadecatetraenoic acid to 3,5,7,10-hexadecatetraenoic acid. When 1-14C-labeled 6,9,12-octadecatrienoic acid or 7,10,13,16-docosatetraenoic acid was incubated without nucleotides, the 3-hydroxy metabolites accumulated, since further degradation requires NAD(+)-dependent 3-hydroxyacyl-CoA dehydrogenase. When [1-14C]5,8,11,14-20:4 was incubated under identical conditions, no polar metabolite was detected, but 2-trans-4,8,11,14-eicosapentaenoic acid accumulated. When NADPH was added to incubations, 3-hydroxy-8,11,14-eicosatrienoic, 2-trans-4,8,11,14-eicosapentaenoic, 2-trans-8,11,14-eicosatetraenoic, and 8,11,14-eicosatrienoic acids were produced. Analogous compounds were formed from [1-14C]5,8-14:2. Our results show that the removal of double bonds from odd-numbered carbons in arachidonic acid thus requires both NADPH-dependent 2,4-dienoyl-CoA reductase and delta 3,5,delta 2,4-dienoyl-CoA isomerase. One complete cycle of 5,8-14:2 and 5,8,11,14-20:4 beta-oxidation yields, respectively, 6-dodecenoic and 6,9,12-octadecatrienoic acids.

Animals↗
PubMed1996

Eukaryotic protein disulfide isomerase complements Escherichia coli dsbA mutants and increases the yield of a heterologous secreted protein with disulfide bonds.

Eukaryotic protein disulfide isomerase (PDI) is a 55-kDa enzyme with cysteine oxidoreductase, chaperone, and antichaperone activities that catalyzes disulfide formation and rearrangement in the eukaryotic endoplasmic reticulum. In Gram-negative bacteria, the formation of disulfide bonds in the periplasm is mediated by DsbA, a strong cysteine oxidase but an inefficient catalyst of disulfide bond isomerization with no known chaperone activity. We show that rat PDI (rPDI) secreted in the periplasmic space of Escherichia coli can catalyze the formation of disulfide bonds and complement several of the phenotypes of dsbA mutants. The function of rPDI was dependent on the dsbB gene, suggesting that the reoxidation of this eukaryotic enzyme involves direct interactions with bacterial redox proteins. Co-expression of rPDI increased the yield of bovine pancreatic trypsin inhibitor (BPTI) severalfold, an effect that was enhanced when reduced glutathione was added to the growth medium. Whereas PDI is thought to function primarily as an isomerase in the eukaryotic endoplasmic reticulum, rPDI failed to decrease the accumulation of two-disulfide folding intermediates of BPTI and thus did not appear to appreciably catalyze the rate-limiting step in the oxidative folding pathway of BPTI. These results demonstrate that expression of eukaryotic foldases in E. coli can be exploited to better understand their function in vivo and also to increase the yield of biotechnologically valuable proteins.

Animals↗