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PubMed1990

Catalytic mechanism of xylose (glucose) isomerase from Clostridium thermosulfurogenes. Characterization of the structural gene and function of active site histidine.

The gene coding for thermophilic xylose (glucose) isomerase of Clostridium thermosulfurogenes was isolated and its complete nucleotide sequence was determined. The structural gene (xylA) for xylose isomerase encodes a polypeptide of 439 amino acids with an estimated molecular weight of 50,474. The deduced amino acid sequence of thermophilic C. thermosulfurogenes xylose isomerase displayed higher homology with those of thermolabile xylose isomerases from Bacillus subtilis (70%) and Escherichia coli (50%) than with those of thermostable xylose isomerases from Ampullariella (22%), Arthrobacter (23%), and Streptomyces violaceoniger (24%). Several discrete regions were highly conserved throughout the amino acid sequences of all these enzymes. To identify the histidine residue of the active site and to elucidate its function during enzymatic xylose or glucose isomerization, histidine residues at four different positions in the C. thermosulfurogenes enzyme were individually modified by site-directed mutagenesis. Substitution of His101 by phenylalanine completely abolished enzyme activity whereas substitution of other histidine residues by phenylalanine had no effect on enzyme activity. When His101 was changed to glutamine, glutamic acid, asparagine, or aspartic acid, approximately 10-16% of wild-type enzyme activity was retained by the mutant enzymes. The Gln101 mutant enzyme was resistant to diethylpyrocarbonate inhibition which completely inactivated the wild-type enzyme, indicating that His101 is the only essential histidine residue involved directly in enzyme catalysis. The constant Vmax values of the Gln101, Glu101, Asn101, and Asp101 mutant enzymes over the pH range of 5.0-8.5 indicate that protonation of His101 is responsible for the reduced Vmax values of the wild-type enzyme at pH below 6.5. Deuterium isotope effects by D-[2-2H]glucose on the rate of glucose isomerization indicated that hydrogen transfer and not substrate ring opening is the rate-determining step for both the wild-type and Gln101 mutant enzymes. These results suggest that the enzymatic sugar isomerization does not involve a histidine-catalyzed proton transfer mechanism. Rather, essential histidine functions to stabilize the transition state by hydrogen bonding to the C5 hydroxyl group of the substrate and this enables a metal-catalyzed hydride shift from C2 to C1.

Aldose-Ketose Isomerases↗
PubMed1978

The shape of L-arabinose isomerase from Escherichia coli.

L-Arabinose isomerase, EC 5.3.1.4, catalyzes the conversion of L-arabinose to L-ribulose, the first step in the catabolism of L-arabinose by Escherichia coli B/r. Patrick and Lee (1969) J. Biol. Chem. 244, 4277--4283) demonstrated that native L-arabinose isomerase is composed of six identical subunits of approximately Mr = 60,000. In this paper we describe an electron microscopy study of the arrangement of the six identical subunits. The isomerase is seen in two distinctly different orientations. The first has three subunits visible, with a 3-fold axis of symmetry, corresponding to a face-on view of two stacked, eclipsed trimers. The second orientation is rectangular in shape with 2-fold symmetry; suggesting a side-on view of the stacked trimers. The six identical subunits are thus arranged with D3 symmetry as in a trigonal prism. Measurements were made on the maximum profile of the three 2-fold axes of symmetry of the face-on orientations, and of both the long and short dimensions of the side-on orientation. The best estimate for the maximum profile of the 2-fold axes of symmetry of the face-on view is 106 +/- 8 A, using glutamine synthetase as an internal size standard. Measurements from micrographs of the isomerase alone, using an external magnification calibration, give the following results: for the maximum profile of the three 2-fold axes of symmetry of the face-on view, 132 +/- 7 A; for the long axis of the side-on view, 136 +/- 10 A; and for the short axis, 105 +/- 6 A. These measurements are consisting with the interpretation of the profiles as representing two different orientations of the L-arabinose isomerase.

Aldose-Ketose Isomerases↗
PubMed1993

Site-directed mutagenesis of putative active-site amino acid residues of 3,2-trans-enoyl-CoA isomerase, conserved within the low-homology isomerase/hydratase enzyme family.

During beta-oxidation of unsaturated fatty acids, mitochondrial 3,2-trans-enoyl-CoA isomerase (mECI) converts 3-cis- or 3-trans-enoyl-CoA intermediates into their 2-trans isomers. The cDNA-derived amino acid sequence of mECI shows weak but significant homologies to the peroxisomal trifunctional enzyme (pTFE), the alpha-subunit of the fatty acid degradation complex from Escherichia coli (FadB), the mitochondrial 2-enoyl-CoA hydratase (mECH), the naphthoate synthase encoded by the menB gene from Bacillus subtilis (MenB), and the 4-chlorobenzoyl-CoA dehalogenase from Pseudomonas sp. (CBDH). These proteins from the isomerase/hydratase enzyme family. Tyr-150, Arg-151, and Asp-211 of the mECI are the only amino acids with protic side chains conserved within the enzymes with isomerase activity (pTFE and FadB). These amino acids are exchanged in the remaining enzymes of the isomerase/hydratase family. Glu-165 is conserved in all enzymes with isomerase and/or hydratase activity (pTFE, FadB, and mECH). We argue that these amino acids are possibly involved in the proton transfer at the active site of mECI. To test this hypothesis, mECI was functionally expressed in E. coli. The recombinant enzyme (rmECI) exhibits the same specific activity as the enzyme from rat liver. Exchange of the candidate active-site amino acids by site-directed mutagenesis revealed that Tyr-150 is not involved in isomerase catalysis. The exchange of Arg-151 and Asp-211 leads to a reduced expression of the recombinant enzyme accompanied by a reduced specific activity. The replacement of Glu-165 by Gln leads to a strongly reduced enzymatic activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1988

Evidence that the fadB gene of the fadAB operon of Escherichia coli encodes 3-hydroxyacyl-coenzyme A (CoA) epimerase, delta 3-cis-delta 2-trans-enoyl-CoA isomerase, and enoyl-CoA hydratase in addition to 3-hydroxyacyl-CoA dehydrogenase.

Genetic complementation of a mutant defective in fatty acid oxidation (fadAB) with plasmids containing DNA inserts from the fadAB region of the Escherichia coli genome was studied. The mutant containing the hybrid plasmid with a 5.2-kilobase (kb) PstI-SalI fragment was found to overproduce 3-hydroxyacyl-coenzyme A (CoA) epimerase and delta 3-cis-delta 2-trans-enoyl-CoA isomerase as well as three other beta-oxidation enzymes by 16- to 18-fold compared with the wild-type parental strain LE392. The purification of a fully functional multienzyme complex of fatty acid oxidation from the transformant ultimately established that the 5.2-kb DNA fragment contained an entire fadAB operon. Since immunotitration of cell extracts with antibodies against the fatty acid oxidation complex proved that all 3-hydroxyacyl-CoA epimerase and delta 3-cis-delta 2-trans-enoyl-CoA isomerase activities were associated with the complex, no genetic loci other than the fadAB operon encoded these two enzymes. Moreover, the binding of antibodies caused parallel inhibition of four component enzymes, whereas 3-ketoacyl-CoA thiolase activity was slightly increased. These findings support the suggestion that the epimerase and isomerase as well as enoyl-CoA hydratase and L-3-hydroxyacyl-CoA dehydrogenase are located on the same polypeptide. The results of this study, together with published data (S.-Y. Yang and H. Schulz, J. Biol. Chem. 258:9780-9785, 1983), lead to the conclusion that 3-hydroxyacyl-CoA epimerase, delta 3-cis-delta 2-trans-enoyl-CoA isomerase, and enoyl-CoA hydratase in addition to 3-hydroxyacyl-CoA dehydrogenase are encoded by the fadB gene.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1963

INDUCTION AND REPRESSION OF L-ARABINOSE ISOMERASE IN PEDIOCOCCUS PENTOSACEUS.

Dobrogosz, Walter J. (University of Illinois, Urbana) and Ralph D. DeMoss. Induction and repression of l-arabinose isomerase in Pediococcus pentosaceus. J. Bacteriol. 85:1350-1355. 1963.-The inducible l-arabinose isomerase of Pediococcus pentosaceus can be rapidly and conveniently measured in whole-cell preparations by use of a standard colorimetric procedure originally developed for studies with cell-free enzyme preparations. The enzyme is measured by its ability to catalyze the isomerization of l-arabinose to l-ribulose. Whole cells suspended in a suitable buffer and pretreated with toluene were shown to exhibit this isomerase activity at a level comparable with that observed in cell-free enzyme preparations. Conditions for optimal induction of l-arabinose isomerase are described. In addition, it was determined that the formation of this enzyme is subject to repression by glucose, i.e., via catabolite repression.

Aldose-Ketose Isomerases↗
PubMed1990

Delta 3,delta 2-enoyl-CoA isomerases. Characterization of the mitochondrial isoenzyme in the rat.

Delta 3,delta 2-Enoyl-CoA isomerase (EC 5.3.3.8), an obligatory auxiliary enzyme for the metabolism of double bonds at odd-numbered positions of fatty acids during their beta-oxidation, was studied in hearts and livers of normal and clofibrate-treated rats. Hepatic peroxisomal and mitochondrial isoenzymes were separable by dye-ligand chromatography. The mitochondrial one was further purified to apparent homogeneity. An isomerase was also purified from heart muscle, a peroxisome-poor tissue. These enzymes were dimeric basic proteins (pI 9.5) with a subunit molecular weight of 30,000. Both cis- and trans-enoyl-CoA served as substrates for the hepatic enzyme studied. The velocity ratio for the C6-, C10-, and C12-trans-3-enoyl substrates was 9:2.5:1. By immunoelectron microscopy the enzyme protein selected for purification was found to be mitochondrial both in liver and heart. Chromatographic evidence, immunoelectron microscopy, and immunoblotting indicated that in the liver but not in the heart, the enzyme underwent an induction of 1 order of magnitude during clofibrate treatment. Antibodies towards the rat isomerases detected cross-reactive proteins in bovine and pig liver and heart and human placenta. The estimated subunit sizes varied from species to species, being 31,000 in bovine liver and heart, 29,000 in pig liver and heart, and 30,000 in human placenta. The data are in accord with the notion of a dual location of the delta 3,delta 2-enoyl-CoA isomerase. Mitochondrial origin of one of the isoenzymes and its tissue-specific induction by clofibrate were verified by immunochemistry and the identity of the peroxisomal one revealed by the chromatographic behavior of the proteins.

Animals↗
PubMed1990

Peroxisomal bifunctional protein from rat liver is a trifunctional enzyme possessing 2-enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and delta 3, delta 2-enoyl-CoA isomerase activities.

Peroxisomal delta 3, delta 2-enoyl-CoA isomerase (EC 5.3.3.8) was studied in the liver of rats treated with clofibrate. The mitochondrial and peroxisomal isoenzymes were separated chromatographically and the peroxisomal isomerase purified to apparent homogeneity. In addition to the isomerization of 3-enoyl-CoA esters, the purified protein also catalyzed hydration of trans-2-enoyl-CoA and oxidation of L-3-hydroxyacyl-CoA. Incubation of the purified protein with trans-3-decenoyl-CoA, NAD+, and Mg2+ resulted in an increase in absorbance at 303 nm, indicating the formation of 3-ketoacyl-CoA. The protein purified was monomeric, with an estimated molecular weight of 78,000. In immunoblotting it was recognized by the antibody to peroxisomal bifunctional protein from rat liver. Comparison of the amino acid sequences of cyanogen bromide cleaved isomerase with the known sequence of the peroxisomal bifunctional protein from the rat identified them as the same molecule. In control experiments, the peroxisomal bifunctional protein purified according to published methods also catalyzed delta 3, delta 2-enoyl-CoA isomerization. This means that the bifunctional protein of rat liver is in fact a trifunctional enzyme possessing delta 3, delta 2-enoyl-CoA isomerase, 2-enoyl-CoA hydratase (EC 4.2.1.17), and L-3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) activities in the same polypeptide.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1970

Hydroperoxide isomerase: a new enzyme of lipid metabolism.

An enzyme has been isolated from flaxseed (Linum usitatissimum) which utilizes the product of lipoxidase for its substrate. The enzyme, termed hydroperoxide isomerase, converts the conjugated diene hydroperoxide of linoleic acid to the corresponding monoenoic ketohydroxy fatty acid. The structure of the latter has been determined by ultraviolet, infrared, and nuclear magnetic resonance spectroscopy; periodate and permangate oxidation; gas chromatography; and thin layer chromatography. Hydroperoxide isomerase activity has also been demonstrated in crude extracts from barley (Hordeum vulgare), wheat germ (Triticum aestivum), mung beans (Phaseolus aureus), and corn (Zea mays) and from partially purified extracts of soybean (Glycine max).The hydroperoxide isomerase enzyme from flaxseed has a pH optimum of 7.0. The enzyme was not inhibited by nordihydroguairetic acid, p-chloromercuribenzoic acid, or cyanide, but it was inhibited by cupric ion. One hundred per cent of the activity was lost by heating the enzyme for 1 minute at 68 C. Both linoleic and linolenic acid hydroperoxides can serve as substrates for the enzyme. The pH optimum for the hydroperoxide isomerase enzyme from barley is 6.2; from wheat germ, 6.1; and from soybean, 6.1.The identification of the hydroperoxide isomerase enzyme clarifies the role of lipoxidase in plant tissue and suggests a participation of lipid in the electron transport system.

Journal Article↗
PubMed1976

Studies on glucose isomerase from a Streptomyces species.

Production and properties of glucose isomerase from a Co2+-sensitive Streptomyces species were studied. After 4 days of shaking cultivation at 30 degrees C and 200 rpm, a maximum of 1.1 enzyme units per ml of broth was obtained. Cell-free glucose isomerase, obtained from mycelia heat-treated in the presence of 0.5 mM Co2+, showed a 3.5-fold increase in specific activity over enzyme obtained from untreated mycelia. The optimum pH and temperature for the glucose isomerase were 7 to 8 and 80 degrees C, respectively. The Michaelis constant for fructose was 0.40 M. Mg2+ was found to enhance the glucose isomerase activity, whereas the effect of Co2+ on enzyme activity depended on the manner in which the enzyme was prepared. This glucose isomerase was quite heat stable, with a half-life of 120 h at 70 degrees C.

Carbohydrate Epimerases↗
PubMed1990

Characterization of thermoanaerobacter glucose isomerase in relation to saccharidase synthesis and development of single-step processes for sweetener production.

Regulation of glucose isomerase synthesis was studied in Thermoanaerobacter strain B6A, which fermented a wide variety of carbohydrates including glucose, xylose, lactose, starch, and xylan. Glucogenic amylase activities and beta-galactosidase were produced constitutively, whereas the synthesis of glucose isomerase was induced by either xylose or xylan. Production of these saccharidase activities was not significantly repressed by the presence of glucose or 2-deoxyglucose in the growth media. Glucose isomerase production was optimized by controlling the culture pH at 5.5 during xylose fermentation. The apparent temperature and pH optima for these cell-bound saccharidase activities were as follows: glucose isomerase, 80 degrees C, pH 7.0 to 7.5; glucogenic amylase, 70 degrees C, pH 5.0 to 5.5; and beta-galactosidase, 60 degrees C, pH 6.0 to 6.5 Glucose isomerase, glucogenic amylase, and beta-galactosidase were produced in xylose-grown cells that were active and stable at 60 to 70 degrees C and pH 6.0 to 6.5. Under single-step process conditions, these saccharidase activities in whole cells or cell extracts converted starch or lactose directly into fructose mixtures. A total of 96% of initial liquefied starch was converted into a 49:51 mixture of glucose and fructose, whereas 85% of initial lactose was converted into a 40:31:29 mixture of galactose, glucose, and fructose.

Journal Article↗
PubMed1979

Salmonella typhimurium newD and Escherichia coli leuC genes code for a functional isopropylmalate isomerase in Salmonella typhimurium-Escherichia coli hybrids.

The supQ newD gene substitution system in Salmonella typhimurium restores leucine prototrophy to leuD mutants by providing the newD gene product which is capable of replacing the missing leuD polypeptide in the isopropylmalate isomerase, a complex of the leuC and leuD gene product. Mutations in the supQ gene are required to make the newD protein available. An Escherichia coli F' factor was constructed which carried supQ- newD+ from S. typhimurium on a P22-specialized transducing genome. This F' pro lac (P22dsupQ394newD) episome was transferred into S. typhimurium strains containing th leuD798-ara deletion; the resulting merodiploid strains had a Leu+ phenotype, indicating that supQ- newD+ is dominant over supQ+ newD+, and eliminating the possibility that the supQ gene codes for a repressor of the newD gene. Furthermore, transfer of the F' pro lac (P22dsupQ39newD) into E. coli leuD deletion strains restored leucine prototrophy, showing that the S. typhimurium newD gene can complment the E. coli leuC gene. Growth rates of the S. typhimurium-E coli hybrid strains indicated that the mutant isopropylmalate isomerase in these strains does not induce a leucine limitation, as it does in S. typhimurium leuD supQ mutants. In vitro activity of the mutant isopropylmalate isomerase was demonstrated; the Km values for alpha-isopropylmalate of both the S. typhimurium leuC-newD isomerase and the S. typhimurium-E. coli hybrid isomerase were as much as 100 times higher than the Km values for alpha-isopropylmalate of the wild-type enzyme, which was 3 x 10(-4) M. Mutagenesis of E. coli leuD deletion strains failed to restore leucine prototrophy, indicating that E. coli does not have genes analogous to the S. typhimurium supQ newD genes, of that, if present, activation of a newD is a rare event or is lethal to the cell.

Escherichia coli↗
PubMed1996

Amino acid sequence of peptidyl-prolyl isomerase a of Fusarium sporotrichioides.

Peptidyl-prolyl-cis-trans isomerase catalyzes the interconversion of the cis and trans isomers of the proline-containing polypeptides and the folding process of proteins. This protein was known to be cyclophilin which has high binding affinity for cyclosporin A, a cyclic undecapeptide of fungal origin with potent immunosuppressive property agent. The two cytosolic peptidyl-prolyl-isomerases were found from Fusarium sporotrichioides. The amino acid sequence of the major peptidyl-prolyl isomerase a was determined by conventional sequencing methods; the protein with a calculated molecular mass of 19.7 kDa consisting of 179 amino acids. The comparison of the amino acid sequence of peptidyl-prolyl-isomerase from Fusarium with that of Nucerospora crassa revealed a significant degree of amino acid sequence homology (82.2%).

Amino Acid Isomerases↗
PubMed1994

Characterization of DsbC, a periplasmic protein of Erwinia chrysanthemi and Escherichia coli with disulfide isomerase activity.

We identified and characterized an Erwinia chrysanthemi gene able to complement an Escherichia coli dsbA mutation that prevents disulfide bond formation in periplasmic proteins. This gene, dsbC, codes for a 24 kDa periplasmic protein that contains a characteristic active site sequence of disulfide isomerases, Phe-X-X-X-X-Cys-X-X-Cys. Besides the active site, DsbC has no homology with DsbA, thioredoxin or eukaryotic protein disulfide isomerase and it could define a new subfamily of disulfide isomerases. Purified DsbC protein is able to catalyse insulin oxidation in a dithiothreitol dependent manner. The E.coli gene xprA codes for a protein functionally equivalent to DsbC. The in vivo function of DsbC seems to be the formation of disulfide bonds in proteins. The presence of XprA could explain the residual disulfide isomerase activity existing in dsbA mutants. Re-oxidation of XprA does not seem to occur through DsbB, the protein that probably re-oxidizes DsbA.

Amino Acid Sequence↗
PubMed1995

Secretion by Trypanosoma cruzi of a peptidyl-prolyl cis-trans isomerase involved in cell infection.

Macrophage infectivity potentiators are membrane proteins described as virulence factors in bacterial intracellular parasites, such as Legionella and Chlamydia. These factors share amino acid homology to eukaryotic peptidyl-prolyl cis-trans isomerases that are inhibited by FK506, an inhibitor of signal transduction in mammalian cells with potent immunosuppressor activity. We report here the characterization of a protein released into the culture medium by the infective stage of the protozoan intracellular parasite Trypanosoma cruzi. The protein possesses a peptidyl-prolyl cis-trans isomerase activity that is inhibited by FK506 and its non-immunosuppressing derivative L-685,818. The corresponding gene presents sequence homology with bacterial macrophage infectivity potentiators. The addition of the protein, produced heterologously in Escherichia coli, to cultures of trypomastigotes and simian epithelial or HeLa cells enhances invasion of the mammalian cells by the parasites. Antibodies raised in mice against the T.cruzi isomerase greatly reduce infectivity. A similar reduction of infectivity is obtained by addition to the cultures of FK506 and L-685,818. We concluded that the T.cruzi isomerase is involved in cell invasion.

Amino Acid Isomerases↗
PubMed1996

Human isopentenyl diphosphate: dimethylallyl diphosphate isomerase: overproduction, purification, and characterization.

Isopentenyl diphosphate (IPP):dimethylallyl diphosphate isomerase catalyzes an essential activation step in the isoprenoid biosynthetic pathway. A human cDNA sequence [J. Xuan, J. Kowalski, A.F. Chambers, and D.T. Denhardt (1994) Genomics 20, 129-131] containing a 684-base-pair open reading frame was recently reported that encoded a protein with a significant degree of similarity to two fungal IPP isomerases [F.M. Hahn and C.D. Poulter (1995) J. Biol. Chem. 270, 11298-11303]. The human cDNA sequence was cloned into expression plasmid pFMH12. The encoded protein was overproduced in Escherichia coli and purified to > 90% homogeneity in two steps by ion-exchange and hydrophobic interaction chromatography. The recombinant protein catalyzed the isomerization of IPP to dimethylallyl diphosphate and was maximally active at pH 7.0 in the presence of Mg2+. The Michaelis constant for IPP was 33 microM, similar to the value of 43 microM reported for yeast IPP isomerase; Vmax = 4.1 mumol min-1 mg-1 for recombinant human IPP isomerase, approximately fivefold less than reported for the yeast enzyme [I.P. Street and C.D. Poulter (1990) Biochemistry 29, 7531-7538].

Amino Acid Sequence↗
PubMed2001

The crystal structure of delta(3)-delta(2)-enoyl-CoA isomerase.

The active-site geometry of the first crystal structure of a Delta(3)-Delta(2)-enoyl-coenzyme A (CoA) isomerase (the peroxisomal enzyme from the yeast Saccharomyces cerevisiae) shows that only one catalytic base, Glu158, is involved in shuttling the proton from the C2 carbon atom of the substrate, Delta(3)-enoyl-CoA, to the C4 atom of the product, Delta(2)-enoyl-CoA. Site-directed mutagenesis has been performed to confirm that this glutamate residue is essential for catalysis. This Delta(3)-Delta(2)-enoyl-CoA isomerase is a hexameric enzyme, consisting of six identical subunits. It belongs to the hydratase/isomerase superfamily of enzymes which catalyze a wide range of CoA-dependent reactions. The members of the hydratase/ isomerase superfamily have only a low level of sequence identity. Comparison of the crystal structure of the Delta(3)-Delta(2)-enoyl-CoA isomerase with the other structures of this superfamily shows only one region of large structural variability, which is in the second turn of the spiral fold and which is involved in defining the shape of the binding pocket.

Amino Acid Sequence↗
PubMed1996

Protein disulfide isomerase: a multifunctional protein of the endoplasmic reticulum.

Protein disulfide isomerase (PDI) is a resident enzyme of the endoplasmic reticulum (ER) that was discovered over three decades ago. Contemporary biochemical and molecular biology techniques have revealed that it is present in all eukaryotic cells studied and retained in the ER via a -KDEL or -HDEL sequence at its C-terminus. However, evidence is accumulating that in certain cell types, PDI can be found in other subcellular compartments, despite possessing an intact retention sequence. A wide range of studies has established that in presence of a redox pair, PDI acts catalytically to both form and reduce disulfide bonds, therefore acting as a disulfide isomerase. Recent studies have focused on the mechanism of the isomerization process and the precise role of the two active site sequences (-CGHC-) in the process. In addition, prokaryotes have been shown to possess a set of proteins that function in a similar fashion, being able to generate disulfide bonds on polypeptides translocated into the periplasmic space. Following the recent discovery that PDI binds peptides, coupled with earlier findings that PDI is a subunit of at least two enzymatic complexes (prolyl 4-hydroxylase and microsomal triglyceride transfer protein), it seems that it may serve functions other than merely that of a disulfide isomerase. In fact, it is now clear that PDI can facilitate protein folding independently of its disulfide isomerase activity. A major challenge for the future is to define mechanistically how it accomplishes isomerization and the relationship between this process and the protein folding steps that culminate in the final, fully mature protein.

Bacterial Proteins↗
PubMed1993

An isoform of protein disulfide isomerase is expressed in the developing acrosome of spermatids during rat spermiogenesis and is transported into the nucleus of mature spermatids and epididymal spermatozoa.

We have purified an isoform of protein disulfide isomerase (EC 5.3.4.1) from rat liver, and raised a specific antibody against the purified protein in rabbit. Immunohistochemical studies using this antibody on rat testis sections, at both light and electron microscopic levels, showed a specific localization of the isoform of protein disulfide isomerase in the developing acrosome of the spermatids. The protein was transferred to the acrosomic vesicle from the Golgi apparatus at late Golgi phase, and remained present in the acrosome of spermatids during cap phase, acrosome phase, and maturation phase. In addition to the acrosome, the protein appeared in the nucleus of spermatids during maturation phase, and was localized in the nucleus of epididymal spermatozoa. By immunoblot analysis, almost all of the isoform of protein disulfide isomerase in the testis was found to be extractable by an isotonic buffer. On the contrary, detergent extraction was required for complete solubilization of the protein in the epididymis. These results suggest that the isoform of protein disulfide isomerase is a new intra-acrosomal soluble protein, and that the protein begins to enter the nucleus of mature spermatids in the testis and tightly binds to the nuclear components in epididymal spermatozoa.

Acrosome↗