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Sample records for peptidyl-prolyl isomerase prsa2

  1. Peptidyl prolyl cis-trans-isomerase activity associated with the lumen of the endoplasmic reticulum.

    PubMed Central

    Bose, S; Freedman, R B

    1994-01-01

    Peptidyl prolyl cis-trans-isomerase (PPI) activity was detected in microsomal fractions from bovine and rat liver. Extensive washing, proteinase and sonication treatments indicated that although some of this activity was due to adsorbed cytosolic enzymes, there was also an active but latent microsomal PPI activity. Density-gradient subfractionation indicated that activity was associated with vesicles derived from both the rough and the smooth endoplasmic reticulum (ER), suggesting that the activity was located within the ER lumen. The luminal PPI activity was inhibited by cyclosporin A and was active towards an unfolded protein substrate as well as towards the standard peptide substrate. PMID:8010971

  2. Structural and Biochemical Characterization of the Human Cyclophilin Family of Peptidyl-Prolyl Isomerases

    SciTech Connect

    Davis, Tara L.; Walker, John R.; Campagna-Slater, Valérie; Finerty, Jr., Patrick J.; Paramanathan, Ragika; Bernstein, Galina; MacKenzie, Farrell; Tempel, Wolfram; Ouyang, Hui; Lee, Wen Hwa; Eisenmesser, Elan Z.; Dhe-Paganon, Sirano

    2011-12-14

    Peptidyl-prolyl isomerases catalyze the conversion between cis and trans isomers of proline. The cyclophilin family of peptidyl-prolyl isomerases is well known for being the target of the immunosuppressive drug cyclosporin, used to combat organ transplant rejection. There is great interest in both the substrate specificity of these enzymes and the design of isoform-selective ligands for them. However, the dearth of available data for individual family members inhibits attempts to design drug specificity; additionally, in order to define physiological functions for the cyclophilins, definitive isoform characterization is required. In the current study, enzymatic activity was assayed for 15 of the 17 human cyclophilin isomerase domains, and binding to the cyclosporin scaffold was tested. In order to rationalize the observed isoform diversity, the high-resolution crystallographic structures of seven cyclophilin domains were determined. These models, combined with seven previously solved cyclophilin isoforms, provide the basis for a family-wide structure:function analysis. Detailed structural analysis of the human cyclophilin isomerase explains why cyclophilin activity against short peptides is correlated with an ability to ligate cyclosporin and why certain isoforms are not competent for either activity. In addition, we find that regions of the isomerase domain outside the proline-binding surface impart isoform specificity for both in vivo substrates and drug design. We hypothesize that there is a well-defined molecular surface corresponding to the substrate-binding S2 position that is a site of diversity in the cyclophilin family. Computational simulations of substrate binding in this region support our observations. Our data indicate that unique isoform determinants exist that may be exploited for development of selective ligands and suggest that the currently available small-molecule and peptide-based ligands for this class of enzyme are insufficient for isoform

  3. Cellular peptidyl-prolyl cis/trans isomerase Pin1 facilitates replication of feline coronavirus.

    PubMed

    Tanaka, Yoshikazu; Amano, Arisa; Morisaki, Masateru; Sato, Yuka; Sasaki, Takashi

    2016-02-01

    Although feline coronavirus (FCoV) causes feline infectious peritonitis (FIP), which is a fatal infectious disease, there are no effective therapeutic medicines or vaccines. Previously, in vitro studies have shown that cyclosporin (CsA) and FK506 inhibit virus replication in diverse coronaviruses. CsA and FK506 are targets of clinically relevant immunosuppressive drugs and bind to cellular cyclophilins (Cyps) or FK506 binding proteins (FKBPs), respectively. Both Cyp and FKBP have peptidyl-prolyl cis-trans isomerase (PPIase) activity. However, protein interacting with NIMA (Pin1), a member of the parvulin subfamily of PPIases that differs from Cyps and FKBPs, is essential for various signaling pathways. Here we demonstrated that genetic silencing or knockout of Pin1 resulted in decreased FCoV replication in vitro. Dipentamethylene thiuram monosulfide, a specific inhibitor of Pin1, inhibited FCoV replication. These data indicate that Pin1 modulates FCoV propagation. PMID:26675666

  4. Peroxide-mediated oxidation and inhibition of the peptidyl-prolyl isomerase Pin1.

    PubMed

    Innes, Brendan T; Sowole, Modupeola A; Gyenis, Laszlo; Dubinsky, Michelle; Konermann, Lars; Litchfield, David W; Brandl, Christopher J; Shilton, Brian H

    2015-05-01

    Pin1 is a phosphorylation-dependent peptidyl-prolyl isomerase that plays a critical role in mediating protein conformational changes involved in signaling processes related to cell cycle control. Pin1 has also been implicated as being neuroprotective in aging-related neurodegenerative disorders including Alzheimer's disease where Pin1 activity is diminished. Notably, recent proteomic analysis of brain samples from patients with mild cognitive impairment revealed that Pin1 is oxidized and also displays reduced activity. Since the Pin1 active site contains a functionally critical cysteine residue (Cys113) with a low predicted pK(a), we hypothesized that Cys113 is sensitive to oxidation. Consistent with this hypothesis, we observed that treatment of Pin1 with hydrogen peroxide results in a 32Da mass increase, likely resulting from the oxidation of Cys113 to sulfinic acid (Cys-SO(2)H). This modification results in loss of peptidyl-prolyl isomerase activity. Notably, Pin1 with Cys113 substituted by aspartic acid retains activity and is no longer sensitive to oxidation. Structural studies by X-ray crystallography revealed increased electron density surrounding Cys113 following hydrogen peroxide treatment. At lower concentrations of hydrogen peroxide, oxidative inhibition of Pin1 can be partially reversed by treatment with dithiothreitol, suggesting that oxidation could be a reversible modification with a regulatory role. We conclude that the loss of Pin1 activity upon oxidation results from oxidative modification of the Cys113 sulfhydryl to sulfenic (Cys-SOH) or sulfinic acid (Cys-SO(2)H). Given the involvement of Pin1 in pathological processes related to neurodegenerative diseases and to cancer, these findings could have implications for the prevention or treatment of disease. PMID:25595659

  5. Kinetic Isotope Effects Support the Twisted Amide Mechanism of Pin1 Peptidyl-Prolyl Isomerase

    PubMed Central

    Mercedes-Camacho, Ana Y.; Mullins, Ashley B.; Mason, Matthew D.; Xu, Guoyan G.; Mahoney, Brendan J.; Wang, Xingsheng; Peng, Jeffrey W.; Etzkorn, Felicia A.

    2013-01-01

    The Pin1 peptidyl-prolyl isomerase (PPIase) catalyzes isomerization of pSer/pThr-Pro motifs in regulating the cell cycle. Peptide substrates, Ac–Phe–Phe–phosphoSer–Pro–Arg–p-nitroaniline, were synthesized in unlabeled form, and with deuterium labeled Ser-d3 and Pro-d7 amino acids. Kinetic data was collected as a function of Pin1 concentration to measure kinetic isotope effects (KIE) on catalytic efficiency (kcat/Km). The normal secondary (2°) KIE value measured for the Ser-d3 substrate (kH/kD = 1.6 ± 0.2) indicates that the serine carbonyl does not rehybridize from sp2 to sp3 in the rate-determining step, ruling out a nucleophilic addition mechanism. The normal 2° KIE can be explained by hyperconjugation between Ser α-C–H/D and C=O, and release of steric strain upon rotation of the amide bond from cis to syn-exo. The inverse 2° KIE value (kH/kD = 0.86 ± 0.08) measured for the Pro-d7 substrate indicates rehybridization of the prolyl nitrogen from sp2 to sp3 during the rate-limiting step of isomerization. No solvent kinetic isotope was measured by NMR exchange spectroscopy (EXSY) (kH2O/kD2O = 0.92 ± 0.12), indicating little or no involvement of exchangeable protons in the mechanism. These results support the formation of a simple twisted-amide transition state as the mechanism for peptidyl prolyl isomerization catalyzed by Pin1. A model of the reaction mechanism is presented using crystal structures of Pin1 with ground state analogues and an inhibitor that resembles a twisted amide transition state. PMID:24116866

  6. Kinetic isotope effects support the twisted amide mechanism of Pin1 peptidyl-prolyl isomerase.

    PubMed

    Mercedes-Camacho, Ana Y; Mullins, Ashley B; Mason, Matthew D; Xu, Guoyan G; Mahoney, Brendan J; Wang, Xingsheng; Peng, Jeffrey W; Etzkorn, Felicia A

    2013-11-01

    The Pin1 peptidyl-prolyl isomerase catalyzes isomerization of pSer/pThr-Pro motifs in regulating the cell cycle. Peptide substrates, Ac-Phe-Phe-phosphoSer-Pro-Arg-p-nitroaniline, were synthesized in unlabeled form, and with deuterium-labeled Ser-d3 and Pro-d7 amino acids. Kinetic data were collected as a function of Pin1 concentration to measure kinetic isotope effects (KIEs) on catalytic efficiency (kcat/Km). The normal secondary (2°) KIE value measured for the Ser-d3 substrate (kH/kD = 1.6 ± 0.2) indicates that the serine carbonyl does not rehybridize from sp(2) to sp(3) in the rate-determining step, ruling out a nucleophilic addition mechanism. The normal 2° KIE can be explained by hyperconjugation between Ser α-C-H/D and C═O and release of steric strain upon rotation of the amide bond from cis to syn-exo. The inverse 2° KIE value (kH/kD = 0.86 ± 0.08) measured for the Pro-d7 substrate indicates rehybridization of the prolyl nitrogen from sp(2) to sp(3) during the rate-limiting step of isomerization. No solvent kinetic isotope was measured by NMR exchange spectroscopy (kH2O/kD2O = 0.92 ± 0.12), indicating little or no involvement of exchangeable protons in the mechanism. These results support the formation of a simple twisted amide transition state as the mechanism for peptidyl prolyl isomerization catalyzed by Pin1. A model of the reaction mechanism is presented using crystal structures of Pin1 with ground state analogues and an inhibitor that resembles a twisted amide transition state. PMID:24116866

  7. Structural and Biochemical Characterization of the Human Cyclophilin Family of Peptidyl-Prolyl Isomerases

    PubMed Central

    Davis, Tara L.; Walker, John R.; Campagna-Slater, Valérie; Finerty, Patrick J.; Paramanathan, Ragika; Bernstein, Galina; MacKenzie, Farrell; Tempel, Wolfram; Ouyang, Hui; Lee, Wen Hwa; Eisenmesser, Elan Z.; Dhe-Paganon, Sirano

    2010-01-01

    Peptidyl-prolyl isomerases catalyze the conversion between cis and trans isomers of proline. The cyclophilin family of peptidyl-prolyl isomerases is well known for being the target of the immunosuppressive drug cyclosporin, used to combat organ transplant rejection. There is great interest in both the substrate specificity of these enzymes and the design of isoform-selective ligands for them. However, the dearth of available data for individual family members inhibits attempts to design drug specificity; additionally, in order to define physiological functions for the cyclophilins, definitive isoform characterization is required. In the current study, enzymatic activity was assayed for 15 of the 17 human cyclophilin isomerase domains, and binding to the cyclosporin scaffold was tested. In order to rationalize the observed isoform diversity, the high-resolution crystallographic structures of seven cyclophilin domains were determined. These models, combined with seven previously solved cyclophilin isoforms, provide the basis for a family-wide structure∶function analysis. Detailed structural analysis of the human cyclophilin isomerase explains why cyclophilin activity against short peptides is correlated with an ability to ligate cyclosporin and why certain isoforms are not competent for either activity. In addition, we find that regions of the isomerase domain outside the proline-binding surface impart isoform specificity for both in vivo substrates and drug design. We hypothesize that there is a well-defined molecular surface corresponding to the substrate-binding S2 position that is a site of diversity in the cyclophilin family. Computational simulations of substrate binding in this region support our observations. Our data indicate that unique isoform determinants exist that may be exploited for development of selective ligands and suggest that the currently available small-molecule and peptide-based ligands for this class of enzyme are insufficient for isoform

  8. Peptidyl-Prolyl Isomerase Pin1 Is a Cellular Factor Required for Hepatitis C Virus Propagation▿

    PubMed Central

    Lim, Yun-Sook; Tran, Huong T. L.; Park, Soo-Je; Yim, Seung-Ae; Hwang, Soon B.

    2011-01-01

    The life cycle of hepatitis C virus (HCV) is highly dependent on cellular factors. Using small interfering RNA (siRNA) library screening, we identified peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) as a host factor involved in HCV propagation. Here we demonstrated that silencing of Pin1 expression resulted in decreases in HCV replication in both HCV replicon cells and cell culture-grown HCV (HCVcc)-infected cells, whereas overexpression of Pin1 increased HCV replication. Pin1 interacted with both the NS5A and NS5B proteins. However, Pin1 expression was increased only by the NS5B protein. Both the protein binding and isomerase activities of Pin1 were required for HCV replication. Juglone, a natural inhibitor of Pin1, inhibited HCV propagation by inhibiting the interplay between the Pin1 and HCV NS5A/NS5B proteins. These data indicate that Pin1 modulates HCV propagation and may contribute to HCV-induced liver pathogenesis. PMID:21680504

  9. Microbial Peptidyl-Prolyl cis/trans Isomerases (PPIases): Virulence Factors and Potential Alternative Drug Targets

    PubMed Central

    2014-01-01

    SUMMARY Initially discovered in the context of immunomodulation, peptidyl-prolyl cis/trans isomerases (PPIases) were soon identified as enzymes catalyzing the rate-limiting protein folding step at peptidyl bonds preceding proline residues. Intense searches revealed that PPIases are a superfamily of proteins consisting of three structurally distinguishable families with representatives in every described species of prokaryote and eukaryote and, recently, even in some giant viruses. Despite the clear-cut enzymatic activity and ubiquitous distribution of PPIases, reports on solely PPIase-dependent biological roles remain scarce. Nevertheless, they have been found to be involved in a plethora of biological processes, such as gene expression, signal transduction, protein secretion, development, and tissue regeneration, underscoring their general importance. Hence, it is not surprising that PPIases have also been identified as virulence-associated proteins. The extent of contribution to virulence is highly variable and dependent on the pleiotropic roles of a single PPIase in the respective pathogen. The main objective of this review is to discuss this variety in virulence-related bacterial and protozoan PPIases as well as the involvement of host PPIases in infectious processes. Moreover, a special focus is given to Legionella pneumophila macrophage infectivity potentiator (Mip) and Mip-like PPIases of other pathogens, as the best-characterized virulence-related representatives of this family. Finally, the potential of PPIases as alternative drug targets and first tangible results are highlighted. PMID:25184565

  10. Peptidyl Prolyl Isomerase PIN1 Directly Binds to and Stabilizes Hypoxia-Inducible Factor-1α.

    PubMed

    Han, Hyeong-Jun; Kwon, Nayoung; Choi, Min-A; Jung, Kyung Oh; Piao, Juan-Yu; Ngo, Hoang Kieu Chi; Kim, Su-Jung; Kim, Do-Hee; Chung, June-Key; Cha, Young-Nam; Youn, Hyewon; Choi, Bu Young; Min, Sang-Hyun; Surh, Young-Joon

    2016-01-01

    Peptidyl prolyl isomerase (PIN1) regulates the functional activity of a subset of phosphoproteins through binding to phosphorylated Ser/Thr-Pro motifs and subsequently isomerization of the phosphorylated bonds. Interestingly, PIN1 is overexpressed in many types of malignancies including breast, prostate, lung and colon cancers. However, its oncogenic functions have not been fully elucidated. Here, we report that PIN1 directly interacts with hypoxia-inducible factor (HIF)-1α in human colon cancer (HCT116) cells. PIN1 binding to HIF-1α occurred in a phosphorylation-dependent manner. We also found that PIN1 interacted with HIF-1α at both exogenous and endogenous levels. Notably, PIN1 binding stabilized the HIF-1α protein, given that their levels were significantly increased under hypoxic conditions. The stabilization of HIF-1α resulted in increased transcriptional activity, consequently upregulating expression of vascular endothelial growth factor, a major contributor to angiogenesis. Silencing of PIN1 or pharmacologic inhibition of its activity abrogated the angiogenesis. By utilizing a bioluminescence imaging technique, we were able to demonstrate that PIN1 inhibition dramatically reduced the tumor volume in a subcutaneous mouse xenograft model and angiogenesis as well as hypoxia-induced transcriptional activity of HIF-1α. These results suggest that PIN1 interacting with HIF-1α is a potential cancer chemopreventive and therapeutic target. PMID:26784107

  11. Peptidyl Prolyl Isomerase PIN1 Directly Binds to and Stabilizes Hypoxia-Inducible Factor-1α

    PubMed Central

    Han, Hyeong-jun; Kwon, Nayoung; Choi, Min-A; Jung, Kyung Oh; Piao, Juan-Yu; Ngo, Hoang Kieu Chi; Kim, Su-Jung; Kim, Do-Hee; Chung, June-Key; Cha, Young-Nam; Youn, Hyewon; Choi, Bu Young; Min, Sang-Hyun; Surh, Young-Joon

    2016-01-01

    Peptidyl prolyl isomerase (PIN1) regulates the functional activity of a subset of phosphoproteins through binding to phosphorylated Ser/Thr-Pro motifs and subsequently isomerization of the phosphorylated bonds. Interestingly, PIN1 is overexpressed in many types of malignancies including breast, prostate, lung and colon cancers. However, its oncogenic functions have not been fully elucidated. Here, we report that PIN1 directly interacts with hypoxia-inducible factor (HIF)-1α in human colon cancer (HCT116) cells. PIN1 binding to HIF-1α occurred in a phosphorylation-dependent manner. We also found that PIN1 interacted with HIF-1α at both exogenous and endogenous levels. Notably, PIN1 binding stabilized the HIF-1α protein, given that their levels were significantly increased under hypoxic conditions. The stabilization of HIF-1α resulted in increased transcriptional activity, consequently upregulating expression of vascular endothelial growth factor, a major contributor to angiogenesis. Silencing of PIN1 or pharmacologic inhibition of its activity abrogated the angiogenesis. By utilizing a bioluminescence imaging technique, we were able to demonstrate that PIN1 inhibition dramatically reduced the tumor volume in a subcutaneous mouse xenograft model and angiogenesis as well as hypoxia-induced transcriptional activity of HIF-1α. These results suggest that PIN1 interacting with HIF-1α is a potential cancer chemopreventive and therapeutic target. PMID:26784107

  12. Determining the roles of a conserved tyrosine residue in a Mip-like peptidyl-prolyl cis-trans isomerase.

    PubMed

    Polley, Soumitra; Chakravarty, Devlina; Chakrabarti, Gopal; Sau, Subrata

    2016-06-01

    The FKBP22 and the related peptidyl-prolyl cis-trans isomerases dimerize using their N-terminal domains. Conversely, their C-terminal domains possess both the substrate and inhibitor binding sites. To delineate the roles of a conserved Tyr residue at their N-terminal domains, we have studied a FKBP22 mutant that carries an Ala in place of the conserved Tyr at position 15. We have demonstrated that the Tyr 15 of FKBP22 is indispensable for preserving its dimerization ability, catalytic activity, and structure. The residue, however, little contributed to its inhibitor binding ability and stability. The mode of action of Tyr 15 has been discussed at length. PMID:26944658

  13. Peptidyl-prolyl cis/trans isomerase-independent functional NifH mutant of Azotobacter vinelandii.

    PubMed

    Gavini, Nara; Tungtur, Sudheer; Pulakat, Lakshmi

    2006-08-01

    Peptidyl-prolyl cis/trans isomerases (PPIases) play a pivotal role in catalyzing the correct folding of many prokaryotic and eukaryotic proteins that are implicated in a variety of biological functions, ranging from cell cycle regulation to bacterial infection. The nif accessory protein NifM, which is essential for the biogenesis of a functional NifH component of nitrogenase, is a PPIase. To understand the nature of the molecular signature that defines the NifM dependence of NifH, we screened a library of nifH mutants in the nitrogen-fixing bacterium Azotobacter vinelandii for mutants that acquired NifM independence. Here, we report that NifH can acquire NifM independence when the conserved Pro258 located in the C-terminal region of NifH, which wraps around the other subunit in the NifH dimer, is replaced by serine. PMID:16885471

  14. Inhibition of the FKBP family of peptidyl prolyl isomerases induces abortive translocation and degradation of the cellular prion protein.

    PubMed

    Stocki, Pawel; Sawicki, Maxime; Mays, Charles E; Hong, Seo Jung; Chapman, Daniel C; Westaway, David; Williams, David B

    2016-03-01

    Prion diseases are fatal neurodegenerative disorders for which there is no effective treatment. Because the cellular prion protein (PrP(C)) is required for propagation of the infectious scrapie form of the protein, one therapeutic strategy is to reduce PrP(C) expression. Recently FK506, an inhibitor of the FKBP family of peptidyl prolyl isomerases, was shown to increase survival in animal models of prion disease, with proposed mechanisms including calcineurin inhibition, induction of autophagy, and reduced PrP(C) expression. We show that FK506 treatment results in a profound reduction in PrP(C) expression due to a defect in the translocation of PrP(C) into the endoplasmic reticulum with subsequent degradation by the proteasome. These phenotypes could be bypassed by replacing the PrP(C) signal sequence with that of prolactin or osteopontin. In mouse cells, depletion of ER luminal FKBP10 was almost as potent as FK506 in attenuating expression of PrP(C). However, this occurred at a later stage, after translocation of PrP(C) into the ER. Both FK506 treatment and FKBP10 depletion were effective in reducing PrP(Sc) propagation in cell models. These findings show the involvement of FKBP proteins at different stages of PrP(C) biogenesis and identify FKBP10 as a potential therapeutic target for the treatment of prion diseases. PMID:26764098

  15. Mycobacterium tuberculosis Peptidyl-Prolyl Isomerases Also Exhibit Chaperone like Activity In-Vitro and In-Vivo

    PubMed Central

    Pandey, Saurabh; Sharma, Ashish; Tripathi, Deeksha; Kumar, Ashutosh; Khubaib, Mohd; Bhuwan, Manish; Chaudhuri, Tapan Kumar; Hasnain, Seyed Ehtesham; Ehtesham, Nasreen Zafar

    2016-01-01

    Peptidyl-prolyl cis-trans isomerases (Ppiases), also known as cyclophilins, are ubiquitously expressed enzymes that assist in protein folding by isomerization of peptide bonds preceding prolyl residues. Mycobacterium tuberculosis (M.tb) is known to possess two Ppiases, PpiA and PpiB. However, our understanding about the biological significance of mycobacterial Ppiases with respect to their pleiotropic roles in responding to stress conditions inside the macrophages is restricted. This study describes chaperone-like activity of mycobacterial Ppiases. We show that recombinant rPpiA and rPpiB can bind to non-native proteins in vitro and can prevent their aggregation. Purified rPpiA and rPpiB exist in oligomeric form as evident from gel filtration chromatography.E. coli cells overexpressing PpiA and PpiB of M.tb could survive thermal stress as compared to plasmid vector control. HEK293T cells transiently expressing M.tb PpiA and PpiB proteins show increased survival as compared to control cells in response to oxidative stress and hypoxic conditions generated after treatment with H2O2 and CoCl2 thereby pointing to their likely role in adaption under host generated oxidative stress and conditions of hypoxia. The chaperone-like function of these M.tuberculosis cyclophilins may possibly function as a stress responder and consequently contribute to virulence. PMID:26981873

  16. Inhibition of the FKBP family of peptidyl prolyl isomerases induces abortive translocation and degradation of the cellular prion protein

    PubMed Central

    Stocki, Pawel; Sawicki, Maxime; Mays, Charles E.; Hong, Seo Jung; Chapman, Daniel C.; Westaway, David; Williams, David B.

    2016-01-01

    Prion diseases are fatal neurodegenerative disorders for which there is no effective treatment. Because the cellular prion protein (PrPC) is required for propagation of the infectious scrapie form of the protein, one therapeutic strategy is to reduce PrPC expression. Recently FK506, an inhibitor of the FKBP family of peptidyl prolyl isomerases, was shown to increase survival in animal models of prion disease, with proposed mechanisms including calcineurin inhibition, induction of autophagy, and reduced PrPC expression. We show that FK506 treatment results in a profound reduction in PrPC expression due to a defect in the translocation of PrPC into the endoplasmic reticulum with subsequent degradation by the proteasome. These phenotypes could be bypassed by replacing the PrPC signal sequence with that of prolactin or osteopontin. In mouse cells, depletion of ER luminal FKBP10 was almost as potent as FK506 in attenuating expression of PrPC. However, this occurred at a later stage, after translocation of PrPC into the ER. Both FK506 treatment and FKBP10 depletion were effective in reducing PrPSc propagation in cell models. These findings show the involvement of FKBP proteins at different stages of PrPC biogenesis and identify FKBP10 as a potential therapeutic target for the treatment of prion diseases. PMID:26764098

  17. Regulation of Estrogen Receptor α N-Terminus Conformation and Function by Peptidyl Prolyl Isomerase Pin1

    PubMed Central

    Rajbhandari, Prashant; Finn, Greg; Solodin, Natalia M.; Singarapu, Kiran K.; Sahu, Sarata C.; Markley, John L.; Kadunc, Kelley J.; Ellison-Zelski, Stephanie J.; Kariagina, Anastasia; Haslam, Sandra Z.; Lu, Kun Ping

    2012-01-01

    Estrogen receptor alpha (ERα), a key driver of growth in the majority of breast cancers, contains an unstructured transactivation domain (AF1) in its N terminus that is a convergence point for growth factor and hormonal activation. This domain is controlled by phosphorylation, but how phosphorylation impacts AF1 structure and function is unclear. We found that serine 118 (S118) phosphorylation of the ERα AF1 region in response to estrogen (agonist), tamoxifen (antagonist), and growth factors results in recruitment of the peptidyl prolyl cis/trans isomerase Pin1. Phosphorylation of S118 is critical for Pin1 binding, and mutation of S118 to alanine prevents this association. Importantly, Pin1 isomerizes the serine118-proline119 bond from a cis to trans isomer, with a concomitant increase in AF1 transcriptional activity. Pin1 overexpression promotes ligand-independent and tamoxifen-inducible activity of ERα and growth of tamoxifen-resistant breast cancer cells. Pin1 expression correlates with proliferation in ERα-positive rat mammary tumors. These results establish phosphorylation-coupled proline isomerization as a mechanism modulating AF1 functional activity and provide insight into the role of a conformational switch in the functional regulation of the intrinsically disordered transactivation domain of ERα. PMID:22064478

  18. The emerging role of peptidyl-prolyl isomerase chaperones in tau oligomerization, amyloid processing and Alzheimer's disease

    PubMed Central

    Blair, Laura J.; Baker, Jeremy D.; Sabbagh, Jonathan J.; Dickey, Chad A.

    2015-01-01

    Peptidyl-prolyl cis/trans isomerases (PPIases), a unique family of molecular chaperones, regulate protein folding at proline residues. These residues are abundant within intrinsically disordered proteins, like the microtubule-associated protein tau. Tau has been shown to become hyperphosphorylated and accumulate as one of the two main pathological hallmarks in Alzheimer's disease (AD), the other being amyloid beta (Aβ). PPIases, including Pin1, FK506-binding protein (FKBP) 52, FKBP51, and FKBP12, have been shown to interact with and regulate tau biology. This interaction is particularly important given the numerous proline-directed phosphorylation sites found on tau and the role phosphorylation has been found to play in pathogenesis. This regulation then affects downstream aggregation and oligomerization of tau. However, many PPIases have yet to be explored for their effects on tau biology, despite the high likelihood of interaction based on proline content. Moreover, Pin1, FKBP12, FKBP52, cyclophilin (Cyp) A, CypB, and CypD have been shown to also regulate Aβ production or the toxicity associated with Aβ pathology. Therefore, PPIases directly and indirectly regulate pathogenic protein multimerization in AD and represent a family rich in targets for modulating the accumulation and toxicity. PMID:25628064

  19. Cytosolic Aryl sulfotransferase 4A1 interacts with the peptidyl prolyl cis-trans isomerase Pin1.

    PubMed

    Mitchell, Deanne J; Minchin, Rodney F

    2009-08-01

    Sulfonation by cytosolic sulfotransferases plays an important role in the metabolism of both endogenous and exogenous compounds. Sulfotransferase 4A1 (SULT4A1) is a novel sulfotransferase found primarily in neurons in the brain. It is highly conserved between species, but no substantial enzyme activity has been identified for the protein. Consequently, little is known about the role of this enzyme in the brain. We performed a yeast two-hybrid screen of a human brain library to isolate potential SULT4A1-interacting proteins that might identify the role or regulation of the sulfotransferase in humans. The screen isolated the peptidyl-prolyl cis-trans isomerase Pin1. Its interaction with SULT4A1 was confirmed by coimmunoprecipitation studies in HeLa cells and by in vitro pull-down of expressed proteins. Moreover, Pin1 binding was dependent on phosphorylation of the SULT4A1 protein. Pin1 destabilized SULT4A1, decreasing its half-life from more than 8 h to approximately 4.5 h. This effect was dependent on the isomerase activity of Pin1 and was inhibited by okadaic acid, suggesting a role for the phosphatase PP2A. Pin1-mediated SULT4A1 degradation did not involve the proteosomes or macroautophagy, but it was inhibited by the calpain antagonists N-acetyl-Leu-Leu-Nle-CHO and Z-Val-Phe-CHO. Finally, Pin1 binding was mapped to two threonine-proline motifs (Thr(8) and Thr(11)) that are not present in any of the other human cytosolic sulfotransferases. Our findings suggest that SULT4A1 is subject to post-translational modification that alters its stability in the cell. These modifications may also be important for enzyme activity, which explains why specific substrates for SULT4A1 have not yet been identified. PMID:19439498

  20. Domain structure and denaturation of a dimeric Mip-like peptidyl-prolyl cis-trans isomerase from Escherichia coli.

    PubMed

    Jana, Biswanath; Bandhu, Amitava; Mondal, Rajkrishna; Biswas, Anindya; Sau, Keya; Sau, Subrata

    2012-02-14

    FKBP22, a protein expressed by Escherichia coli, possesses PPIase (peptidyl-prolyl cis-trans isomerase) activity, binds FK506 (an immunosuppressive drug), and shares homology with Legionella Mip (a virulence factor) and its related proteins. To understand the domain structure and the folding-unfolding mechanism of Mip-like proteins, we investigated a recombinant E. coli FKBP22 (His-FKBP22) as a model protein. Limited proteolysis indicated that His-FKBP22 harbors an N-terminal domain (NTD), a C-terminal domain (CTD), and a long flexible region linking the two domains. His-FKBP22, NTD(+) (NTD with the entire flexible region), and CTD(+) (CTD with a truncated flexible region) were unfolded by a two-state mechanism in the presence of urea. Urea induced the swelling of dimeric His-FKBP22 molecules at the pretransition state but dissociated it at the early transition state. In contrast, guanidine hydrochloride (GdnCl)-induced equilibrium unfolding of His-FKBP22 or NTD(+) and CTD(+) seemed to follow three-step and two-step mechanisms, respectively. Interestingly, the intermediate formed during the unfolding of His-FKBP22 with GdnCl was not a molten globule but was thought to be composed of the partially unfolded dimeric as well as various multimeric His-FKBP22 molecules. Dimeric His-FKBP22 did not dissociate gradually with increasing concentrations of GdnCl. Very low GdnCl concentrations also had little effect on the molecular dimensions of His-FKBP22. Unfolding with either denaturant was found to be reversible, as refolding of the unfolded His-FKBP22 completely, or nearly completely, restored the structure and function of the protein. Additionally, denaturation of His-FKBP22 appeared to begin at the CTD(+). PMID:22263615

  1. Peptidyl-prolyl cis/trans-isomerase A1 (Pin1) is a target for modification by lipid electrophiles.

    PubMed

    Aluise, Christopher D; Rose, Kristie; Boiani, Mariana; Reyzer, Michelle L; Manna, Joseph D; Tallman, Keri; Porter, Ned A; Marnett, Lawrence J

    2013-02-18

    Oxidation of membrane phospholipids is associated with inflammation, neurodegenerative disease, and cancer. Oxyradical damage to phospholipids results in the production of reactive aldehydes that adduct proteins and modulate their function. 4-Hydroxynonenal (HNE), a common product of oxidative damage to lipids, adducts proteins at exposed Cys, His, or Lys residues. Here, we demonstrate that peptidyl-prolyl cis/trans-isomerase A1 (Pin1), an enzyme that catalyzes the conversion of the peptide bond of pSer/pThr-Pro moieties in signaling proteins from cis to trans, is highly susceptible to HNE modification. Incubation of purified Pin1 with HNE followed by MALDI-TOF/TOF mass spectrometry resulted in detection of Michael adducts at the active site residues His-157 and Cys-113. Time and concentration dependencies indicate that Cys-113 is the primary site of HNE modification. Pin1 was adducted in MDA-MB-231 breast cancer cells treated with 8-alkynyl-HNE as judged by click chemistry conjugation with biotin followed by streptavidin-based pulldown and Western blotting with anti-Pin1 antibody. Furthermore, orbitrap MS data support the adduction of Cys-113 in the Pin1 active site upon HNE treatment of MDA-MB-231 cells. siRNA knockdown of Pin1 in MDA-MB-231 cells partially protected the cells from HNE-induced toxicity. Recent studies indicate that Pin1 is an important molecular target for the chemopreventive effects of green tea polyphenols. The present study establishes that it is also a target for electrophilic modification by products of lipid peroxidation. PMID:23231502

  2. Cyclophilin J Is a Novel Peptidyl-Prolyl Isomerase and Target for Repressing the Growth of Hepatocellular Carcinoma

    PubMed Central

    Chen, Jian; Chen, Shuai; Wang, Jiahui; Zhang, Mingjun; Gong, Zhaohua; Wei, Youheng; Li, Li; Zhang, Yuanyuan; Zhao, Xuemei; Jiang, Songmin; Yu, Long

    2015-01-01

    Cyclophilin J (CYPJ) is a new member of the peptidyl-prolyl cis/trans-isomerase (PPIase) identified with upregulated expression in human glioma. However, the biological function of CYPJ remained unclear. We aimed to study the role of CYPJ in hepatocellular carcinoma (HCC) carcinogenesis and its therapeutic potential. We determined the expression of CYPJ in HCC/adjacent normal tissues using Western blot, Northern blot and semi-quantitative RT-PCR, analyzed the biochemical characteristics of CYPJ, and resolved the 3D-structure of CYPJ/Cyclosporin A (CsA) complex. We also studied the roles of CYPJ in cell cycle, cyclin D1 regulation, in vitro and in vivo tumor growth. We found that CYPJ expression was upregulated in over 60% HCC tissues. The PPIase activity of CYPJ could be inhibited by the widely used immunosuppressive drug CsA. CYPJ was found expressed in the whole cell of HCC with preferential location at the cell nucleus. CYPJ promoted the transition of cells from G1 phase to S phase in a PPIase-dependent manner by activating cyclin D1 promoter. CYPJ overexpression accelerated liver cell growth in vitro (cell growth assay, colony formation) and in vivo (xenograft tumor formation). Inhibition of CYPJ by its inhibitor CsA or CYPJ-specific RNAi diminished the growth of liver cancer cells in vitro and in vivo. In conclusion, CYPJ could facilitate HCC growth by promoting cell cycle transition from G1 to S phase through the upregulation of cyclin D1. Suppression of CYPJ could repress the growth of HCC, which makes CYPJ a potential target for the development of new strategies to treat this malignancy. PMID:26020957

  3. Rapamycin sensitivity in Saccharomyces cerevisiae is mediated by a peptidyl-prolyl cis-trans isomerase related to human FK506-binding protein.

    PubMed Central

    Koltin, Y; Faucette, L; Bergsma, D J; Levy, M A; Cafferkey, R; Koser, P L; Johnson, R K; Livi, G P

    1991-01-01

    Rapamycin is a macrolide antifungal agent with structural similarity to FK506. It exhibits potent immunosuppressive properties analogous to those of both FK506 and cyclosporin A (CsA). Unlike FK506 and CsA, however, rapamycin does not inhibit the transcription of early T-cell activation genes, including interleukin-2, but instead appears to block downstream events leading to T-cell activation. FK506 and CsA receptor proteins (FKBP and cyclophilin, respectively) have been identified and shown to be distinct members of a class of enzymes that possess peptidyl-prolyl cis-trans isomerase (PPIase) activity. Despite the apparent differences in their mode of action, rapamycin and FK506 act as reciprocal antagonists in vivo and compete for binding to FKBP. As a means of rapidly identifying a target protein for rapamycin in vivo, we selected and genetically characterized rapamycin-resistant mutants of Saccharomyces cerevisiae and isolated a yeast genomic fragment that confers drug sensitivity. We demonstrate that the resonse to rapamycin in yeast cells is mediated by a gene encoding a 114-amino-acid, approximately 13-kDa protein which has a high degree of sequence homology with human FKBP; we designated this gene RBP1 (for rapamycin-binding protein). The RBP1 protein (RBP) was expressed in Escherichia coli, purified to homogeneity, and shown to catalyze peptidyl-prolyl isomerization of a synthetic peptide substrate. PPIase activity was completely inhibited by rapamycin and FK506 but not by CsA, indicating that both macrolides bind to the recombinant protein. Expression of human FKBP in rapamycin-resistant mutants restored rapamycin sensitivity, indicating a functional equivalence between the yeast and human enzymes. Images PMID:1996117

  4. Characterization of Peptidyl-Prolyl Cis-Trans Isomerase- and Calmodulin-Binding Activity of a Cytosolic Arabidopsis thaliana Cyclophilin AtCyp19-3

    PubMed Central

    Kaur, Gundeep; Singh, Supreet; Singh, Harpreet; Chawla, Mrinalini; Dutta, Tanima; Kaur, Harsimran; Bender, Kyle; Snedden, W. A.; Kapoor, Sanjay; Pareek, Ashwani; Singh, Prabhjeet

    2015-01-01

    Cyclophilins, which bind to immunosuppressant cyclosporin A (CsA), are ubiquitous proteins and constitute a multigene family in higher organisms. Several members of this family are reported to catalyze cis-trans isomerisation of the peptidyl-prolyl bond, which is a rate limiting step in protein folding. The physiological role of these proteins in plants, with few exceptions, is still a matter of speculation. Although Arabidopsis genome is predicted to contain 35 cyclophilin genes, biochemical characterization, imperative for understanding their cellular function(s), has been carried only for few of the members. The present study reports the biochemical characterization of an Arabidopsis cyclophilin, AtCyp19-3, which demonstrated that this protein is enzymatically active and possesses peptidyl-prolyl cis-trans isomerase (PPIase) activity that is specifically inhibited by CsA with an inhibition constant (Ki) of 18.75 nM. The PPIase activity of AtCyp19-3 was also sensitive to Cu2+, which covalently reacts with the sulfhydryl groups, implying redox regulation. Further, using calmodulin (CaM) gel overlay assays it was demonstrated that in vitro interaction of AtCyp19-3 with CaM is Ca2+-dependent, and CaM-binding domain is localized to 35–70 amino acid residues in the N-terminus. Bimolecular fluorescence complementation assays showed that AtCyp19-3 interacts with CaM in vivo also, thus, validating the in vitro observations. However, the PPIase activity of the Arabidopsis cyclophilin was not affected by CaM. The implications of these findings are discussed in the context of Ca2+ signaling and cyclophilin activity in Arabidopsis. PMID:26317213

  5. Enhancement of antibody fragment secretion into the Escherichia coli periplasm by co-expression with the peptidyl prolyl isomerase, FkpA, in the cytoplasm.

    PubMed

    Levy, Raphael; Ahluwalia, Kiran; Bohmann, David J; Giang, Hoa M; Schwimmer, Lauren J; Issafras, Hassan; Reddy, Nithin B; Chan, Chung; Horwitz, Arnold H; Takeuchi, Toshihiko

    2013-08-30

    Improper protein folding or aggregation can frequently be responsible for low expression and poor functional activity of antibody fragments secreted into the Escherichia coli periplasm. Expression issues also can affect selection of antibody candidates from phage libraries, since antibody fragments displayed on phage also are secreted into the E. coli periplasm. To improve secretion of properly folded antibody fragments into the periplasm, we have developed a novel approach that involves co-expressing the antibody fragments with the peptidyl prolyl cis-trans isomerase, FkpA, lacking its signal sequence (cytFkpA) which consequently is expressed in the E. coli cytosol. Cytoplasmic expression of cytFkpA improved secretion of functional Fab fragments into the periplasm, exceeding even the benefits from co-expressing Fab fragments with native, FkpA localized in the periplasm. In addition, panning and subsequent screening of large Fab and scFv naïve phage libraries in the presence of cytFkpA significantly increased the number of unique clones selected, as well as their functional expression levels and diversity. PMID:23624043

  6. Isolation and characterization of a 17-kDa FKBP-type peptidyl-prolyl cis/trans isomerase from Vibrio anguillarum.

    PubMed

    Jo, Geon-A; Lee, Jong Min; No, Gyuyou; Kang, Dong Seop; Kim, So-Hyun; Ahn, Sun-Hee; Kong, In-Soo

    2015-06-01

    Peptidyl-prolyl cis/trans isomerase (PPIase) catalyzes the isomerization of peptide bonds to achieve conformational changes in native folded proteins. An FKBP-type PPIase with an approximate molecular weight of 17kDa was isolated from Vibrio anguillarum O1 and named VaFKBP17. To investigate its biochemical properties, the ppi gene from V. anguillarum O1 was isolated and overexpressed in Escherichia coli. A protease-coupled assay for isomerization activity, using Succinyl-Ala-Phe-Pro-Phe-p nitroanilide as substrate, indicated that the activity of VaFKBP17 was highest at low temperature (5°C) and alkaline conditions (pH 10). The immunosuppressant FK506 inhibited the isomerization activity of VaFKBP17. The chaperone activity of VaFKBP17 was assessed using a citrate synthase thermal aggregation activity assay. To evaluate its ability to catalyze protein refolding, the effect of VaFKBP17 on inclusion bodies was investigated during a dilution process. In this assay, VaFKBP17 was able to assist protein refolding. These results provide evidence that VaFKBP17 possesses chaperone-like activity. The structural homology of VaFKBP17 relative to other known bacterial FKBPs was also examined. PMID:25747528

  7. Nerve Growth Factor Stimulates Interaction of Cayman Ataxia Protein BNIP-H/Caytaxin with Peptidyl-Prolyl Isomerase Pin1 in Differentiating Neurons

    PubMed Central

    Buschdorf, Jan Paul; Chew, Li Li; Soh, Unice Jim Kim; Liou, Yih-Cherng; Low, Boon Chuan

    2008-01-01

    Mutations in ATCAY that encodes the brain-specific protein BNIP-H (or Caytaxin) lead to Cayman cerebellar ataxia. BNIP-H binds to glutaminase, a neurotransmitter-producing enzyme, and affects its activity and intracellular localization. Here we describe the identification and characterization of the binding between BNIP-H and Pin1, a peptidyl-prolyl cis/trans isomerase. BNIP-H interacted with Pin1 after nerve growth factor-stimulation and they co-localized in the neurites and cytosol of differentiating pheochromocytoma PC12 cells and the embryonic carcinoma P19 cells. Deletional mutagenesis revealed two cryptic binding sites within the C-terminus of BNIP-H such that single point mutants affecting the WW domain of Pin1 completely abolished their binding. Although these two sites do not contain any of the canonical Pin1-binding motifs they showed differential binding profiles to Pin1 WW domain mutants S16E, S16A and W34A, and the catalytically inert C113A of its isomerase domain. Furthermore, their direct interaction would occur only upon disrupting the ability of BNIP-H to form an intramolecular interaction by two similar regions. Furthermore, expression of Pin1 disrupted the BNIP-H/glutaminase complex formation in PC12 cells under nerve growth factor-stimulation. These results indicate that nerve growth factor may stimulate the interaction of BNIP-H with Pin1 by releasing its intramolecular inhibition. Such a mechanism could provide a post-translational regulation on the cellular activity of BNIP-H during neuronal differentiation. (213 words) PMID:18628984

  8. FK506-Binding Protein 22 from a Psychrophilic Bacterium, a Cold Shock-Inducible Peptidyl Prolyl Isomerase with the Ability to Assist in Protein Folding

    PubMed Central

    Budiman, Cahyo; Koga, Yuichi; Takano, Kazufumi; Kanaya, Shigenori

    2011-01-01

    Adaptation of microorganisms to low temperatures remains to be fully elucidated. It has been previously reported that peptidyl prolyl cis-trans isomerases (PPIases) are involved in cold adaptation of various microorganisms whether they are hyperthermophiles, mesophiles or phsycrophiles. The rate of cis-trans isomerization at low temperatures is much slower than that at higher temperatures and may cause problems in protein folding. However, the mechanisms by which PPIases are involved in cold adaptation remain unclear. Here we used FK506-binding protein 22, a cold shock protein from the psychrophilic bacterium Shewanella sp. SIB1 (SIB1 FKBP22) as a model protein to decipher the involvement of PPIases in cold adaptation. SIB1 FKBP22 is homodimer that assumes a V-shaped structure based on a tertiary model. Each monomer consists of an N-domain responsible for dimerization and a C-catalytic domain. SIB1 FKBP22 is a typical cold-adapted enzyme as indicated by the increase of catalytic efficiency at low temperatures, the downward shift in optimal temperature of activity and the reduction in the conformational stability. SIB1 FKBP22 is considered as foldase and chaperone based on its ability to catalyze refolding of a cis-proline containing protein and bind to a folding intermediate protein, respectively. The foldase and chaperone activites of SIB1 FKBP22 are thought to be important for cold adaptation of Shewanella sp. SIB1. These activities are also employed by other PPIases for being involved in cold adaptation of various microorganisms. Despite other biological roles of PPIases, we proposed that foldase and chaperone activities of PPIases are the main requirement for overcoming the cold-stress problem in microorganisms due to folding of proteins. PMID:21954357

  9. Single-Domain Peptidyl-Prolyl cis/trans Isomerase FkpA from Corynebacterium glutamicum Improves the Biomass Yield at Increased Growth Temperatures.

    PubMed

    Kallscheuer, Nicolai; Bott, Michael; van Ooyen, Jan; Polen, Tino

    2015-11-01

    Peptidyl-prolyl cis/trans isomerases (PPIases) catalyze the rate-limiting protein folding step at peptidyl bonds preceding proline residues and were found to be involved in several biological processes, including gene expression, signal transduction, and protein secretion. Representative enzymes were found in almost all sequenced genomes, including Corynebacterium glutamicum, a facultative anaerobic Gram-positive and industrial workhorse for the production of amino acids. In C. glutamicum, a predicted single-domain FK-506 (tacrolimus) binding protein (FKBP)-type PPIase (FkpA) is encoded directly downstream of gltA, which encodes citrate synthase (CS). This gene cluster is also present in other Actinobacteria. Here we carried out in vitro and in vivo experiments to study the function and influence of predicted FkpA in C. glutamicum. In vitro, FkpA indeed shows typical PPIase activity with artificial substrates and is inhibited by FK-506. Furthermore, FkpA delays the aggregation of CS, which is also inhibited by FK-506. Surprisingly, FkpA has a positive effect on the activity and temperature range of CS in vitro. Deletion of fkpA causes a 50% reduced biomass yield compared to that of the wild type when grown at 37°C, whereas there is only a 10% reduced biomass yield at the optimal growth temperature of 30°C accompanied by accumulation of 7 mM l-glutamate and 22 mM 2-oxoglutarate. Thus, FkpA may be exploited for improved product formation in biotechnical processes. Comparative transcriptome analysis revealed 69 genes which exhibit ≥2-fold mRNA level changes in C. glutamicum ΔfkpA, giving insight into the transcriptional response upon mild heat stress when FkpA is absent. PMID:26341203

  10. Identification and Comparative Analysis of the Peptidyl-Prolyl cis/trans Isomerase Repertoires of H. sapiens, D. melanogaster, C. elegans, S. cerevisiae and Sz. pombe

    PubMed Central

    Kay, John E.

    2005-01-01

    The peptidyl-prolyl cis/trans isomerase (PPIase) class of proteins comprises three member families that are found throughout nature and are present in all the major compartments of the cell. Their numbers appear to be linked to the number of genes in their respective genomes, although we have found the human repertoire to be smaller than expected due to a reduced cyclophilin repertoire. We show here that whilst the members of the cyclophilin family (which are predominantly found in the nucleus and cytoplasm) and the parvulin family (which are predominantly nuclear) are largely conserved between different repertoires, the FKBPs (which are predominantly found in the cytoplasm and endoplasmic reticulum) are not. It therefore appears that the cyclophilins and parvulins have evolved to perform conserved functions, while the FKBPs have evolved to fill ever-changing niches within the constantly evolving organisms. Many orthologous subgroups within the different PPIase families appear to have evolved from a distinct common ancestor, whereas others, such as the mitochondrial cyclophilins, appear to have evolved independently of one another. We have also identified a novel parvulin within Drosophila melanogaster that is unique to the fruit fly, indicating a recent evolutionary emergence. Interestingly, the fission yeast repertoire, which contains no unique cyclophilins and parvulins, shares no PPIases solely with the budding yeast but it does share a majority with the higher eukaryotes in this study, unlike the budding yeast. It therefore appears that, in comparison with Schizosaccharomyces pombe, Saccharomyces cerevisiae is a poor representation of the higher eukaryotes for the study of PPIases. PMID:18629211

  11. Single-Domain Peptidyl-Prolyl cis/trans Isomerase FkpA from Corynebacterium glutamicum Improves the Biomass Yield at Increased Growth Temperatures

    PubMed Central

    Bott, Michael; van Ooyen, Jan

    2015-01-01

    Peptidyl-prolyl cis/trans isomerases (PPIases) catalyze the rate-limiting protein folding step at peptidyl bonds preceding proline residues and were found to be involved in several biological processes, including gene expression, signal transduction, and protein secretion. Representative enzymes were found in almost all sequenced genomes, including Corynebacterium glutamicum, a facultative anaerobic Gram-positive and industrial workhorse for the production of amino acids. In C. glutamicum, a predicted single-domain FK-506 (tacrolimus) binding protein (FKBP)-type PPIase (FkpA) is encoded directly downstream of gltA, which encodes citrate synthase (CS). This gene cluster is also present in other Actinobacteria. Here we carried out in vitro and in vivo experiments to study the function and influence of predicted FkpA in C. glutamicum. In vitro, FkpA indeed shows typical PPIase activity with artificial substrates and is inhibited by FK-506. Furthermore, FkpA delays the aggregation of CS, which is also inhibited by FK-506. Surprisingly, FkpA has a positive effect on the activity and temperature range of CS in vitro. Deletion of fkpA causes a 50% reduced biomass yield compared to that of the wild type when grown at 37°C, whereas there is only a 10% reduced biomass yield at the optimal growth temperature of 30°C accompanied by accumulation of 7 mM l-glutamate and 22 mM 2-oxoglutarate. Thus, FkpA may be exploited for improved product formation in biotechnical processes. Comparative transcriptome analysis revealed 69 genes which exhibit ≥2-fold mRNA level changes in C. glutamicum ΔfkpA, giving insight into the transcriptional response upon mild heat stress when FkpA is absent. PMID:26341203

  12. Identification of Conserved and Species-Specific Functions of the Listeria monocytogenes PrsA2 Secretion Chaperone.

    PubMed

    Cahoon, Laty A; Freitag, Nancy E

    2015-10-01

    The Gram-positive bacterium Listeria monocytogenes is a facultative intracellular pathogen that relies on the regulated secretion and activity of a variety of proteins that sustain life within diverse environments. PrsA2 has recently been identified as a secreted peptidyl-prolyl cis/trans isomerase and chaperone that is dispensable for bacterial growth in broth culture but essential for L. monocytogenes virulence. Following host infection, PrsA2 contributes to the proper folding and activity of secreted proteins that are required for bacterial replication within the host cytosol and for bacterial spread to adjacent cells. PrsA2 is one member of a family of Gram-positive secretion chaperones that appear to play important roles in bacterial physiology; however, it is not known how these proteins recognize their substrate proteins or the degree to which their function is conserved across diverse Gram-positive species. We therefore examined PrsA proteins encoded by a variety of Gram-positive bacteria for functional complementation of L. monocytogenes mutants lacking prsA2. PrsA homologues encoded by Bacillus subtilis, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus mutans, Staphylococcus aureus, and Lactococcus lactis were examined for functional complementation of a variety of L. monocytogenes PrsA2-associated phenotypes central to L. monocytogenes pathogenesis and bacterial cell physiology. Our results indicate that while selected aspects of PrsA2 function are broadly conserved among diverse Gram-positive bacteria, PrsA2 exhibits unique specificity for L. monocytogenes target proteins required for pathogenesis. The L. monocytogenes PrsA2 chaperone thus appears evolutionarily optimized for virulence factor secretion within the host cell cytosol while still maintaining aspects of activity relevant to more general features of Gram-positive protein translocation. PMID:26216425

  13. Utilizing the peptidyl-prolyl cis-trans isomerase pin1 as a probe of its phosphorylated target proteins. Examples of binding to nuclear proteins in a human kidney cell line and to tau in Alzheimer's diseased brain.

    PubMed

    Thorpe, J R; Morley, S J; Rulten, S L

    2001-01-01

    The human parvulin Pin1 is a member of the peptidyl-prolyl cis-trans isomerase group of proteins, which modulate the assembly, folding, activity, and transport of essential cellular proteins. Pin1 is a mitotic regulator interacting with a range of proteins that are phosphorylated before cell division. In addition, an involvement of Pin1 in the tau-related neurodegenerative brain disorders has recently been shown. In this context, Pin1 becomes depleted from the nucleus in Alzheimer's disease (AD) neurons when it is redirected to the large amounts of hyperphosphorylated tau associated with the neurofibrillary tangles. This depletion from the nucleus may ultimately contribute to neuron cell death. Recently we have devised a novel methodology in which exogenous Pin1 is used as a TEM probe for its target proteins. Here we extend this methodology to provide further evidence that Pin1 binds at enhanced levels to mitotic nuclear proteins and to hyperphosphorylated tau in AD brain. We suggest that exogenous Pin1 labeling can be used to elucidate the phosphorylation status of its target proteins in general and could specifically provide important insights into the development of tau-related neurodegenerative brain disorders. PMID:11118482

  14. NF-κB transcriptional activity is modulated by FK506-binding proteins FKBP51 and FKBP52: a role for peptidyl-prolyl isomerase activity.

    PubMed

    Erlejman, Alejandra G; De Leo, Sonia A; Mazaira, Gisela I; Molinari, Alejandro M; Camisay, María Fernanda; Fontana, Vanina; Cox, Marc B; Piwien-Pilipuk, Graciela; Galigniana, Mario D

    2014-09-19

    Hsp90 binding immunophilins FKBP51 and FKBP52 modulate steroid receptor trafficking and hormone-dependent biological responses. With the purpose to expand this model to other nuclear factors that are also subject to nuclear-cytoplasmic shuttling, we analyzed whether these immunophilins modulate NF-κB signaling. It is demonstrated that FKBP51 impairs both the nuclear translocation rate of NF-κB and its transcriptional activity. The inhibitory action of FKBP51 requires neither the peptidylprolyl-isomerase activity of the immunophilin nor its association with Hsp90. The TPR domain of FKBP51 is essential. On the other hand, FKBP52 favors the nuclear retention time of RelA, its association to a DNA consensus binding sequence, and NF-κB transcriptional activity, the latter effect being strongly dependent on the peptidylprolyl-isomerase activity and also on the TPR domain of FKBP52, but its interaction with Hsp90 is not required. In unstimulated cells, FKBP51 forms endogenous complexes with cytoplasmic RelA. Upon cell stimulation with phorbol ester, the NF-κB soluble complex exchanges FKBP51 for FKBP52, and the NF-κB biological effect is triggered. Importantly, FKBP52 is functionally recruited to the promoter region of NF-κB target genes, whereas FKBP51 is released. Competition assays demonstrated that both immunophilins antagonize one another, and binding assays with purified proteins suggest that the association of RelA and immunophilins could be direct. These observations suggest that the biological action of NF-κB in different cell types could be positively regulated by a high FKBP52/FKBP51 expression ratio by favoring NF-κB nuclear retention, recruitment to the promoter regions of target genes, and transcriptional activity. PMID:25104352

  15. Calcineurin Undergoes a Conformational Switch Evoked via Peptidyl-Prolyl Isomerization

    PubMed Central

    Guasch, Alicia; Aranguren-Ibáñez, Álvaro; Pérez-Luque, Rosa; Aparicio, David; Martínez-Høyer, Sergio; Mulero, M. Carmen; Serrano-Candelas, Eva

    2015-01-01

    A limited repertoire of PPP family of serine/threonine phosphatases with a highly conserved catalytic domain acts on thousands of protein targets to orchestrate myriad central biological roles. A major structural reorganization of human calcineurin, a ubiquitous Ser/Thr PPP regulated by calcium and calmodulin and targeted by immunosuppressant drugs cyclosporin A and FK506, is unveiled here. The new conformation involves trans- to cis- isomerization of proline in the SAPNY sequence, highly conserved across PPPs, and remodels the main regulatory site where NFATc transcription factors bind. Transitions between cis- and trans- conformations may involve peptidyl prolyl isomerases such as cyclophilin A and FKBP12, which are known to physically interact with and modulate calcineurin even in the absence of immunosuppressant drugs. Alternative conformations in PPPs provide a new perspective on interactions with substrates and other protein partners and may foster development of more specific inhibitors as drug candidates. PMID:26248042

  16. Internal Activation of Peptidyl Prolyl Thioesters in Native Chemical Ligation.

    PubMed

    Gui, Yue; Qiu, Lingqi; Li, Yaohao; Li, Hongxing; Dong, Suwei

    2016-04-13

    Prolyl thioesters have shown significantly lower reactivities in native chemical ligation (NCL) in comparison to that of the alanyl thioester. This report describes a mild and efficient internal activation protocol of peptidyl prolyl thioesters in NCL without using any thiol-based additives, where the introduction of a 4-mercaptan substituent on the C-terminal proline significantly improves the reactivity of prolyl thioesters via the formation of a bicyclic thiolactone intermediate. The kinetic data indicate that the reaction rate is comparable to that of the reported data of alanyl thioesters, and the mechanistic studies suggest that the ligation of two peptide segments proceeds through an NCL-like pathway instead of a direct aminolysis, which ensures the chemoselectivity and compatibility of various amino acid side chains. This 4-mercaptoprolyl thioester-based protocol also allows an efficient one-pot ligation-desulfurization procedure. The utility of this method has been further demonstrated in the synthesis of a proline-rich region of Wilms tumor protein 1. PMID:26982082

  17. Folding of barstar C40A/C82A/P27A and catalysis of the peptidyl-prolyl cis/trans isomerization by human cytosolic cyclophilin (Cyp18).

    PubMed Central

    Golbik, R.; Fischer, G.; Fersht, A. R.

    1999-01-01

    Refolding of b*C40A/C82A/P27A is comprised of several kinetically detectable folding phases. The slowest phase in refolding originates from trans-->cis isomerization of the Tyr47-Pro48 peptide bond being in cis conformation in the native state. This refolding phase can be accelerated by the peptidyl-prolyl cis/trans isomerase human cytosolic cyclophilin (Cyp18) with a kcat/K(M) of 254,000 M(-1) s(-1). The fast refolding phase is not influenced by the enzyme. PMID:10422840

  18. A structural comparison of Listeria monocytogenes protein chaperones PrsA1 and PrsA2 reveals molecular features required for virulence.

    PubMed

    Cahoon, Laty A; Freitag, Nancy E; Prehna, Gerd

    2016-07-01

    Listeria monocytogenes is a Gram-positive environmental bacterium that lives within soil but transitions into a pathogen upon contact with a mammalian host. The transition of L. monocytogenes from soil dweller to cytosolic pathogen is dependent upon secreted virulence factors that mediate cell invasion and intracellular growth. PrsA1 and PrsA2 are secreted bacterial lipoprotein chaperones that contribute to the folding of proteins translocated across the bacterial membrane; PrsA2 is required for L. monocytogenes virulence, whereas the function of PrsA1 remains to be determined. We have solved an X-ray crystal structure of PrsA1 and have used this model to guide comparison structure-based mutagenesis studies with PrsA2. Targeted mutagenesis of PrsA2 demonstrates that oligomerization of PrsA2 as well as molecular features of the foldase domain are required for protein secretion and virulence, whereas a functional role was uncovered for PrsA1 in bacterial resistance to alcohol. Interestingly, PrsA2 membrane localization is not required for all PrsA2-dependent activities, suggesting that the lipoprotein retains function when released from the bacterial cell. PrsA chaperones are thus multifaceted proteins with distinct domains adapted to accommodate the functional needs of a diverse array of secreted substrates. PMID:27007641

  19. Chaperone function of FkpA, a heat shock prolyl isomerase, in the periplasm of Escherichia coli.

    PubMed

    Arié, J P; Sassoon, N; Betton, J M

    2001-01-01

    The nature of molecular chaperones in the periplasm of Escherichia coli that assist newly translocated proteins to reach their native state has remained poorly defined. Here, we show that FkpA, a heat shock periplasmic peptidyl-prolyl cis/trans isomerase (PPIase), suppresses the formation of inclusion bodies from a defective-folding variant of the maltose-binding protein, MalE31. This chaperone-like activity of FkpA, which is independent of its PPIase activity, requires a full-length structure of the protein. In vitro, FkpA does not catalyse a slow rate-limiting step in the refolding of MalE31, but prevents its aggregation at stoichiometric amounts and promotes the reactivation of denaturated citrate synthase. We propose that FkpA functions as a chaperone for envelope proteins in the bacterial periplasm. PMID:11123702

  20. Phosphorylation of human immunodeficiency virus type 1 capsid protein at serine 16, required for peptidyl-prolyl isomerase-dependent uncoating, is mediated by virion-incorporated extracellular signal-regulated kinase 2.

    PubMed

    Dochi, Takeo; Nakano, Takashi; Inoue, Mutsumi; Takamune, Nobutoki; Shoji, Shozo; Sano, Kouichi; Misumi, Shogo

    2014-05-01

    We reported previously that Pin1 facilitates human immunodeficiency virus type 1 (HIV-1) uncoating by interacting with the capsid core through the phosphorylated Ser(16)-Pro(17) motif. However, the specific kinase responsible for Ser(16) phosphorylation has remained unknown. Here, we showed that virion-associated extracellular signal-regulated kinase 2 (ERK2) phosphorylates Ser(16). The characterization of immature virions produced by exposing chronically HIV-1LAV-1-infected CEM/LAV-1 cells to 10 µM saquinavir indicated that Ser(16) is phosphorylated after the initiation of Pr55(Gag) processing. Furthermore, a mass spectrometry-based in vitro kinase assay demonstrated that ERK2 specifically phosphorylated the Ser(16) residue in the Ser(16)-Pro(17) motif-containing substrate. The treatment of CEM/LAV-1 cells with the ERK2 inhibitor sc-222229 decreased the Ser(16) phosphorylation level inside virions, and virus partially defective in Ser(16) phosphorylation showed impaired reverse transcription and attenuated replication owing to attenuated Pin1-dependent uncoating. Furthermore, the suppression of ERK2 expression by RNA interference in CEM/LAV-1 cells resulted in suppressed ERK2 packaging inside virions and decreased the Ser(16) phosphorylation level inside virions. Interestingly, the ERK2-packaging-defective virus showed impaired reverse transcription and attenuated HIV-1 replication. Taken together, these findings provide insights into the as-yet-obscure processes in Pin1-dependent HIV-1 uncoating. PMID:24509437

  1. ShaPINg Cell Fate Upon DNA Damage: Role of Pin1 Isomerase in DNA Damage-Induced Cell Death and Repair.

    PubMed

    Polonio-Vallon, Tilman; Krüger, Daniel; Hofmann, Thomas G

    2014-01-01

    The peptidyl-prolyl cis/trans isomerase Pin1 acts as a molecular timer in proline-directed Ser/Thr kinase signaling and shapes cellular responses based on recognition of phosphorylation marks and implementing conformational changes in its substrates. Accordingly, Pin1 has been linked to numerous phosphorylation-controlled signaling pathways and cellular processes such as cell cycle progression, proliferation, and differentiation. In addition, Pin1 plays a pivotal role in DNA damage-triggered cell fate decisions. Whereas moderate DNA damage is balanced by DNA repair, cells confronted with massive genotoxic stress are eliminated by the induction of programed cell death or cellular senescence. In this review, we summarize and discuss the current knowledge on how Pin1 specifies cell fate through regulating key players of the apoptotic and the repair branch of the DNA-damage response. PMID:24982848

  2. Roles of Prolyl Isomerases in RNA-Mediated Gene Expression

    PubMed Central

    Thapar, Roopa

    2015-01-01

    The peptidyl-prolyl cis-trans isomerases (PPIases) that include immunophilins (cyclophilins and FKBPs) and parvulins (Pin1, Par14, Par17) participate in cell signaling, transcription, pre-mRNA processing and mRNA decay. The human genome encodes 19 cyclophilins, 18 FKBPs and three parvulins. Immunophilins are receptors for the immunosuppressive drugs cyclosporin A, FK506, and rapamycin that are used in organ transplantation. Pin1 has also been targeted in the treatment of Alzheimer’s disease, asthma, and a number of cancers. While these PPIases are characterized as molecular chaperones, they also act in a nonchaperone manner to promote protein-protein interactions using surfaces outside their active sites. The immunosuppressive drugs act by a gain-of-function mechanism by promoting protein-protein interactions in vivo. Several immunophilins have been identified as components of the spliceosome and are essential for alternative splicing. Pin1 plays roles in transcription and RNA processing by catalyzing conformational changes in the RNA Pol II C-terminal domain. Pin1 also binds several RNA binding proteins such as AUF1, KSRP, HuR, and SLBP that regulate mRNA decay by remodeling mRNP complexes. The functions of ribonucleoprotein associated PPIases are largely unknown. This review highlights PPIases that play roles in RNA-mediated gene expression, providing insight into their structures, functions and mechanisms of action in mRNP remodeling in vivo. PMID:25992900

  3. Theileria parasites secrete a prolyl isomerase to maintain host leukocyte transformation

    PubMed Central

    Marsolier, J.; Perichon, M.; DeBarry, JD.; Villoutreix, BO.; Chluba, J.; Lopez, T.; Garrido, C.; Zhou, XZ.; Lu, KP.; Fritsch, L.; Ait-Si-Ali, S.; Mhadhbi, M; Medjkane, S.; Weitzman, JB.

    2014-01-01

    Infectious agents develop intricate mechanisms to interact with host cell pathways and hijack the genetic and epigenetic machinery to change phenotypic states. Amongst the Apicomplexa phylum of obligate intracellular parasites which cause veterinary and human diseases, Theileria is the only genus which transforms its mammalian host cells1. Theileria infection of bovine leukocytes induces proliferative and invasive phenotypes associated with activated signalling pathways, notably JNK and AP-12. The transformed phenotypes are reversed by treatment with the theilericidal drug Buparvaquone3. We used comparative genomics to identify a homologue of the Peptidyl Prolyl Isomerase Pin1 (designated TaPin1) in T. annulata which is secreted into the host cell and modulates oncogenic signalling pathways. Here we show that TaPin1 is a bona fide prolyl isomerase and that it interacts with the host ubiquitin ligase FBW7 leading to its degradation and subsequent stabilization of c-Jun which promotes transformation. We performed in vitro analysis and in vivo zebrafish xenograft experiments to demonstrate that TaPin1 is directly inhibited by the anti-parasite drug Buparvaquone (and other known Pin1 inhibitors) and is mutated in a drug-resistant strain. Prolyl isomerisation is thus a conserved mechanism which is important in cancer and is used by Theileria parasites to manipulate host oncogenic signaling. PMID:25624101

  4. Downregulation of Peptidylprolyl isomerase A promotes cell death and enhances doxorubicin-induced apoptosis in hepatocellular carcinoma.

    PubMed

    Cheng, Shaobing; Luo, Mengchao; Ding, Chaofeng; Peng, Chuanhui; Lv, Zhen; Tong, Rongliang; Xiao, Heng; Xie, Haiyang; Zhou, Lin; Wu, Jian; Zheng, Shusen

    2016-10-10

    Peptidylprolyl isomerase A (PPIA) is a peptidyl-prolyl cis-trans isomerase that is known to play a critical role in the development of many human cancers. However, the precise biological function of PPIA in hepatocellular carcinoma (HCC) remains largely unclear. In this study, lentiviral overexpression vectors and small interfering RNA knockdown methods were employed to investigate the biological effects of PPIA in HCC. PPIA levels in HCC tissues and peritumoral tissues were detected by real-time Polymerase Chain Reaction (RT-PCR), Western blotting, and immunohistochemistry. Our results indicate that PPIA levels were significantly higher in the HCC tissues compared to the matched peritumoral tissues. Moreover, PPIA expression was significantly associated with tumor size in these tissues. Interestingly, serum PPIA (sPPIA) levels were significantly higher in healthy controls compared to the HCC patients. Knockdown or overexpression of PPIA was shown to downregulate and upregulate cell growth, respectively. Moreover, PPIA siRNA knockdown appears to promote doxorubicin-induced apoptosis in HCC cells, altering the expression of downstream apoptotic factors. In summary, our results indicate that PPIA may play a pivotal role in HCC by regulating cell growth and could serve as a novel marker and therapeutic molecular target for HCC patients. PMID:27397650

  5. Phosphate–Induced Renal Fibrosis Requires the Prolyl Isomerase Pin1

    PubMed Central

    Shiizaki, Kazuhiro; Kuro-o, Makoto; Malter, James S.

    2016-01-01

    Tubulo-interstitial fibrosis is a common, destructive endpoint for a variety of kidney diseases. Fibrosis is well correlated with the loss of kidney function in both humans and rodents. The identification of modulators of fibrosis could provide novel therapeutic approaches to reducing disease progression or severity. Here, we show that the peptidyl-prolyl isomerase Pin1 is an important molecular contributor that facilitates renal fibrosis in a well-characterized animal model. While wild-type mice fed a high phosphate diet (HPD) for 8–12 weeks developed calcium deposition, macrophage infiltration and extracellular matrix (ECM) accumulation in the kidney interstitium, Pin1 null mice showed significantly less pathology. The serum Pi in both WT and KO mice were significantly increased by the HPD, but the serum Ca was slightly decreased in KO compared to WT. In addition, both WT and KO HPD mice had less weight gain but exhibited normal organ mass (kidney, lung, spleen, liver and heart). Unexpectedly, renal function was not initially impaired in either genotype irrespective of the HPD. Our results suggest that diet containing high Pi induces rapid renal fibrosis before a significant impact on renal function and that Pin1 plays an important role in the fibrotic process. PMID:26914452

  6. Prolyl isomerase Pin1 and protein kinase HIPK2 cooperate to promote cortical neurogenesis by suppressing Groucho/TLE:Hes1-mediated inhibition of neuronal differentiation.

    PubMed

    Ciarapica, R; Methot, L; Tang, Y; Lo, R; Dali, R; Buscarlet, M; Locatelli, F; del Sal, G; Rota, R; Stifani, S

    2014-02-01

    The Groucho/transducin-like Enhancer of split 1 (Gro/TLE1):Hes1 transcriptional repression complex acts in cerebral cortical neural progenitor cells to inhibit neuronal differentiation. The molecular mechanisms that regulate the anti-neurogenic function of the Gro/TLE1:Hes1 complex during cortical neurogenesis remain to be defined. Here we show that prolyl isomerase Pin1 (peptidyl-prolyl cis-trans isomerase NIMA-interacting 1) and homeodomain-interacting protein kinase 2 (HIPK2) are expressed in cortical neural progenitor cells and form a complex that interacts with the Gro/TLE1:Hes1 complex. This association depends on the enzymatic activities of both HIPK2 and Pin1, as well as on the association of Gro/TLE1 with Hes1, but is independent of the previously described Hes1-activated phosphorylation of Gro/TLE1. Interaction with the Pin1:HIPK2 complex results in Gro/TLE1 hyperphosphorylation and weakens both the transcriptional repression activity and the anti-neurogenic function of the Gro/TLE1:Hes1 complex. These results provide evidence that HIPK2 and Pin1 work together to promote cortical neurogenesis, at least in part, by suppressing Gro/TLE1:Hes1-mediated inhibition of neuronal differentiation. PMID:24270405

  7. Cyclophilin40 isomerase activity is regulated by a temperature-dependent allosteric interaction with Hsp90

    PubMed Central

    Blackburn, Elizabeth A.; Wear, Martin A.; Landré, Vivian; Narayan, Vikram; Ning, Jia; Erman, Burak; Ball, Kathryn L.; Walkinshaw, Malcolm D.

    2015-01-01

    Cyclophilin 40 (Cyp40) comprises an N-terminal cyclophilin domain with peptidyl-prolyl isomerase (PPIase) activity and a C-terminal tetratricopeptide repeat (TPR) domain that binds to the C-terminal–EEVD sequence common to both heat shock protein 70 (Hsp70) and Hsp90. We show in the present study that binding of peptides containing the MEEVD motif reduces the PPIase activity by ∼30%. CD and fluorescence assays show that the TPR domain is less stable than the cyclophilin domain and is stabilized by peptide binding. Isothermal titration calorimetry (ITC) shows that the affinity for the–MEEVD peptide is temperature sensitive in the physiological temperature range. Results from these biophysical studies fit with the MD simulations of the apo and holo (peptide-bound) structures which show a significant reduction in root mean square (RMS) fluctuation in both TPR and cyclophilin domains when–MEEVD is bound. The MD simulations of the apo-protein also highlight strong anti-correlated motions between residues around the PPIase-active site and a band of residues running across four of the seven helices in the TPR domain. Peptide binding leads to a distortion in the shape of the active site and a significant reduction in these strongly anti-correlated motions, providing an explanation for the allosteric effect of ligand binding and loss of PPIase activity. Together the experimental and MD results suggest that on heat shock, dissociation of Cyp40 from complexes mediated by the TPR domain leads to an increased pool of free Cyp40 capable of acting as an isomerase/chaperone in conditions of cellular stress. PMID:26330616

  8. A dual inhibitor against prolyl isomerase Pin1 and cyclophilin discovered by a novel real-time fluorescence detection method

    SciTech Connect

    Mori, Tadashi; Hidaka, Masafumi; Lin, Yi-Chin; Yoshizawa, Ibuki; Okabe, Takayoshi; Egashira, Shinichiro; Kojima, Hirotatsu; Nagano, Tetsuo; Koketsu, Mamoru; Takamiya, Mari; Uchida, Takafumi

    2011-03-18

    Research highlights: {yields} A Pin1 (prolyl isomerase) inhibitor, TME-001, has been discovered by using a new established high-throughput screening method. {yields} The TME-001 showed a cell-active inhibition with lower cytotoxic effect than known Pin1 inhibitors. {yields} Kinetic analyses revealed that the TME-001 is the first compound that exhibits dual inhibition of Pin1 and another type of prolyl isomerase, cyclophilin. {yields} Thus, similarities of structure and reaction mechanism between Pin1 and cyclophilin are proposed. -- Abstract: Pin1, a peptidyl prolyl cis/trans isomerase (PPIase), is a potential target molecule for cancer, infectious disease, and Alzheimer's disease. We established a high-throughput screening method for Pin1 inhibitors, which employs a real-time fluorescence detector. This screening method identified 66 compounds that inhibit Pin1 out of 9756 compounds from structurally diverse chemical libraries. Further evaluations of surface plasmon resonance methods and a cell proliferation assay were performed. We discovered a cell-active inhibitor, TME-001 (2-(3-chloro-4-fluoro-phenyl)-isothiazol-3-one). Surprisingly, kinetic analyses revealed that TME-001 is the first compound that exhibits dual inhibition of Pin1 (IC{sub 50} = 6.1 {mu}M) and cyclophilin, another type of PPIase, (IC{sub 50} = 13.7 {mu}M). This compound does not inhibit FKBP. This finding suggests the existence of similarities of structure and reaction mechanism between Pin1 and cyclophilin, and may lead to a more complete understanding of the active sites of PPIases.

  9. A high-throughput screen for inhibitors of the prolyl isomerase, Pin1, identifies a seaweed polyphenol that reduces adipose cell differentiation.

    PubMed

    Mori, Tadashi; Hidaka, Masafumi; Ikuji, Hiroko; Yoshizawa, Ibuki; Toyohara, Haruhiko; Okuda, Toru; Uchida, Chiyoko; Asano, Tomoichiro; Yotsu-Yamashita, Mari; Uchida, Takafumi

    2014-01-01

    The peptidyl prolyl cis/trans isomerase Pin1 enhances the uptake of triglycerides and the differentiation of fibroblasts into adipose cells in response to insulin stimulation. Pin1 downregulation could be a potential approach to prevent and treat obesity-related disorders. In order to identify an inhibitor of Pin1 that exhibited minimal cytotoxicity, we established a high-throughput screen for Pin1 inhibitors and used this method to identify an inhibitor from 1,056 crude fractions of two natural product libraries. The candidate, a phlorotannin called 974-B, was isolated from the seaweed, Ecklonia kurome. 974-B inhibited the differentiation of mouse embryonic fibroblasts and 3T3-L1 cells into adipose cells without inducing cytotoxicity. We discovered the Pin1 inhibitor, 974-B, from the seaweed, E. kurome, and showed that it blocks the differentiation of fibroblasts into adipose cells, suggesting that 974-B could be a lead drug candidate for obesity-related disorders. PMID:25035986

  10. Prolyl isomerase Pin1 negatively regulates the stability of SUV39H1 to promote tumorigenesis in breast cancer.

    PubMed

    Khanal, Prem; Kim, Garam; Lim, Sung-Chul; Yun, Hyo-Jeong; Lee, Kwang Youl; Choi, Hoo-Kyun; Choi, Hong Seok

    2013-11-01

    Pin1, a conserved eukaryotic peptidyl-prolyl cis/trans isomerase, has profound effects on numerous key-signaling molecules, and its deregulation contributes to disease, particularly cancer. Although Pin1-mediated prolyl isomerization of protein servers as a regulatory switch in signaling pathways, the significance of proline isomerase activity in chromatin modifying complex remains unclear. Here, we identify Pin1 as a key negative regulator for suppressor of variegation 3-9 homologue 1 (SUV39H1) stability, a major methyltransferase responsible for histone H3 trimethylation on Lys9 (H3K9me3). Pin1 interacts with SUV39H1 in a phosphorylation-dependent manner and promotes ubiquitination-mediated degradation of SUV39H1. Consequently, Pin1 reduces SUV39H1 abundance and suppresses SUV39H1 ability to induce H3K9me3. In contrast, depletion of Pin1 in cancer cells leads to elevated SUV39H1 expression, which subsequently increases H3K9me3, inhibiting tumorigenecity of cancer cells. In a xenograft model with 4T1 metastatic mouse breast carcinoma cells, Pin1 overexpression increases tumor growth, whereas SUV39H1 overexpression abrogates it. In human breast cancer patients, immunohistochemical staining shows that Pin1 levels are negatively correlated with SUV39H1 as well as H3K9me3 levels. Thus, Pin1-mediated reduction of SUV39H1 stability contributes to convey oncogenic signals for aggressiveness of human breast cancer, suggesting that Pin1 may be a promising drug target for anticancer therapy. PMID:23934277

  11. Uncoating of human immunodeficiency virus type 1 requires prolyl isomerase Pin1.

    PubMed

    Misumi, Shogo; Inoue, Mutsumi; Dochi, Takeo; Kishimoto, Naoki; Hasegawa, Naomi; Takamune, Nobutoki; Shoji, Shozo

    2010-08-13

    The process by which the human immunodeficiency virus type 1 (HIV-1) conical core dissociates is called uncoating, but not much is known about this process. Here, we show that the uncoating process requires the interaction of the capsid (CA) protein with the peptidyl-prolyl isomerase Pin1 that specifically recognizes the phosphorylated serine/threonine residue followed by proline. We found that the HIV-1 core is composed of some isoforms of the CA protein with different isoelectric points, and one isoform is preferentially phosphorylated in the Ser(16)-Pro(17) motif. The mutant virus S16A/P17A shows a severely attenuated HIV-1 replication and an impaired reverse transcription. The S16A/P17A change increased the amount of particulate CA cores in the cytosol of target cells and correlated with the restriction of HIV-1 infection. Glutathione S-transferase pulldown assays demonstrated a direct interaction between Pin1 and the HIV-1 core via the Ser(16)-Pro(17) motif. Suppression of Pin1 expression by RNA interference in a target cell results in an attenuated HIV-1 replication and increases the amount of particulate CA cores in the cytosol of target cells. Furthermore, heat-inactivated, inhibitor-treated, or W34A/K63A Pin1 causes an attenuated in vitro uncoating of the HIV-1 core. The Pin1-dependent uncoating is inhibited by antisera raised against a CA peptide phosphorylated at Ser(16) or treatment of the HIV-1 core with alkaline phosphatase. These findings provide insights into this obscure uncoating process in the HIV-1 life cycle and a new cellular target for HIV-1 drug development. PMID:20529865

  12. Glucose-6-phosphate isomerase.

    PubMed

    Achari, A; Marshall, S E; Muirhead, H; Palmieri, R H; Noltmann, E A

    1981-06-26

    Glucose-6-phosphate isomerase (EC 5.3.1.9) is a dimeric enzyme of molecular mass 132000 which catalyses the interconversion of D-glucose-6-phosphate and D-fructose-6-phosphate. The crystal structure of the enzyme from pig muscle has been determined at a nominal resolution of 2.6 A. The structure is of the alpha/beta type. Each subunit consists of two domains and the active site is in both the domain interface and the subunit interface (P.J. Shaw & H. Muirhead (1976), FEBS Lett. 65, 50-55). Each subunit contains 13 methionine residues so that cyanogen bromide cleavage will produce 14 fragments, most of which have been identified and at least partly purified. Sequence information is given for about one-third of the molecule from 5 cyanogen bromide fragments. One of the sequences includes a modified lysine residue. Modification of this residue leads to a parallel loss of enzymatic activity. A tentative fit of two of the peptides to the electron density map has been made. It seems possible that glucose-6-phosphate isomerase, triose phosphate isomerase and pyruvate kinase all contain a histidine and a glutamate residue at the active site. PMID:6115414

  13. Genetics Home Reference: glucose phosphate isomerase deficiency

    MedlinePlus

    ... Me Understand Genetics Home Health Conditions GPI deficiency glucose phosphate isomerase deficiency Enable Javascript to view the ... boxes. Download PDF Open All Close All Description Glucose phosphate isomerase (GPI) deficiency is an inherited disorder ...

  14. Development of Novel Sugar Isomerases by Optimization of Active Sites in Phosphosugar Isomerases for Monosaccharides

    PubMed Central

    Yeom, Soo-Jin; Kim, Yeong-Su

    2013-01-01

    Phosphosugar isomerases can catalyze the isomerization of not only phosphosugar but also of monosaccharides, suggesting that the phosphosugar isomerases can be used as sugar isomerases that do not exist in nature. Determination of active-site residues of phosphosugar isomerases, including ribose-5-phosphate isomerase from Clostridium difficile (CDRPI), mannose-6-phosphate isomerase from Bacillus subtilis (BSMPI), and glucose-6-phosphate isomerase from Pyrococcus furiosus (PFGPI), was accomplished by docking of monosaccharides onto the structure models of the isomerases. The determinant residues, including Arg133 of CDRPI, Arg192 of BSMPI, and Thr85 of PFGPI, were subjected to alanine substitutions and found to act as phosphate-binding sites. R133D of CDRPI, R192 of BSMPI, and T85Q of PFGPI displayed the highest catalytic efficiencies for monosaccharides at each position. These residues exhibited 1.8-, 3.5-, and 4.9-fold higher catalytic efficiencies, respectively, for the monosaccharides than the wild-type enzyme. However, the activities of these 3 variant enzymes for phosphosugars as the original substrates disappeared. Thus, R133D of CDRPI, R192 of BSMPI, and T85Q of PFGPI are no longer phosphosugar isomerases; instead, they are changed to a d-ribose isomerase, an l-ribose isomerase, and an l-talose isomerase, respectively. In this study, we used substrate-tailored optimization to develop novel sugar isomerases which are not found in nature based on phosphosugar isomerases. PMID:23204422

  15. Genetics Home Reference: triosephosphate isomerase deficiency

    MedlinePlus

    ... of triosephosphate isomerase deficiency. Eur J Haematol. 2011 Mar;86(3):265-7. doi: 10.1111/j.1600-0609.2010.01484.x. Citation on PubMed Orosz F, Oláh J, Ovádi J. Triosephosphate isomerase deficiency: facts and doubts. IUBMB Life. 2006 Dec;58(12):703-15. Review. Citation ...

  16. A Tale of Two Isomerases: Compact versus Extended Active Sites in Ketosteroid Isomerase and Phosphoglucose Isomerase

    SciTech Connect

    Somarowthu, Srinivas; Brodkin, Heather R.; D’Aquino, J. Alejandro; Ringe, Dagmar; Ondrechen, Mary Jo; Beuning, Penny J.

    2012-07-11

    Understanding the catalytic efficiency and specificity of enzymes is a fundamental question of major practical and conceptual importance in biochemistry. Although progress in biochemical and structural studies has enriched our knowledge of enzymes, the role in enzyme catalysis of residues that are not nearest neighbors of the reacting substrate molecule is largely unexplored experimentally. Here computational active site predictors, THEMATICS and POOL, were employed to identify functionally important residues that are not in direct contact with the reacting substrate molecule. These predictions then guided experiments to explore the active sites of two isomerases, Pseudomonas putida ketosteroid isomerase (KSI) and human phosphoglucose isomerase (PGI), as prototypes for very different types of predicted active sites. Both KSI and PGI are members of EC 5.3 and catalyze similar reactions, but they represent significantly different degrees of remote residue participation, as predicted by THEMATICS and POOL. For KSI, a compact active site of mostly first-shell residues is predicted, but for PGI, an extended active site in which residues in the first, second, and third layers around the reacting substrate are predicted. Predicted residues that have not been previously tested experimentally were investigated by site-directed mutagenesis and kinetic analysis. In human PGI, single-point mutations of the predicted second- and third-shell residues K362, H100, E495, D511, H396, and Q388 show significant decreases in catalytic activity relative to that of the wild type. The results of these experiments demonstrate that, as predicted, remote residues are very important in PGI catalysis but make only small contributions to catalysis in KSI.

  17. An endoplasmic reticulum-specific cyclophilin.

    PubMed Central

    Hasel, K W; Glass, J R; Godbout, M; Sutcliffe, J G

    1991-01-01

    Cyclophilin is a ubiquitously expressed cytosolic peptidyl-prolyl cis-trans isomerase that is inhibited by the immunosuppressive drug cyclosporin A. A degenerate oligonucleotide based on a conserved cyclophilin sequence was used to isolate cDNA clones representing a ubiquitously expressed mRNA from mice and humans. This mRNA encodes a novel 20-kDa protein, CPH2, that shares 64% sequence identity with cyclophilin. Bacterially expressed CPH2 binds cyclosporin A and is a cyclosporin A-inhibitable peptidyl-prolyl cis-trans isomerase. Cell fractionation of rat liver followed by Western blot (immunoblot) analysis indicated that CPH2 is not cytosolic but rather is located exclusively in the endoplasmic reticulum. These results suggest that cyclosporin A mediates its effect on cells through more than one cyclophilin and that cyclosporin A-induced misfolding of T-cell membrane proteins normally mediated by CPH2 plays a role in immunosuppression. Images PMID:1710767

  18. 21 CFR 862.1720 - Triose phosphate isomerase test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... isomerase test system is a device intended to measure the activity of the enzyme triose phosphate isomerase in erythrocytes (red blood cells). Triose phosphate isomerase is an enzyme important in glycolysis... this device are used in the diagnosis and treatment of congenital triose phosphate isomerase...

  19. 21 CFR 862.1720 - Triose phosphate isomerase test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... isomerase test system is a device intended to measure the activity of the enzyme triose phosphate isomerase in erythrocytes (red blood cells). Triose phosphate isomerase is an enzyme important in glycolysis... this device are used in the diagnosis and treatment of congenital triose phosphate isomerase...

  20. 21 CFR 862.1720 - Triose phosphate isomerase test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... isomerase test system is a device intended to measure the activity of the enzyme triose phosphate isomerase in erythrocytes (red blood cells). Triose phosphate isomerase is an enzyme important in glycolysis... this device are used in the diagnosis and treatment of congenital triose phosphate isomerase...

  1. 21 CFR 862.1720 - Triose phosphate isomerase test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... isomerase test system is a device intended to measure the activity of the enzyme triose phosphate isomerase in erythrocytes (red blood cells). Triose phosphate isomerase is an enzyme important in glycolysis... this device are used in the diagnosis and treatment of congenital triose phosphate isomerase...

  2. 21 CFR 862.1720 - Triose phosphate isomerase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... isomerase test system is a device intended to measure the activity of the enzyme triose phosphate isomerase in erythrocytes (red blood cells). Triose phosphate isomerase is an enzyme important in glycolysis... this device are used in the diagnosis and treatment of congenital triose phosphate isomerase...

  3. Essential role of Pro 74 in stefin B amyloid-fibril formation: dual action of cyclophilin A on the process.

    PubMed

    Smajlović, Aida; Berbić, Selma; Schiene-Fischer, Cordelia; Tusek-Znidaric, Magda; Taler, Ajda; Jenko-Kokalj, Sasa; Turk, Dusan; Zerovnik, Eva

    2009-04-01

    We report that Pro74 in human stefin B is critical for fibril formation and that proline isomerization plays an important role. The stefin B P74S mutant did not fibrillate over the time of observation at 25 degrees C, and it exhibited a prolonged lag phase at 30 degrees C and 37 degrees C. The peptidyl prolyl cis/trans isomerase cyclophilin A, when added to the wild-type protein, exerted two effects: it prolonged the lag phase and increased the yield and length of the fibrils. Addition of the inactive cyclophilin A R55A variant still resulted in a prolonged lag phase but did not mediate the increase of the final fibril yield. These results demonstrate that peptidyl prolyl cis/trans isomerism is rate-limiting in stefin B fibril formation. PMID:19265692

  4. 21 CFR 184.1372 - Insoluble glucose isomerase enzyme preparations.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Insoluble glucose isomerase enzyme preparations... RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS § 184.1372 Insoluble glucose isomerase enzyme preparations. (a) Insoluble glucose isomerase enzyme preparations are used in the production...

  5. 21 CFR 184.1372 - Insoluble glucose isomerase enzyme preparations.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Insoluble glucose isomerase enzyme preparations... RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS § 184.1372 Insoluble glucose isomerase enzyme preparations. (a) Insoluble glucose isomerase enzyme preparations are used in the production...

  6. 21 CFR 184.1372 - Insoluble glucose isomerase enzyme preparations.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Insoluble glucose isomerase enzyme preparations... Substances Affirmed as GRAS § 184.1372 Insoluble glucose isomerase enzyme preparations. (a) Insoluble glucose isomerase enzyme preparations are used in the production of high fructose corn syrup described in §...

  7. Thermoinactivation Mechanism of Glucose Isomerase

    NASA Astrophysics Data System (ADS)

    Lim, Leng Hong; Saville, Bradley A.

    In this article, the mechanisms of thermoinactivation of glucose isomerase (GI) from Streptomyces rubiginosus (in soluble and immobilized forms) were investigated, particularly the contributions of thiol oxidation of the enzyme's cysteine residue and a "Maillard-like" reaction between the enzyme and sugars in high fructose corn syrup (HFCS). Soluble GI (SGI) was successfully immobilized on silica gel (13.5 μm particle size), with an activity yield between 20 and 40%. The immobilized GI (IGI) has high enzyme retention on the support during the glucose isomerization process. In batch reactors, SGI (half-life =145 h) was more stable than IGI (half-life=27 h) at 60°C in HFCS, whereas at 80°C, IGI (half-life=12 h) was more stable than SGI (half-life=5.2 h). IGI was subject to thiol oxidation at 60°C, which contributed to the enzyme's deactivation. IGI was subject to thiol oxidation at 80°C, but this did not contribute to the deactivation of the enzyme. SGI did not undergo thiol oxidation at 60°C, but at 80°C SGI underwent severe precipitation and thiol oxidation, which caused the enzyme to deactivate. Experimental results show that immobilization suppresses the destablizing effect of thiol oxidation on GI. A "Maillard-like" reaction between SGI and the sugars also caused SGI thermoinactivation at 60, 70, and 80°C, but had minimal effect on IGI. At 60 and 80°C, IGI had higher thermostability in continuous reactors than in batch reactors, possibily because of reduced contact with deleterious compounds in HFCS.

  8. Dynamical role of phosphorylation on serine/threonine-proline Pin1 substrates from constant force molecular dynamics simulations

    NASA Astrophysics Data System (ADS)

    Velazquez, Hector A.; Hamelberg, Donald

    2015-02-01

    Cis-trans isomerization of peptidyl-prolyl bonds of the protein backbone plays an important role in numerous biological processes. Cis-trans isomerization can be the rate-limiting step due its extremely slow dynamics, compared to the millisecond time scale of many processes, and is catalyzed by a widely studied family of peptidyl-prolyl cis-trans isomerase enzymes. Also, mechanical forces along the peptide chain can speed up the rate of isomerization, resulting in "mechanical catalysis," and have been used to study peptidyl-prolyl cis-trans isomerization and other mechanical properties of proteins. Here, we use constant force molecular dynamics simulations to study the dynamical effects of phosphorylation on serine/threonine-proline protein motifs that are involved in the function of many proteins and have been implicated in many aberrant biological processes. We show that the rate of cis-trans isomerization is slowed down by phosphorylation, in excellent agreement with experiments. We use a well-grounded theory to describe the force dependent rate of isomerization. The calculated rates at zero force are also in excellent agreement with experimentally measured rates, providing additional validation of the models and force field parameters. Our results suggest that the slowdown in the rate upon phosphorylation is mainly due to an increase in the friction along the peptidyl-prolyl bond angle during isomerization. Our results provide a microscopic description of the dynamical effects of post-translational phosphorylation on cis-trans isomerization and insights into the properties of proteins under tension.

  9. 21 CFR 184.1372 - Insoluble glucose isomerase enzyme preparations.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Insoluble glucose isomerase enzyme preparations... enzyme preparations. (a) Insoluble glucose isomerase enzyme preparations are used in the production of... additional requirements for enzyme preparations in the Food Chemicals Codex, 3d Ed. (1981), p. 107, which...

  10. 21 CFR 184.1372 - Insoluble glucose isomerase enzyme preparations.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Insoluble glucose isomerase enzyme preparations... enzyme preparations. (a) Insoluble glucose isomerase enzyme preparations are used in the production of... additional requirements for enzyme preparations in the Food Chemicals Codex, 3d Ed. (1981), p. 107, which...

  11. Characterization of triosephosphate isomerase from Mycoplasma gallisepticum.

    PubMed

    Bao, Shijun; Chen, Danqing; Yu, Shengqing; Chen, Hongjun; Tan, Lei; Hu, Meirong; Qiu, Xusheng; Song, Cuiping; Ding, Chan

    2015-09-01

    Triosephosphate isomerase (Tpi) is a glycolytic enzyme that is essential for efficient energy production in many pathogens. However, its function in Mycoplasma gallisepticum has not been fully elucidated. In this study, the mga0357 gene of M. gallisepticum, which encodes TpiA (MGTpiA), was amplified and expressed in Escherichia coli by IPTG induction. The purified recombinant MGTpiA protein exhibited catalytic activity that was similar to TPI from rabbit muscle, reducing NAD(+) to NADH. The MGTpiA was also found to be a surface-exposed protein by western blotting and immunofluorescence assays. In addition, cytadherence inhibition assays confirmed that the cytadherence of M. gallisepticum to the DF-1 cells was significantly inhibited by the anti-MGTpiA serum. The results of the study suggested that MGTpiA plays an important role in the metabolism and closely related to the M. gallisepticum pathogenicity. PMID:26319024

  12. Molecular and industrial aspects of glucose isomerase.

    PubMed Central

    Bhosale, S H; Rao, M B; Deshpande, V V

    1996-01-01

    Glucose isomerase (GI) (D-xylose ketol-isomerase; EC. 5.3.1.5) catalyzes the reversible isomerization of D-glucose and D-xylose to D-fructose and D-xylulose, respectively. The enzyme has the largest market in the food industry because of its application in the production of high-fructose corn syrup (HFCS). HFCS, an equilibrium mixture of glucose and fructose, is 1.3 times sweeter than sucrose and serves as a sweetener for use by diabetics. Interconversion of xylose to xylulose by GI serves a nutritional requirement in saprophytic bacteria and has a potential application in the bioconversion of hemicellulose to ethanol. The enzyme is widely distributed in prokaryotes. Intensive research efforts are directed toward improving its suitability for industrial application. Development of microbial strains capable of utilizing xylan-containing raw materials for growth or screening for constitutive mutants of GI is expected to lead to discontinuation of the use of xylose as an inducer for the production of the enzyme. Elimination of Co2+ from the fermentation medium is desirable for avoiding health problems arising from human consumption of HFCS. Immobilization of GI provides an efficient means for its easy recovery and reuse and lowers the cost of its use. X-ray crystallographic and genetic engineering studies support a hydride shift mechanism for the action of GI. Cloning of GI in homologous as well as heterologous hosts has been carried out, with the prime aim of overproducing the enzyme and deciphering the genetic organization of individual genes (xylA, xylB, and xylR) in the xyl operon of different microorganisms. The organization of xylA and xylB seems to be highly conserved in all bacteria. The two genes are transcribed from the same strand in Escherichia coli and Bacillus and Lactobacillus species, whereas they are transcribed divergently on different strands in Streptomyces species. A comparison of the xylA sequences from several bacterial sources revealed the

  13. Anomeric specificity of D-xylose isomerase.

    PubMed

    Collyer, C A; Goldberg, J D; Viehmann, H; Blow, D M; Ramsden, N G; Fleet, G W; Montgomery, F J; Grice, P

    1992-12-01

    Crystal structures of complexes of D-xylose isomerase with deoxysugars have been determined. Deoxynojirimycin is a structural analogue of alpha-pyranose and mimics the binding of these aldose substrates. The structure of this complex supports the hypothesis that an imidazole group catalyzes ring opening of the pyranose. The steric restrictions in the active site of the enzyme prevent a beta-pyranose from binding in the same way. For the reverse reaction with ketoses, the anomeric specificity is less certain. Dideoxyimino-D-glucitol is a structural analogue of the ketose alpha-D-furanose. The binding of the inhibitor dideoxyimino-D-glucitol to the crystals of the enzyme does not mimic the binding of the reactive alpha-D-fructofuranose. Superposition of the nonphysiological substrate alpha-D-fructofuranose onto the atomic positions of dideoxyimino-D-glucitol is not possible due to the steric restrictions of the active site. However, by utilizing the approximate 2-fold symmetry of the sugar, a stereochemically sensible model is produced which is consistent with other data. In addition to reaction with alpha-D-furanose, the enzyme probably reacts with open ring keto sugars which are present at significant concentrations. Other sugars which resemble furanoses either do not inhibit significantly or are not observed in the crystals bound in a single conformation. PMID:1457418

  14. Overexpression of protein disulfide isomerase in Aspergillus.

    PubMed

    El-Adawi, H; Khanh, N Q; Gassen, H

    2000-10-01

    One of the major problems with the production of biotechnologically valuable proteins has been the purification of the product. For Escherichia coli and Saccharomyces cerevisiae, there are several techniques for the purification of intracellular proteins, but these are time consuming and often result in poor yields. Purification can be considerably facilitated, if the product is secreted from the host cell. In the work presented, we have constructed an expression vector (pSGNH2) for the secretion of protein disulfide isomerase (PDI; EC 5.3.4.1) from Aspergillus niger, in which the retention signal His-Asp-Glu-Leu (H-D-E-L) was modified to Ala-Leu-Glu-Gln (A-L-E-Q) via the polymerase chain reaction (PCR) method. The PDI gene was placed under the control of the A. oryzae alpha-amylase promoter. This expression vector was transformed into A. niger NRRL3, resulting in PDI secretion into the medium. The catalytic activity of overexpressed PDI from A. niger was indistinguishable from that of PDI isolated from bovine liver. With further strain improvement and optimization of culture conditions, it could be possible to raise the PDI production to the bioprocessing scale. PMID:10977899

  15. Extracellular Thiol Isomerases and Their Role in Thrombus Formation

    PubMed Central

    Schulman, Sol; Bendapudi, Pavan; Sharda, Anish; Chen, Vivien; Bellido-Martin, Lola; Jasuja, Reema; Furie, Barbara C.; Flaumenhaft, Robert

    2016-01-01

    Abstract Significance: The mammalian endoplasmic reticulum (ER) houses a large family of twenty thioredoxin-like proteins of which protein disulfide isomerase (PDI) is the archetypal member. Although the PDI family is best known for its role in oxidative protein folding of secretory proteins in the ER, these thioredoxin-like proteins fulfill ever-expanding roles, both within the secretory pathway and beyond. Recent Advances: Secreted PDI family proteins have now been shown to serve a critical role in platelet thrombus formation and fibrin generation. Utilizing intravital microscopy to visualize thrombus formation in mice, we have demonstrated the presence of extracellular PDI antigen during thrombus formation following injury of the vascular wall. Inhibition of PDI abrogates thrombus formation in vivo (16, 26, 46, 55). These observations have been extended to other PDI family members, including ERp57 (39, 116, 118, 123) and ERp5 (77). The vascular thiol isomerases are those PDI family members secreted from platelets and/or endothelium (40): PDI, ERp57, ERp5, ERp72, ERp44, ERp29, and TMX3. We focus here on PDI (16, 46, 55), ERp57 (39, 116, 118, 123), and ERp5 (77), which have been implicated in thrombus formation in vivo. Critical Issues: It would appear that a system of thiol isomerase redox catalysts has been hijacked from the ER to regulate thrombus formation in the vasculature. Future Directions: How this redox system is trafficked to and regulated at the cell surface, the identity of extracellular substrates, why so many thiol isomerases are required, and which thiol isomerase functions are necessary are critical unanswered questions in understanding the role of thiol isomerases in thrombus formation. Antioxid. Redox Signal. 24, 1–15. PMID:26467859

  16. Protein disulfide isomerase a multifunctional protein with multiple physiological roles

    NASA Astrophysics Data System (ADS)

    Ali Khan, Hyder; Mutus, Bulent

    2014-08-01

    Protein disulfide isomerase (PDI), is a member of the thioredoxin superfamily of redox proteins. PDI has three catalytic activities including, thiol-disulfide oxireductase, disulfide isomerase and redox-dependent chaperone. Originally, PDI was identified in the lumen of the endoplasmic reticulum and subsequently detected at additional locations, such as cell surfaces and the cytosol. This review will provide an overview of the recent advances in relating the structural features of PDI to its multiple catalytic roles as well as its physiological and pathophysiological functions related to redox regulation and protein folding.

  17. The structure of rhamnose isomerase from Escherichia coli and its relation with xylose isomerase illustrates a change between inter and intra-subunit complementation during evolution.

    PubMed

    Korndörfer, I P; Fessner, W D; Matthews, B W

    2000-07-21

    Using a new expression construct, rhamnose isomerase from Escherichia coli was purified and crystallized. The crystal structure was solved by multiple isomorphous replacement and refined to a crystallographic residual of 17.4 % at 1.6 A resolution. Rhamnose isomerase is a tight tetramer of four (beta/alpha)(8)-barrels. A comparison with other known structures reveals that rhamnose isomerase is most similar to xylose isomerase. Alignment of the sequences of the two enzymes based on their structures reveals a hitherto undetected sequence identity of 13 %, suggesting that the two enzymes evolved from a common precursor. The structure and arrangement of the (beta/alpha)(8)-barrels of rhamnose isomerase are very similar to xylose isomerase. Each enzyme does, however, have additional alpha-helical domains, which are involved in tetramer association, and largely differ in structure. The structures of complexes of rhamnose isomerase with the inhibitor l-rhamnitol and the natural substrate l-rhamnose were determined and suggest that an extended loop, which is disordered in the native enzyme, becomes ordered on substrate binding, and may exclude bulk solvent during catalysis. Unlike xylose isomerase, this loop does not extend across a subunit interface but contributes to the active site of its own subunit. It illustrates how an interconversion between inter and intra-subunit complementation can occur during evolution. In the crystal structure (although not necessarily in vivo) rhamnose isomerase appears to bind Zn(2+) at a "structural" site. In the presence of substrate the enzyme also binds Mn(2+) at a nearby "catalytic" site. An array of hydrophobic residues, not present in xylose isomerase, is likely to be responsible for the recognition of l-rhamnose as a substrate. The available structural data suggest that a metal-mediated hydride-shift mechanism, which is generally favored for xylose isomerase, is also feasible for rhamnose isomerase. PMID:10891278

  18. Ribose 5-Phosphate Isomerase Investigations for the Undergraduate Biochemistry Laboratory

    ERIC Educational Resources Information Center

    Jewett, Kathy; Sandwick, Roger K.

    2011-01-01

    The enzyme ribose 5-phosphate isomerase (RpiA) has many features that make it attractive as a focal point of a semester-long, advanced biochemistry laboratory for undergraduate students. The protein can easily and inexpensively be isolated from spinach using traditional purification techniques. Characterization of RpiA enzyme activity can be…

  19. Evidence for distinct dehydrogenase and isomerase sites within a single 3. beta. -hydroxysteroid dehydrogenase/5-ene-4-ene isomerase protein

    SciTech Connect

    Luu-The, V.; Takahashi, Masakazu; de Launoit, Y.; Dumont, M.; Lachance, Y.; Labrie, F. )

    1991-09-10

    Complementary DNA encoding human 3{beta}-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase (3-{beta}-HSD) has been expressed in transfected GH{sub 4}C{sub 1} with use of the cytomegalovirus promoter. The activity of the expressed protein clearly shows that both dehydrogenase and isomerase enzymatic activities are present within a single protein. However, such findings do not indicate whether the two activities reside within one or two closely related catalytic sites. With use of ({sup 3}H)-5-androstenedione, the intermediate compound in dehydroepiandrosterone (DHEA) transformation into 4-androstenedione by 3{beta}-HSD, the present study shows that 4MA (N,N-diethyl-4-methyl-3-oxo-4-aza-5{alpha}-androstane-17{beta}-carboxamide) and its analogues of 5-androstenedione to 4-androstenedione with an approximately 1,000-fold higher K{sub i} value. The present results thus strongly suggest that dehydrogenase and isomerase activities are present at separate sites on the 3-{beta}-HSD protein. Such data suggest that the irreversible step in the transformation of DHEA to 4-androstenedione is due to a separate site possessing isomerase activity that converts the 5-ene-3-keto to a much more stable 4-ene-3-keto configuration.

  20. A role for trigger factor and an rgg-like regulator in the transcription, secretion and processing of the cysteine proteinase of Streptococcus pyogenes.

    PubMed Central

    Lyon, W R; Gibson, C M; Caparon, M G

    1998-01-01

    The ability of numerous microorganisms to cause disease relies upon the highly regulated expression of secreted proteinases. In this study, mutagenesis with a novel derivative of Tn4001 was used to identify genes required for the expression of the secreted cysteine proteinase (SCP) of the pathogenic Gram-positive bacterium Streptococcus pyogenes. Designated as Rop loci (regulation of proteinase), ropB is a rgg-like transcriptional activator required for transcription of the gene which encodes the proteinase. In contrast, ropA contributes post-transcriptionally to the secretion and processing of SCP and encodes a homologue of Trigger Factor, a peptidyl-prolyl isomerase and putative chaparone which is highly conserved in most bacterial species, but of unknown function. Analysis of additional ropA mutants demonstrated that RopA acts both to assist in targeting SCP to the secretory pathway and to promote the ability of the proprotein to establish an active conformation upon secretion. This latter function was dependent upon the peptidyl-prolyl isomerase domain of RopA and mutants that lacked this domain exhibited a bipartite deficiency manifested as a kinetic defect in autologous processing of the proprotein to the mature proteinase, and as a catalytic defect in the mature proteinase. These results provide insight into the function of Trigger Factor, the regulation of proteinase activity and the mechanism of secretion in Gram-positive bacteria. PMID:9799235

  1. A role for trigger factor and an rgg-like regulator in the transcription, secretion and processing of the cysteine proteinase of Streptococcus pyogenes.

    PubMed

    Lyon, W R; Gibson, C M; Caparon, M G

    1998-11-01

    The ability of numerous microorganisms to cause disease relies upon the highly regulated expression of secreted proteinases. In this study, mutagenesis with a novel derivative of Tn4001 was used to identify genes required for the expression of the secreted cysteine proteinase (SCP) of the pathogenic Gram-positive bacterium Streptococcus pyogenes. Designated as Rop loci (regulation of proteinase), ropB is a rgg-like transcriptional activator required for transcription of the gene which encodes the proteinase. In contrast, ropA contributes post-transcriptionally to the secretion and processing of SCP and encodes a homologue of Trigger Factor, a peptidyl-prolyl isomerase and putative chaparone which is highly conserved in most bacterial species, but of unknown function. Analysis of additional ropA mutants demonstrated that RopA acts both to assist in targeting SCP to the secretory pathway and to promote the ability of the proprotein to establish an active conformation upon secretion. This latter function was dependent upon the peptidyl-prolyl isomerase domain of RopA and mutants that lacked this domain exhibited a bipartite deficiency manifested as a kinetic defect in autologous processing of the proprotein to the mature proteinase, and as a catalytic defect in the mature proteinase. These results provide insight into the function of Trigger Factor, the regulation of proteinase activity and the mechanism of secretion in Gram-positive bacteria. PMID:9799235

  2. Exploring the chemistry and evolution of the isomerases

    PubMed Central

    2016-01-01

    Isomerization reactions are fundamental in biology, and isomers usually differ in their biological role and pharmacological effects. In this study, we have cataloged the isomerization reactions known to occur in biology using a combination of manual and computational approaches. This method provides a robust basis for comparison and clustering of the reactions into classes. Comparing our results with the Enzyme Commission (EC) classification, the standard approach to represent enzyme function on the basis of the overall chemistry of the catalyzed reaction, expands our understanding of the biochemistry of isomerization. The grouping of reactions involving stereoisomerism is straightforward with two distinct types (racemases/epimerases and cis-trans isomerases), but reactions entailing structural isomerism are diverse and challenging to classify using a hierarchical approach. This study provides an overview of which isomerases occur in nature, how we should describe and classify them, and their diversity. PMID:26842835

  3. Exploring the chemistry and evolution of the isomerases.

    PubMed

    Martínez Cuesta, Sergio; Rahman, Syed Asad; Thornton, Janet M

    2016-02-16

    Isomerization reactions are fundamental in biology, and isomers usually differ in their biological role and pharmacological effects. In this study, we have cataloged the isomerization reactions known to occur in biology using a combination of manual and computational approaches. This method provides a robust basis for comparison and clustering of the reactions into classes. Comparing our results with the Enzyme Commission (EC) classification, the standard approach to represent enzyme function on the basis of the overall chemistry of the catalyzed reaction, expands our understanding of the biochemistry of isomerization. The grouping of reactions involving stereoisomerism is straightforward with two distinct types (racemases/epimerases and cis-trans isomerases), but reactions entailing structural isomerism are diverse and challenging to classify using a hierarchical approach. This study provides an overview of which isomerases occur in nature, how we should describe and classify them, and their diversity. PMID:26842835

  4. Substrate product equilibrium on a reversible enzyme, triosephosphate isomerase

    PubMed Central

    Rozovsky, Sharon; McDermott, Ann E.

    2007-01-01

    The highly efficient glycolytic enzyme, triosephosphate isomerase, is expected to differentially stabilize the proposed stable reaction species: ketone, aldehyde, and enediol(ate). The identity and steady-state populations of the chemical entities bound to triosephosphate isomerase have been probed by using solid- and solution-state NMR. The 13C-enriched ketone substrate, dihydroxyacetone phosphate, was bound to the enzyme and characterized at steady state over a range of sample conditions. The ketone substrate was observed to be the major species over a temperature range from −60°C to 15°C. Thus, there is no suggestion that the enzyme preferentially stabilizes the reactive intermediate or the product. The predominance of dihydroxyacetone phosphate on the enzyme would support a mechanism in which the initial proton abstraction in the reaction from dihydroxyacetone phosphate to d-glyceraldehyde 3-phosphate is significantly slower than the subsequent chemical steps. PMID:17287353

  5. Methods of measuring Protein Disulfide Isomerase activity: a critical overview

    NASA Astrophysics Data System (ADS)

    Watanabe, Monica; Laurindo, Francisco; Fernandes, Denise

    2014-09-01

    Protein disulfide isomerase is an essential redox chaperone from the endoplasmic reticulum (ER) and is responsible for correct disulfide bond formation in nascent proteins. PDI is also found in other cellular locations in the cell, particularly the cell surface. Overall, PDI contributes to ER and global cell redox homeostasis and signaling. The knowledge about PDI structure and function progressed substantially based on in vitro studies using recombinant PDI and chimeric proteins. In these experimental scenarios, PDI reductase and chaperone activities are readily approachable. In contrast, assays to measure PDI isomerase activity, the hallmark of PDI family, are more complex. Assessment of PDI roles in cells and tissues mainly relies on gain- or loss-of-function studies. However, there is limited information regarding correlation of experimental readouts with the distinct types of PDI activities. In this mini-review, we evaluate the main methods described for measuring the different kinds of PDI activity: thiol reductase, thiol oxidase, thiol isomerase and chaperone. We emphasize the need to use appropriate controls and the role of critical interferents (e.g., detergent, presence of reducing agents). We also discuss the translation of results from in vitro studies with purified recombinant PDI to cellular and tissue samples, with critical comments on the interpretation of results.

  6. Roles of triosephosphate isomerase and aerobic metabolism in Trypanosoma brucei.

    PubMed Central

    Helfert, S; Estévez, A M; Bakker, B; Michels, P; Clayton, C

    2001-01-01

    Kinetoplastid protozoa compartmentalize the first seven enzymes of glycolysis and two enzymes of glycerol metabolism in a microbody, the glycosome. While in its mammalian host, Trypanosoma brucei depends entirely on glucose for ATP generation. Under aerobic conditions, most of the glucose is metabolized to pyruvate. Aerobic metabolism depends on the activities of glycosomal triosephosphate isomerase and a mitochondrial glycerophosphate oxidase, and on glycerophosphate<-->dihydroxyacetone phosphate exchange across the glycosomal membrane. Using a combination of genetics and computer modelling, we show that triosephosphate isomerase is probably essential for bloodstream trypanosome survival, but not for the insect-dwelling procyclics, which preferentially use amino acids as an energy source. When the enzyme level decreased to about 15% of that of the wild-type, the growth rate was halved. Below this level, a lethal rise in dihydroxyacetone phosphate was predicted. Expression of cytosolic triosephosphate isomerase inhibited cell growth. Attempts to knockout the trypanosome alternative oxidase genes (which are needed for glycerophosphate oxidase activity) were unsuccessful, but when we lowered the level of the corresponding mRNA by expressing a homologous double-stranded RNA, oxygen consumption was reduced fourfold and the rate of trypanosome growth was halved. PMID:11415442

  7. Fermentation of xylulose to ethanol using xylose isomerase and yeasts

    SciTech Connect

    Jeffries, T.W.

    1981-01-01

    In a survey of 35 organisms, predominantly yeasts, about 40% were capable of fermenting xylulose to ethanol. Two species, Candida tropicalis and Schizosaccharomyces pombe, did so at good rates and without an initial lag. Saccharomyces cerevisiae strains that fermented glucose rapidly fermented xylulose at a slower rate. Ten yeasts and three strains of the bacterium Zymomonas mobilis were weak or negative for xylulose, even though they fermented glucose under the conditions employed. C. tropicalis was able to form 1.0 M ethanol from 1.0 M xylose if the fermentation broth was recycled over immobilized xylose isomerase.

  8. Cyclophilins of a novel subfamily interact with SNW/SKIP coregulator in Dictyostelium discoideum and Schizosaccharomyces pombe.

    PubMed

    Skruzný, M; Ambrozková, M; Fuková, I; Martínková, K; Blahůsková, A; Hamplová, L; Půta, F; Folk, P

    2001-10-31

    We screened the Dictyostelium discoideum two-hybrid cDNA library with the SNW/SKIP transcription coregulator SnwA and identified a novel cyclophilin CypE. Independently, the Schizosaccharomyces pombe cDNA library was screened with the SnwA ortholog Snw1 and the ortholog of CypE (named Cyp2) was found. Both cyclophilins bind the respective SNW protein in their autologous systems. The interaction was localized to the N-terminal part of SnwA as well as of Snw1. CypE was confirmed in vitro to be a cyclosporin A-sensitive peptidyl-prolyl cis-trans isomerase. Remarkably, both SNW proteins bind the cyclophilins in a cyclosporin A independent manner, possibly serving as adaptors for these novel isomerases. These results are the first characterization of the members of a novel cyclophilin subfamily, which includes the human CGI-124/PPIL1 protein. PMID:11690648

  9. Characterization of a mutant glucose isomerase from Thermoanaerobacterium saccharolyticum.

    PubMed

    Xu, Heng; Shen, Dong; Wu, Xue-Qiang; Liu, Zhi-Wei; Yang, Qi-He

    2014-10-01

    A series of site-directed mutant glucose isomerase at tryptophan 139 from Thermoanaerobacterium saccharolyticum strain B6A were purified to gel electrophoretic homogeneity, and the biochemical properties were determined. W139F mutation is the most efficient mutant derivative with a tenfold increase in its catalytic efficiency toward glucose compared with the native GI. With a maximal activity at 80 °C of 59.58 U/mg on glucose, this mutant derivative is the most active type ever reported. The enzyme activity was maximal at 90 °C and like other glucose isomerase, this mutant enzyme required Co(2+) or Mg(2+) for enzyme activity and thermal stability (stable for 20 h at 80 °C in the absence of substrate). Its optimum pH was around 7.0, and it had 86 % of its maximum activity at pH 6.0 incubated for 12 h at 60 °C. This enzyme was determined as thermostable and weak-acid stable. These findings indicated that the mutant GI W139F from T. saccharolyticum strain B6A is appropriate for use as a potential candidate for high-fructose corn syrup producing enzyme. PMID:25139657

  10. Protein disulfide isomerase mediates glutathione depletion-induced cytotoxicity.

    PubMed

    Okada, Kazushi; Fukui, Masayuki; Zhu, Bao-Ting

    2016-08-26

    Glutathione depletion is a distinct cause underlying many forms of pathogenesis associated with oxidative stress and cytotoxicity. Earlier studies showed that glutamate-induced glutathione depletion in immortalized murine HT22 hippocampal neuronal cells leads to accumulation of reactive oxygen species (ROS) and ultimately cell death, but the precise mechanism underlying these processes is not clear. Here we show that during the induction of glutathione depletion, nitric oxide (NO) accumulation precedes ROS accumulation. While neuronal NO synthase (nNOS) in untreated HT22 cells exists mostly as a monomer, glutathione depletion results in increased formation of the dimer nNOS, accompanied by increases in the catalytic activity. We identified that nNOS dimerization is catalyzed by protein disulfide isomerase (PDI). Inhibition of PDI's isomerase activity effectively abrogates glutathione depletion-induced conversion of monomer nNOS into dimer nNOS, accumulation of NO and ROS, and cytotoxicity. Furthermore, we found that PDI is present in untreated cells in an inactive S-nitrosylated form, which becomes activated following glutathione depletion via S-denitrosylation. These results reveal a novel role for PDI in mediating glutathione depletion-induced oxidative cytotoxicity, as well as its role as a valuable therapeutic target for protection against oxidative cytotoxicity. PMID:27317486

  11. The amino acid sequence of rabbit muscle triose phosphate isomerase.

    PubMed Central

    Corran, P H; Waley, S G

    1975-01-01

    The amino acid sequence of rabbit muscle triose phosphate isomerase was deduced by characterizing peptides that overlap the tryptic peptides. Thiol groups were modified by oxidation, carboxymethylation or aminoen. About 50 peptides that provided information about overlaps were isolated; the peptides were mostly characterized by their compositions and N-terminal residues. The peptide chains contain 248 amino acid residues, and no evidence for dissimilarity of the two subunits that comprise the native enzyme was found. The sequence of the rabbit muscle enzyme may be compared with that of the coelacanth enzyme (Kolb et al., 1974): 84% of the residues are in identical positions. Similarly, comparison of the sequence with that inferred for the chicken enzyme (Furth et al., 1974) shows that 87% of the residues are in identical positions. Limited though these comparisons are, they suggest that triose phosphate isomerase has one of the lowest rates of evolutionary change. An extended version of the present paper has been deposited as Supplementary Publication SUP 50040 (42 pages) at the British Library (Lending Division) (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms given in Biochem. J. (1975) 145, 5. PMID:1171682

  12. Converting a Sulfenic Acid Reductase into a Disulfide Bond Isomerase

    PubMed Central

    Chatelle, Claire; Kraemer, Stéphanie; Ren, Guoping; Chmura, Hannah; Marechal, Nils; Boyd, Dana; Roggemans, Caroline; Ke, Na; Riggs, Paul; Bardwell, James

    2015-01-01

    Abstract Aims: Posttranslational formation of disulfide bonds is essential for the folding of many secreted proteins. Formation of disulfide bonds in a protein with more than two cysteines is inherently fraught with error and can result in incorrect disulfide bond pairing and, consequently, misfolded protein. Protein disulfide bond isomerases, such as DsbC of Escherichia coli, can recognize mis-oxidized proteins and shuffle the disulfide bonds of the substrate protein into their native folded state. Results: We have developed a simple blue/white screen that can detect disulfide bond isomerization in vivo, using a mutant alkaline phosphatase (PhoA*) in E. coli. We utilized this screen to isolate mutants of the sulfenic acid reductase (DsbG) that allowed this protein to act as a disulfide bond isomerase. Characterization of the isolated mutants in vivo and in vitro allowed us to identify key amino acid residues responsible for oxidoreductase properties of thioredoxin-like proteins such as DsbC or DsbG. Innovation and Conclusions: Using these key residues, we also identified and characterized interesting environmental homologs of DsbG with novel properties, thus demonstrating the capacity of this screen to discover and elucidate mechanistic details of in vivo disulfide bond isomerization. Antioxid. Redox Signal. 23, 945–957. PMID:26191605

  13. Sequence of the Ampullariella sp. strain 3876 gene coding for xylose isomerase.

    PubMed Central

    Saari, G C; Kumar, A A; Kawasaki, G H; Insley, M Y; O'Hara, P J

    1987-01-01

    The nucleotide sequence of the gene coding for xylose isomerase from Ampullariella sp. strain 3876, a gram-positive bacterium, has been determined. A clone of a fragment of strain 3876 DNA coding for a xylose isomerase activity was identified by its ability to complement a xylose isomerase-defective Escherichia coli strain. One such complementation positive fragment, 2,922 nucleotides in length, was sequenced in its entirety. There are two open reading frames 1,182 and 1,242 nucleotides in length, on opposite strands of this fragment, each of which could code for a protein the expected size of xylose isomerase. The 1,182-nucleotide open reading frame was identified as the coding sequence for the protein from the sequence analysis of the amino-terminal region and selected internal peptides. The gene initiates with GTG and has a high guanine and cytosine content (70%) and an exceptionally strong preference (97%) for guanine or cytosine in the third position of the codons. The gene codes for a 43,210-dalton polypeptide composed of 393 amino acids. The xylose isomerase from Ampullariella sp. strain 3876 is similar in size to other bacterial xylose isomerases and has limited amino acid sequence homology to the available sequences from E. coli, Bacillus subtilis, and Streptomyces violaceus-ruber. In all cases yet studied, the bacterial gene for xylulose kinase is downstream from the gene for xylose isomerase. We present evidence suggesting that in Ampullariella sp. strain 3876 these genes are similarly arranged. PMID:3027039

  14. Regulation of isopropylmalate isomerase synthesis in Neurospora crassa.

    PubMed Central

    Reichenbecher, V E; Fischer, M; Gross, S R

    1978-01-01

    The capacity to synthetize isopropylmalate isomerase (EC 4.2.1.33) by Neurospora crassa increased during induction in the presence of cycloheximide but was inhibited by proflavine and other inhibitors of RNA synthesis. Turnover of the enzyme once formed appeared negligible, but the message (measured as enzyme-forming capacity) had a half-life of 4 to 8 min. A comparison of the kinetics of induction in the wild type and a newly isolated alpha-isopropylmalate-permeable strain suggested strongly that feedback control by leucine of alpha-isopropylmalate production can adequately serve as the primary physiological regulator of endogenous inducer concentration. Genetic data are presented which implicate the involvement of two unlinked genes, ipm-1 and ipm-2, in determining permeation of alpha-isopropylmalate. PMID:146702

  15. Human glucose phosphate isomerase: Exon mapping and gene structure

    SciTech Connect

    Xu, Weiming; Lee, Pauline; Beutler, E.

    1995-10-10

    The structure of the gene for human glucose phosphate isomerase (GPI) has been determined. Three GPI clones were isolated from a human genomic library by using a full-length GPI cDNA probe and were characterized. Oligonucleotides based on the known cDNA sequence were used as primers in amplification and sequence analyses. This led to the identification of the exon-intron junctions. By this approach, 18 exons and 17 introns have been identified. The exons range in size from 44 to 431 nucleotides. The intronic sequences surrounding the exons provide useful information for the identification of mutations that give rise to human GPI deficiency associated with chronic hemolytic anemia. 13 refs., 4 figs., 1 tab.

  16. Mammalian peptide isomerase: platypus-type activity is present in mouse heart.

    PubMed

    Koh, Jennifer M S; Chow, Stephanie J P; Crossett, Ben; Kuchel, Philip W

    2010-06-01

    Male platypus (Ornithorhynchus anatinus) venom has a peptidyl aminoacyl L/D-isomerase (hereafter called peptide isomerase) that converts the second amino acid residue in from the N-terminus from the L- to the D-form, and vice versa. A reversed-phase high-performance liquid chromatography (RP-HPLC) assay has been developed to monitor the interconversion using synthetic hexapeptides derived from defensin-like peptide-2 (DLP-2) and DLP-4 as substrates. It was hypothesised that animals other than the platypus would have peptide isomerase with the same substrate specificity. Accordingly, eight mouse tissues were tested and heart was shown to have the activity. This is notable for being the first evidence of a peptide isomerase being present in a higher mammal and heralds finding the activity in man. PMID:20564672

  17. Structural features of normal and complemented forms of the Neurospora isopropylmalate isomerase.

    PubMed Central

    Reichenbecher, V E; Gross, S R

    1978-01-01

    The isopropylmalate isomerase (EC 4.2.1.33) of Neurospora crassa is a globular protein consisting of a single polypeptide chain with a molecular weight of about 90,000. The isomerase cannot easily be freed of a contaminating protease which cleaves the enzyme into two major fragments, one of approximately 56,000 and the other 37,000 daltons. This suggests that the folded polypeptide chain may contain some hinge point or loop exposed on the surface which makes it susceptible to proteolytic attack. Most of the isomerase activity extracted from the wild-type strain is in monomer form. However, a small fraction of the activity in crude extracts is found in multimeric aggregates, and the active isomerase extracted from complementing leu-2 heterokaryons consists entirely of dimers and higher multimers. These observations suggest that, though active as a monomer, a significant fraction of the normal enzyme might be organized in multimeric form within the cell. Images PMID:146703

  18. INDUCTION AND REPRESSION OF l-ARABINOSE ISOMERASE IN PEDIOCOCCUS PENTOSACEUS1

    PubMed Central

    Dobrogosz, Walter J.; DeMoss, Ralph D.

    1963-01-01

    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. PMID:14047229

  19. Ribose 5-Phosphate Isomerase B Knockdown Compromises Trypanosoma brucei Bloodstream Form Infectivity

    PubMed Central

    Loureiro, Inês; Faria, Joana; Clayton, Christine; Macedo-Ribeiro, Sandra; Santarém, Nuno; Roy, Nilanjan; Cordeiro-da-Siva, Anabela; Tavares, Joana

    2015-01-01

    Ribose 5-phosphate isomerase is an enzyme involved in the non-oxidative branch of the pentose phosphate pathway, and catalyzes the inter-conversion of D-ribose 5-phosphate and D-ribulose 5-phosphate. Trypanosomatids, including the agent of African sleeping sickness namely Trypanosoma brucei, have a type B ribose-5-phosphate isomerase. This enzyme is absent from humans, which have a structurally unrelated ribose 5-phosphate isomerase type A, and therefore has been proposed as an attractive drug target waiting further characterization. In this study, Trypanosoma brucei ribose 5-phosphate isomerase B showed in vitro isomerase activity. RNAi against this enzyme reduced parasites' in vitro growth, and more importantly, bloodstream forms infectivity. Mice infected with induced RNAi clones exhibited lower parasitaemia and a prolonged survival compared to control mice. Phenotypic reversion was achieved by complementing induced RNAi clones with an ectopic copy of Trypanosoma cruzi gene. Our results present the first functional characterization of Trypanosoma brucei ribose 5-phosphate isomerase B, and show the relevance of an enzyme belonging to the non-oxidative branch of the pentose phosphate pathway in the context of Trypanosoma brucei infection. PMID:25568941

  20. Detection of platypus-type L/D-peptide isomerase activity in aqueous extracts of papaya fruit.

    PubMed

    Arakawa, Kensuke; Koh, Jennifer M S; Crossett, Ben; Torres, Allan M; Kuchel, Philip W

    2012-09-01

    Peptide isomerase catalyses the post-translational isomerisation of the L: - to the D: -form of an amino acid residue around the N/C-termini of substrate peptides. To date, some peptide isomerases have been found in a limited number of animal secretions and cells. We show here that papaya extracts have weak peptide isomerase activity. The activity was detected in each 30-100 kDa fraction of the flesh and the seed extracts of unripe and ripe papaya fruit. The definitive activity was confirmed in the ripe papaya extracts, but even then it was much less active than that of the other peptide isomerases previously reported. The activity was markedly inhibited by methanol, and partly so by amastatin and diethyl pyrocarbonate. This is the first report of peptide isomerase activity in a plant and suggests that perhaps every living organism may have some peptide isomerase activity. PMID:22648682

  1. Metabolic phenotype of phosphoglucose isomerase mutants of Corynebacterium glutamicum.

    PubMed

    Marx, Achim; Hans, Stephan; Möckel, Bettina; Bathe, Brigitte; de Graaf, Albert A; McCormack, Ashling C; Stapleton, Cliona; Burke, Kevin; O'Donohue, Michael; Dunican, L K

    2003-09-01

    A series of experiments reported in the literature using fluxomics as an efficient functional genomics tool revealed that the L-lysine production of the Corynebacterium glutamicum strain MH20-22B correlates with the extent of intracellular NADPH supply. Some alternative metabolic engineering strategies to increase intracellular NADPH supply in the C. glutamicum strain DSM5715 were considered and finally the redirection of carbon flux through the pentose phosphate pathway with two NADPH generating enzymatic reactions was favored. Elsewhere, the construction of a phosphoglucose isomerase (Pgi) null mutant of the C. glutamicum strain DSM5715 has been described by utilizing genetic engineering as well as some aspects of its metabolic phenotype. Most interestingly, it was shown that not only could the L-lysine formation be increased by 1.7-fold but the by-product concentration for the null mutant strain was also able to be drastically reduced. In this publication we discuss this metabolic phenotype in detail and present additional data on by-product formation as well as yield considerations. Results from isotope based metabolic flux analysis in combination with considerations on NADPH metabolism clearly exclude the existence of Pgi isoenzymes in C. glutamicum strain DSM5715. The genome region containing the pgi gene was analyzed. It cannot be excluded that polar effects might have been caused by the disruption of the pgi gene and might have contributed to the observed metabolic phenotype of C. glutamicum Pgi mutants. We illustrate growth characteristics of a Pgi mutant of an industrial L-lysine production strain. A reduced growth rate and a biphasic growth behavior was observed. The importance of NADPH reoxidation for well balanced growth in Pgi mutants is discussed. Another phosphoglucose isomerase mutant of C. glutamicum has been described in literature with which an increase in L-lysine yield from 42 to 52% was observed. This finding highlights the general potential

  2. Linoleic acid isomerase from Propionibacterium acnes: purification, characterization, molecular cloning, and heterologous expression.

    PubMed

    Deng, Ming-De; Grund, Alan D; Schneider, Kenneth J; Langley, Kim M; Wassink, Sarah L; Peng, Susan S; Rosson, Reinhardt A

    2007-12-01

    Propionibacterium acnes strain ATCC 6919 catalyzes the isomerization of the double bond at the C9 position in linoleic acid (c9,c12, 18:2) to form t10,c12 conjugated linoleic acid (CLA, 18:2). CLA has significant health benefits in animal and human. The linoleic acid C9 isomerase was purified to an apparent homogeneity by successive chromatography on diethylaminoethyl (DEAE) anion exchange, hydrophobic interaction, and chromatofocusing columns. Two degenerated oligonucleotide primers were synthesized according to the N-terminal peptide sequence to clone, by polymerase chain reaction (PCR), a short nucleotide sequence (62 bp) of the isomerase gene. The linoleic acid isomerase gene (lai) was subsequently cloned by inverse PCR. The amino acid sequence deduced from the lai coding sequence predicts a protein of 424 amino acid residues (48 kDa), excluding the N-terminal methionine, which was absent in the polypeptide purified from the native host. The isomerase shares no significant sequence homology to other enzymes except a flavin-binding domain in the N-terminal region. The recombinant isomerase purified from Escherichia coli showed a typical ultraviolet spectrum for FAD-bound proteins. The recombinant enzyme produced a single isomer of t10,c12-CLA from linoleic acid, as demonstrated by gas chromatography and gas chromatography-mass spectrum analysis. The recombinant isomerase protein was expressed at high levels in E. coli, but it was almost totally sequestered in inclusion bodies. The level of active isomerase was increased 376-fold by medium and process optimization in bench-scale fermentors. PMID:18057448

  3. Secretion of protein disulphide isomerase AGR2 confers tumorigenic properties

    PubMed Central

    Fessart, Delphine; Domblides, Charlotte; Avril, Tony; Eriksson, Leif A; Begueret, Hugues; Pineau, Raphael; Malrieux, Camille; Dugot-Senant, Nathalie; Lucchesi, Carlo; Chevet, Eric; Delom, Frederic

    2016-01-01

    The extracellular matrix (ECM) plays an instrumental role in determining the spatial orientation of epithelial polarity and the formation of lumens in glandular tissues during morphogenesis. Here, we show that the Endoplasmic Reticulum (ER)-resident protein anterior gradient-2 (AGR2), a soluble protein-disulfide isomerase involved in ER protein folding and quality control, is secreted and interacts with the ECM. Extracellular AGR2 (eAGR2) is a microenvironmental regulator of epithelial tissue architecture, which plays a role in the preneoplastic phenotype and contributes to epithelial tumorigenicity. Indeed, eAGR2, is secreted as a functionally active protein independently of its thioredoxin-like domain (CXXS) and of its ER-retention domain (KTEL), and is sufficient, by itself, to promote the acquisition of invasive and metastatic features. Therefore, we conclude that eAGR2 plays an extracellular role independent of its ER function and we elucidate this gain-of-function as a novel and unexpected critical ECM microenvironmental pro-oncogenic regulator of epithelial morphogenesis and tumorigenesis. DOI: http://dx.doi.org/10.7554/eLife.13887.001 PMID:27240165

  4. Catalytic mechanism of a retinoid isomerase essential for vertebrate vision

    DOE PAGESBeta

    Kiser, Philip D.; Zhang, Jianye; Badiee, Mohsen; Li, Qingjiang; Shi, Wuxian; Sui, Xuewu; Golczak, Marcin; Tochtrop, Gregory P.; Palczewski, Krzysztof

    2015-04-20

    Visual function in vertebrates is dependent on the membrane-bound retinoid isomerase RPE65, an essential component of the retinoid cycle pathway that regenerates 11-cis-retinal for rod and cone opsins. The mechanism by which RPE65 catalyzes stereoselective retinoid isomerization has remained elusive because of uncertainty about how retinoids bind to its active site. Here we present crystal structures of RPE65 in complex with retinoid-mimetic compounds, one of which is in clinical trials for the treatment of age-related macular degeneration. The structures reveal the active site retinoid-binding cavity located near the membrane-interacting surface of the enzyme as well as an Fe-bound palmitate ligandmore » positioned in an adjacent pocket. With the geometry of the RPE65–substrate complex clarified, we delineate a mechanism of catalysis that reconciles the extensive biochemical and structural research on this enzyme. Finally, these data provide molecular foundations for understanding a key process in vision and pharmacological inhibition of RPE65 with small molecules.« less

  5. Catalytic mechanism of a retinoid isomerase essential for vertebrate vision

    SciTech Connect

    Kiser, Philip D.; Zhang, Jianye; Badiee, Mohsen; Li, Qingjiang; Shi, Wuxian; Sui, Xuewu; Golczak, Marcin; Tochtrop, Gregory P.; Palczewski, Krzysztof

    2015-04-20

    Visual function in vertebrates is dependent on the membrane-bound retinoid isomerase RPE65, an essential component of the retinoid cycle pathway that regenerates 11-cis-retinal for rod and cone opsins. The mechanism by which RPE65 catalyzes stereoselective retinoid isomerization has remained elusive because of uncertainty about how retinoids bind to its active site. Here we present crystal structures of RPE65 in complex with retinoid-mimetic compounds, one of which is in clinical trials for the treatment of age-related macular degeneration. The structures reveal the active site retinoid-binding cavity located near the membrane-interacting surface of the enzyme as well as an Fe-bound palmitate ligand positioned in an adjacent pocket. With the geometry of the RPE65–substrate complex clarified, we delineate a mechanism of catalysis that reconciles the extensive biochemical and structural research on this enzyme. Finally, these data provide molecular foundations for understanding a key process in vision and pharmacological inhibition of RPE65 with small molecules.

  6. ALS-linked protein disulfide isomerase variants cause motor dysfunction.

    PubMed

    Woehlbier, Ute; Colombo, Alicia; Saaranen, Mirva J; Pérez, Viviana; Ojeda, Jorge; Bustos, Fernando J; Andreu, Catherine I; Torres, Mauricio; Valenzuela, Vicente; Medinas, Danilo B; Rozas, Pablo; Vidal, Rene L; Lopez-Gonzalez, Rodrigo; Salameh, Johnny; Fernandez-Collemann, Sara; Muñoz, Natalia; Matus, Soledad; Armisen, Ricardo; Sagredo, Alfredo; Palma, Karina; Irrazabal, Thergiory; Almeida, Sandra; Gonzalez-Perez, Paloma; Campero, Mario; Gao, Fen-Biao; Henny, Pablo; van Zundert, Brigitte; Ruddock, Lloyd W; Concha, Miguel L; Henriquez, Juan P; Brown, Robert H; Hetz, Claudio

    2016-04-15

    Disturbance of endoplasmic reticulum (ER) proteostasis is a common feature of amyotrophic lateral sclerosis (ALS). Protein disulfide isomerases (PDIs) areERfoldases identified as possibleALSbiomarkers, as well as neuroprotective factors. However, no functional studies have addressed their impact on the disease process. Here, we functionally characterized fourALS-linked mutations recently identified in two majorPDIgenes,PDIA1 andPDIA3/ERp57. Phenotypic screening in zebrafish revealed that the expression of thesePDIvariants induce motor defects associated with a disruption of motoneuron connectivity. Similarly, the expression of mutantPDIs impaired dendritic outgrowth in motoneuron cell culture models. Cellular and biochemical studies identified distinct molecular defects underlying the pathogenicity of thesePDImutants. Finally, targetingERp57 in the nervous system led to severe motor dysfunction in mice associated with a loss of neuromuscular synapses. This study identifiesERproteostasis imbalance as a risk factor forALS, driving initial stages of the disease. PMID:26869642

  7. Catalytic mechanism of a retinoid isomerase essential for vertebrate vision

    PubMed Central

    Kiser, Philip D.; Zhang, Jianye; Badiee, Mohsen; Li, Qingjiang; Shi, Wuxian; Sui, Xuewu; Golczak, Marcin; Tochtrop, Gregory P.; Palczewski, Krzysztof

    2015-01-01

    Visual function in vertebrates is dependent on the membrane-bound retinoid isomerase, RPE65, an essential component of the retinoid cycle pathway that regenerates 11-cis-retinal for rod and cone opsins. The mechanism by which RPE65 catalyzes stereoselective retinoid isomerization has remained elusive due to uncertainty about how retinoids bind to its active site. Here we present crystal structures of RPE65 in complex with retinoid-mimetic compounds, one of which is in clinical trials for treatment of age-related macular degeneration. The structures reveal the active site retinoid-binding cavity located near the membrane-interacting surface of the enzyme as well as an Fe-bound palmitate ligand positioned in an adjacent pocket. With the geometry of the RPE65-substrate complex clarified we delineate a mechanism of catalysis that reconciles the extensive biochemical and structural research on this enzyme. These data provide molecular foundations for understanding a key process in vision and pharmacological inhibition of RPE65 with small molecules. PMID:25894083

  8. Structural and functional characterization of Mycobacterium tuberculosis triosephosphate isomerase

    SciTech Connect

    Connor, Sean E.; Capodagli, Glenn C.; Deaton, Michelle K.; Pegan, Scott D.

    2012-04-18

    Tuberculosis (TB) is a major infectious disease that accounts for over 1.7 million deaths every year. Mycobacterium tuberculosis, the causative agent of tuberculosis, enters the human host by the inhalation of infectious aerosols. Additionally, one third of the world's population is likely to be infected with latent TB. The incidence of TB is on the rise owing in part to the emergence of multidrug-resistant strains. As a result, there is a growing need to focus on novel M. tuberculosis enzyme targets. M. tuberculosis triosephosphate isomerase (MtTPI) is an essential enzyme for gluconeogenetic pathways, making it a potential target for future therapeutics. In order to determine its structure, the X-ray crystal structure of MtTPI has been determined, as well as that of MtTPI bound with a reaction-intermediate analog. As a result, two forms of the active site were revealed. In conjunction with the kinetic parameters obtained for the MtTPI-facilitated conversion of dihydroxyacetone phosphate (DHAP) to D-glyceraldehyde-3-phosphate (D-GAP), this provides a greater structural and biochemical understanding of this enzyme. Additionally, isothermal titration calorimetry was used to determine the binding constant for a reaction-intermediate analog bound to the active site of MtTPI.

  9. Plant phosphomannose isomerase as a selectable marker for rice transformation

    PubMed Central

    Hu, Lei; Li, Hao; Qin, Ruiying; Xu, Rongfang; Li, Juan; Li, Li; Wei, Pengcheng; Yang, Jianbo

    2016-01-01

    The E. coli phosphomannose isomerase (EcPMI) gene is widely used as a selectable marker gene (SMG) in mannose (Man) selection-based plant transformation. Although some plant species exhibit significant PMI activity and active PMIs were even identified in Man-sensitive plants, whether plant PMIs can be used as SMGs remains unclear. In this study, we isolated four novel PMI genes from Chlorella variabilis and Oryza sativa. Their isoenzymatic activities were examined in vitro and compared with that of EcPMI. The active plant PMIs were separately constructed into binary vectors as SMGs and then transformed into rice via Agrobacterium. In both Indica and Japonica subspecies, our results indicated that the plant PMIs could select and produce transgenic plants in a pattern similar to that of EcPMI. The transgenic plants exhibited an accumulation of plant PMI transcripts and enhancement of the in vivo PMI activity. Furthermore, a gene of interest was successfully transformed into rice using the plant PMIs as SMGs. Thus, novel SMGs for Man selection were isolated from plants, and our analysis suggested that PMIs encoding active enzymes might be common in plants and could potentially be used as appropriate genetic elements in cisgenesis engineering. PMID:27174847

  10. Genetic and functional aspects of linoleate isomerase in Lactobacillus acidophilus.

    PubMed

    Macouzet, Martin; Robert, Normand; Lee, Byong H

    2010-08-01

    While the remarkable health effects of conjugated linoleic acid (CLA) catalyzed from alpha-linoleic acid by the enzyme linoleate isomerase (LI, EC 5.2.1.5) are well recognized, how widely this biochemical activity is present and the mechanisms of its regulation in lactic acid bacteria are unknown. Although certain strains of Lactobacillus acidophilus can enrich CLA in fermented dairy products, it is unknown if other strains share this capacity. Due to its immense economic importance, this work aimed to investigate genetic aspects of CLA production in L. acidophilus for the first time. The genomic DNA from industrial and type strains of L. acidophilus were subjected to PCR and immunoblot analyses using the putative LI gene of L. reuteri ATCC 55739 as probe. The CLA production ability was estimated by gas chromatography of the biomass extracts. The presumptive LI gene from L. acidophilus ATCC 832 was isolated and sequenced. The resulting sequence shared 71% identity with that of L. reuteri and at least 99% with reported sequences from other L. acidophilus strains. All the strains accumulated detectable levels of CLA and tested positive by PCR and immunoblotting. However, no apparent correlation was observed between the yields and the hybridization patterns. The results suggest that LI activity might be common among L. acidophilus and related species and provide a new tool for screening potential CLA producers. PMID:20461509

  11. Identification of disulfide bond isomerase substrates reveals bacterial virulence factors

    PubMed Central

    Ren, Guoping; Champion, Matthew M.; Huntley, Jason F.

    2014-01-01

    Summary Bacterial pathogens are exposed to toxic molecules inside the host and require efficient systems to form and maintain correct disulfide bonds for protein stability and function. The intracellular pathogen Francisella tularensis encodes a disulfide bond formation protein ortholog, DsbA, which previously was reported to be required for infection of macrophages and mice. However, the molecular mechanisms by which F. tularensis DsbA contributes to virulence are unknown. Here, we demonstrate that F. tularensis DsbA is a bifunctional protein that oxidizes and, more importantly, isomerizes complex disulfide connectivity in substrates. A single amino acid in the conserved cis-proline loop of the DsbA thioredoxin domain was shown to modulate both isomerase activity and F. tularensis virulence. Trapping experiments in F. tularensis identified over 50 F. tularensis DsbA substrates, including outer membrane proteins, virulence factors, and many hypothetical proteins. Six of these hypothetical proteins were randomly selected and deleted, revealing two novel proteins, FTL_1548 and FTL_1709, which are required for F. tularensis virulence. We propose that the extreme virulence of F. tularensis is partially due to the bifunctional nature of DsbA, that many of the newly-identified substrates are required for virulence, and that the development of future DsbA inhibitors could have broad anti-bacterial implications. PMID:25257164

  12. The Oligomeric Nature of Triosephosphate Isomerase. Studies of Monomerization

    NASA Astrophysics Data System (ADS)

    Zárate-Pérez, Francisco; Vázquez-Contreras, Edgar

    2008-02-01

    In this work, we report the implications of the monomerization of triosephosphate isomerase (TIM) from T. cruzi (TcTIM). A monomeric mutant (monoTcTIM) of this species was constructed by genetic engineering, shortening the main loop of interdigitation, which is fundamental for its dimerization. The properties of monoTcTIM were compared with those of the other TIM monomeric versions: genetically engineered mutants, or equilibrium intermediates obtained by chemical denaturation. The stability for almost all the monomeric variants so far reported appears in the same range, and also presents similar structural characteristics. Regarding the catalytic activity of monomeric mutants, when it is present is in several orders of magnitude lower than those observed in their respective wild-type enzyme. The change in the hydrophobic surface of TcTIM after and before monomerization was also studied and corresponds to an extensive area of the interface region, which becomes exposed when monomers are dissociated. This fact could be related with conformational changes in the local environment of the catalytic amino acids responsible for the isomerization of the substrate and consequently the inactivation of its catalytic properties. Comparison of the structural, folding and unfolding properties, as well as stability studies could give answers on why this enzyme is an obligate oligomer.

  13. Human triosephosphate isomerase deficiency resulting from mutation of Phe-240

    SciTech Connect

    Minling Chang; Xiaoyun Wu; Maquat, L.E. ); Artymiuk, P.J. ); Hollan, S. ); Lammi, A. )

    1993-06-01

    Triosephosphate isomerase (TPI; D-glyceraldehyde-3-phosphate ketolisomerase [E.C.5.3.1.1]) deficiency is an autosomal recessive disorder that typically results in chronic, nonspherocytic hemolytic anemia and in neuromuscular impairment. The molecular basis of this disease was analyzed for one Hungarian family and for two Australian families by localizing the defects in TPI cDNA and by determining how each defect affects TPI gene expression. The Hungarian family is noteworthy in having the first reported case of an individual, A. Jo., who harbors two defective TPI alleles but who does not manifest neuromuscular disabilities. This family was characterized by two mutations that have never been described. One is a missense mutation within codon 240 (TTC [Phe][r arrow]CTC [Leu]), which creates a thermolabile protein, as indicated by the results of enzyme activity assays using cell extracts. This substitution, which changes a phylogenetically conserved amino acid, may affect enzyme activity by dusrupting intersubunit contacts or substrate binding, as deduced from enzyme structural studies. The other mutation has yet to be localized but reduces the abundance of TPI mRNA 10--20-fold. Each of the Australian families was characterized by a previously described mutation within codon 104 (GAG [Glu][r arrow]GAC [Asp]), which also results in thermolabile protein. 49 refs., 6 figs., 1 tab.

  14. Purification and Partial characterization of Trypanosoma cruzi triosephosphate isomerase.

    PubMed

    Bourguignon, S C; Meirelles, M N; Pacheco, R S; De Simone, S G

    1998-01-01

    The enzyme triosephosphate isomerase (TPI, EC 5.3.1.1) was purified from extracts of epimastigote forms of Trypanosoma cruzi. The purification steps included: hydrophobic interaction chromatography on phenyl-Sepharose, CM-Sepharose, and high performance liquid gel filtration chromatography. The CM-Sepharose material contained two bands (27 and 25 kDa) with similar isoelectric points (pI 9.3-9.5) which could be separated by gel filtration in high performance liquid chromatography. Polyclonal antibodies raised against the porcine TPI detected one single polypeptide on western blot with a molecular weight (27 kDa) identical to that purified from T. cruzi. These antibodies also recognized only one band of identical molecular weight in western blots of several other trypanosomatids (Blastocrithidia culicis, Crithidia desouzai, Phytomonas serpens, Herpertomonas samuelpessoai). The presence of only one enzymatic form of TPI in T. cruzi epimastigotes was confirmed by agarose gel activity assay and its localization was established by immunocytochemical analysis. The T. cruzi purified TPI (as well as other trypanosomatid' TPIs) is a dimeric protein, composed of two identical subunits with an approximate mw of 27,000 and it is resolved on two dimensional gel electrophoresis with a pI of 9.3. Sequence analysis of the N-terminal portion of the 27 kDa protein revealed a high homology to Leishmania mexicana and T. brucei proteins. PMID:9698898

  15. Plant phosphomannose isomerase as a selectable marker for rice transformation.

    PubMed

    Hu, Lei; Li, Hao; Qin, Ruiying; Xu, Rongfang; Li, Juan; Li, Li; Wei, Pengcheng; Yang, Jianbo

    2016-01-01

    The E. coli phosphomannose isomerase (EcPMI) gene is widely used as a selectable marker gene (SMG) in mannose (Man) selection-based plant transformation. Although some plant species exhibit significant PMI activity and active PMIs were even identified in Man-sensitive plants, whether plant PMIs can be used as SMGs remains unclear. In this study, we isolated four novel PMI genes from Chlorella variabilis and Oryza sativa. Their isoenzymatic activities were examined in vitro and compared with that of EcPMI. The active plant PMIs were separately constructed into binary vectors as SMGs and then transformed into rice via Agrobacterium. In both Indica and Japonica subspecies, our results indicated that the plant PMIs could select and produce transgenic plants in a pattern similar to that of EcPMI. The transgenic plants exhibited an accumulation of plant PMI transcripts and enhancement of the in vivo PMI activity. Furthermore, a gene of interest was successfully transformed into rice using the plant PMIs as SMGs. Thus, novel SMGs for Man selection were isolated from plants, and our analysis suggested that PMIs encoding active enzymes might be common in plants and could potentially be used as appropriate genetic elements in cisgenesis engineering. PMID:27174847

  16. Psychrophilic, mesophilic, and thermophilic triosephosphate isomerases from three clostridial species.

    PubMed Central

    Shing, Y W; Akagi, J M; Himes, R H

    1975-01-01

    Triosephosphate isomerase was purified to homogeneity as judged by analytical gel electrophoresis from clostridium sp. strain 69, clostridium pasteurianum, and C. thermosaccharolyticum, which grow optimally at 18, 37, and 55 C, respectively. Comparative studies on these purified proteins showed that they had the same molecular weight (53,000) and subunit molecular weight (26,500). They were equally susceptible to the active site-directed inhibitor, glycidol phosphate. However, their temperature and pH optima, as well as their stabilities to heat, urea, and sodium dodecyl sulfate, differed. The proteins also had different mobilities in acrylamide gel electrophoresis. This difference in ionic character was also reflected in the elution behavior of the enzymes from hydroxyapatite and in the isoelectric points determined by isoelectric focusing in acrylamide gel. The amino acid composition of these proteins showed that the thermophilic enzyme contains a greater amount of proline than the other enzymes. The ratio of acidic amino acids to basic amino acids was 1.79, 1.38, and 1.66 for the thermophilic mesophilic and psychrophilic enzymes, respectively. This is consistent with the relative isoelectric point values of these three enzymes. Images PMID:235509

  17. The tryptic peptides of rabbit muscle triose phosphate isomerase

    PubMed Central

    Corran, P. H.; Waley, S. G.

    1974-01-01

    1. The peptides obtained by tryptic digestion of S-[14C]carboxymethylated rabbit muscle triose phosphate isomerase have been studied. 2. The first step in the fractionation of the tryptic digest was gel filtration on coupled columns of Sephadex G-25 and G-50. Further fractionation was carried out by paper electrophoresis and paper chromatography. 3. The digest contained 26 peptides and three free amino acids. The sizes of the peptides ranged from two to 29 residues. 4. The sequences of the peptides have been determined. 5. The length of the polypeptide chains is about 250 amino acid residues. 6. The variant sequences encountered were due to partial deamidation; this may be one of the reasons for multiple forms of the enzyme. 7. The chicken and rabbit enzymes are compared. 8. Detailed evidence for the sequences of the tryptic peptides has been deposited as Supplementary Publication SUP 50024 at the British Library, Lending Division (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms given in Biochem. J. (1973) 131, 5. PMID:4618774

  18. Crystal Structure of Triosephosphate Isomerase from Trypanosoma cruzi in Hexane

    NASA Astrophysics Data System (ADS)

    Gao, Xiu-Gong; Maldonado, Ernesto; Perez-Montfort, Ruy; Garza-Ramos, Georgina; Tuena de Gomez-Puyou, Marietta; Gomez-Puyou, Armando; Rodriguez-Romero, Adela

    1999-08-01

    To gain insight into the mechanisms of enzyme catalysis in organic solvents, the x-ray structure of some monomeric enzymes in organic solvents was determined. However, it remained to be explored whether the structure of oligomeric proteins is also amenable to such analysis. The field acquired new perspectives when it was proposed that the x-ray structure of enzymes in nonaqueous media could reveal binding sites for organic solvents that in principle could represent the starting point for drug design. Here, a crystal of the dimeric enzyme triosephosphate isomerase from the pathogenic parasite Trypanosoma cruzi was soaked and diffracted in hexane and its structure solved at 2- angstrom resolution. Its overall structure and the dimer interface were not altered by hexane. However, there were differences in the orientation of the side chains of several amino acids, including that of the catalytic Glu-168 in one of the monomers. No hexane molecules were detected in the active site or in the dimer interface. However, three hexane molecules were identified on the surface of the protein at sites, which in the native crystal did not have water molecules. The number of water molecules in the hexane structure was higher than in the native crystal. Two hexanes localized at <4 angstrom from residues that form the dimer interface; they were in close proximity to a site that has been considered a potential target for drug design.

  19. Purification and characterization of corticosteroid side chain isomerase

    SciTech Connect

    Marandici, A.; Monder, C. )

    1990-02-06

    Corticosteroid side chain isomerase of rat liver catalyzes the interconversion of the ketol (20-oxo-21-ol) and (20-hydroxy-21-al) forms of the corticosteroid side chain. The enzyme has now been purified to apparent homogeneity from rat liver cytosol by sequential chromatography on anionic, hydroxylapatite, and gel filtration columns. Ketol-aldol isomerization is followed by measuring the exchange of tritium from 21-tritiated steroids with water. The native enzyme is a dimer of MW 44,000. The isoelectric point is 4.8 {plus minus} 0.1 pH units. The purified enzyme is stimulated by Co{sup 3+} or Ni{sup 2+}. The enzyme utilizes 11-deoxycorticosterone, corticosterone, and 17-deoxycortisol as substrate but not cortisol, tetrahydrocortisol, and prednisolone. Tritium-water exchange of (21S)-(21-{sup 3}H)DOC is a pseudo-first-order reaction; 21-{sup 3}H exchange from the 21R isomer proceeds with first-order kinetics only after a lag associated with its epimerization to the 21S form.

  20. Catalytic mechanism of a retinoid isomerase essential for vertebrate vision

    SciTech Connect

    Kiser, Philip D.; Zhang, Jianye; Badiee, Mohsen; Li, Qingjiang; Shi, Wuxian; Sui, Xuewu; Golczak, Marcin; Tochtrop, Gregory P.; Palczewski, Krzysztof

    2015-04-20

    Visual function in vertebrates is dependent on the membrane-bound retinoid isomerase RPE65, an essential component of the retinoid cycle pathway that regenerates 11-cis-retinal for rod and cone opsins. The mechanism by which RPE65 catalyzes stereoselective retinoid isomerization has remained elusive because of uncertainty about how retinoids bind to its active site. Here we present crystal structures of RPE65 in complex with retinoid-mimetic compounds, one of which is in clinical trials for the treatment of age-related macular degeneration. The structures reveal the active site retinoid-binding cavity located near the membrane-interacting surface of the enzyme as well as an Fe-bound palmitate ligand positioned in an adjacent pocket. With the geometry of the RPE65–substrate complex clarified, we delineate a mechanism of catalysis that reconciles the extensive biochemical and structural research on this enzyme. These data provide molecular foundations for understanding a key process in vision and pharmacological inhibition of RPE65 with small molecules.

  1. Cell surface thiol isomerases may explain the platelet-selective action of S-nitrosoglutathione.

    PubMed

    Xiao, Fang; Gordge, Michael P

    2011-10-30

    S-nitrosoglutathione (GSNO) at low concentration inhibits platelet aggregation without causing vasodilation, suggesting platelet-selective nitric oxide delivery. The mechanism of this selectivity is unknown, but may involve cell surface thiol isomerases, in particular protein disulphide isomerase (csPDI) (EC 5.3.4.1). We have now compared csPDI expression and activity on platelets, endothelial cells and vascular smooth muscle cells, and the dependence on thiol reductase activity of these cell types for NO uptake from GSNO. csPDI expression was measured by flow cytometry and its reductase activity using the pseudosubstrate dieosin glutathione disulphide. This activity assay was adapted and validated for 96-well plate format. Flow cytometry revealed csPDI on all three cell types, but percentage positivity of expression was higher on platelets than on vascular cells. Consistent with this, thiol isomerase-related reductase activity was higher on platelets (P<0.01), and cellular activation (with either phorbol myristate acetate or ionomycin) increased csPDI activity on both platelets and smooth muscle cells, but not on endothelium. Intracellular NO delivery from GSNO was greater in platelets than in vascular cells (P<0.002), and was more sensitive to thiol isomerase inhibition using phenylarsine oxide (P<0.05). Increased surface thiol isomerase activity on platelets, compared with cells of the vascular wall, may explain the platelet-selective actions of GSNO and help define its antithrombotic potential. PMID:21642008

  2. The innate immune roles of host factors TRIM5α and Cyclophilin A on HIV-1 replication.

    PubMed

    Kuang, Yi-Qun; Liu, Hong-Liang; Zheng, Yong-Tang

    2015-10-01

    During the long-term evolutionary history, the interaction between virus and host has driven the first-line barrier, innate immunity, to invading pathogens. Innate immune factor TRIM5α and host peptidyl-prolyl cis-trans isomerase Cyclophilin A are two key players in the interaction between HIV-1 and host. Interestingly, Cyclophilin A is retrotransposed into the critical host gene, TRIM5, locus via LINE-1 element in some primate species including New World monkeys and Old World monkeys. This review aims to comprehensively discuss the sensing and immune activation procedures of TRIM5α innate signaling pathway through Cyclophilin A. It will then present the production of TRIMCyp chimeric gene and the different fusion patterns in primates. Finally, it will summarize the distinct restriction activity of TRIMCyp from different primates and explain the current understanding on the innate immune mechanisms involved in the early phase of the viral life cycle during HIV-1 replication. PMID:25894765

  3. Solution characterization of the extracellular region of CD147 and its interaction with its enzyme ligand cyclophilin-A

    SciTech Connect

    Schlegel, Jennifer; Redzic, Jasmina S.; Porter, Christopher; Yurchenko, Vyacheslav; Bukrinsky, Michael; Labeikovsky, Wladimir; Armstrong, Geoffrey S.; Zhang, Fengli; Isern, Nancy G.; Degregori, James; Hodges, Robert; Eisenmesser, Elan Z.

    2009-08-21

    The CD147 receptor plays an integral role in numerous diseases by stimulating the expression of several protein families and serving as the receptor for extracellular cyclophilins, however, neither CD147 nor its interactions with its cyclophilin ligands have been well characterized in solution. CD147 is a unique protein in that it can function both at the cell membrane and after being released from cells where it continues to retain activity. Thus, the CD147 receptor functions through at least two mechanisms that include both cyclophilin-independent and cyclophilin-dependent modes of action. In regard to CD147 cyclophilin-independent activity, CD147 homophilic interactions are thought to underlie its activity. In regard to CD147 cyclophilin-dependent activity, cyclophilin/CD147 interactions may represent a novel means of signaling since cyclophilins are also peptidyl-prolyl isomerases.

  4. Twin-arginine-dependent translocation of SufI in the absence of cytosolic helper proteins.

    PubMed

    Holzapfel, Eva; Moser, Michael; Schiltz, Emile; Ueda, Takuya; Betton, Jean-Michel; Müller, Matthias

    2009-06-16

    The twin-arginine translocation (Tat) machinery present in bacterial and thylakoidal membranes is able to transport fully folded proteins. Folding of some Tat precursor proteins requires dedicated chaperones that also sequester the signal sequence during the maturation process. Whether or not signal sequence-binding chaperones are a general prerequisite for all Tat substrate proteins is not known. Here, we have studied the propensity of Tat signal sequences of Escherichia coli to interact with general chaperones and peptidyl-prolyl-cis,trans-isomerases. Site-specific photocross-linking revealed a clear specificity for FK506-binding proteins. Nevertheless transport of the Tat substrate SufI into inverted inner membrane vesicles of E. coli was found to occur in the bona fide absence of any cytosolic chaperone. Our results suggest that in E. coli, cytosolic chaperones are not essential for the twin-arginine-dependent export of cofactor-less substrates. PMID:19432418

  5. Further delineation of FKBP14-related Ehlers-Danlos syndrome: A patient with early vascular complications and non-progressive kyphoscoliosis, and literature review.

    PubMed

    Dordoni, Chiara; Ciaccio, Claudia; Venturini, Marina; Calzavara-Pinton, Piergiacomo; Ritelli, Marco; Colombi, Marina

    2016-08-01

    FKBP14-related Ehlers-Danlos syndrome (EDS) is an extremely rare recessive connective tissue disorder described for the first time in 2012 by Baumann and coworkers. The causal gene, FKBP14, encodes a member of the F506-binding family of peptidyl-prolyl cis-trans isomerases. The paucity of patients described so far makes this disorder poorly defined at clinical level. Here, we report an additional pediatric patient, who is compound heterozygous for a recurrent and a novel FKBP14 mutation, and compare his phenotype with those available in literature. This evaluation confirms that kyphoscoliosis (either progressive or non-progressive), myopathy, joint hypermobility, and congenital hearing loss (sensorineural, conductive, or mixed) are the typical features of the syndrome. Since the patient showed a severe cardiovascular event in childhood and atlantoaxial instability, this report expands the phenotype of the disorder and the allelic repertoire of FKBP14. © 2016 Wiley Periodicals, Inc. PMID:27149304

  6. Spliceosomal Immunophilins

    PubMed Central

    Mesa, Annia; Somarelli, Jason A.; Herrera, Rene J.

    2008-01-01

    The spliceosome is a dynamic, macromolecular complex, which removes non-protein-coding introns from pre-mRNA to form mature mRNA in a process known as splicing. This ribonucleoprotein assembly is comprised of five uridine-rich small nuclear RNAs (snRNAs) as well as over 300 proteins. In humans, several of the known splicing factors are members of the immunophilin superfamily. Immunophilins are peptidyl-prolyl cis-trans isomerases that catalyze the conversion of proteins from cis to trans at Xaa-Pro bonds. Our review of the data portrays a picture of this protein family as activators of spliceosomal proteins by way of folding and transport. PMID:18544344

  7. Cyclophilin and Viruses: Cyclophilin as a Cofactor for Viral Infection and Possible Anti-Viral Target

    PubMed Central

    Watashi, Koichi; Shimotohno, Kunitada

    2007-01-01

    Cyclophilin (CyP) is a peptidyl prolyl cis/trans isomerase, catalyzing the cis-trans isomerization of proline residues in proteins. CyP plays key roles in several different aspects of cellular physiology including the immune response, transcription, mitochondrial function, cell death, and chemotaxis. In addition to these cellular events, a number of reports demonstrated that CyP plays a critical role in the life cycle of viruses, especially human immunodeficiency virus (HIV) and hepatitis C virus (HCV). These two viruses are significant causes of morbidity and mortality worldwide, but current therapies are often insufficient. CyP may provide a novel therapeutic target for the management and/or cure of these diseases, in particular HCV. PMID:21901058

  8. The cyclophilins

    PubMed Central

    Wang, Ping; Heitman, Joseph

    2005-01-01

    Summary: Cyclophilins (Enzyme Commission (EC) number 5.1.2.8) belong to a group of proteins that have peptidyl-prolyl cis-trans isomerase activity; such proteins are collectively known as immunophilins and also include the FK-506-binding proteins and the parvulins. Cyclophilins are found in all cells of all organisms studied, in both prokaryotes and eukaryotes; humans have a total of 16 cyclophilin proteins, Arabidopsis up to 29 and Saccharomyces 8. The first member of the cyclophilins to be identified in mammals, cyclophilin A, is the major cellular target for, and thus mediates the actions of, the immunosuppressive drug cyclosporin A. Cyclophilin A forms a ternary complex with cyclosporin A and the calcium-calmodulin-activated serine/threonine-specific protein phosphatase calcineurin; formation of this complex prevents calcineurin from regulating cytokine gene transcription. Recent studies have implicated a diverse array of additional cellular functions for cyclophilins, including roles as chaperones and in cell signaling. PMID:15998457

  9. What are the pros and cons of the use of host-targeted agents against hepatitis C?

    PubMed

    Pawlotsky, Jean-Michel

    2014-05-01

    Hepatitis C virus (HCV) therapy is living a revolution. Host-targeted agents (HTAs) block HCV production by interacting with host cell components. Because they target conserved host proteins, not variable viral proteins, HTAs have the potential for pangenotypic antiviral activity and a high barrier to resistance. Only two HTAs have reached clinical development, including specific inhibitors of cyclophilin A peptidyl-prolyl cis/trans isomerase activity and antagonists of microRNA-122. Cyclophilin inhibitors have proven to be relatively well tolerated and can be confidently used as backbones of all-oral, interferon-free regimens. In addition, HTAs such as cyclophilin inhibitors offer opportunities for "panviral" approaches when they target mechanisms common to viruses of the same or different families. This article forms part of a symposium in Antiviral Research on "Hepatitis C: next steps toward global eradication." PMID:24583032

  10. Interaction with Ppil3 leads to the cytoplasmic localization of Apoptin in tumor cells

    SciTech Connect

    Huo Dehua; Yi Lina; Yang Jine

    2008-07-18

    Apoptin, a small protein encoded by chicken anemia virus (CAV), induces cell death specifically in cancer cells. In normal cells, Apoptin remains in the cytoplasm; whereas in cancerous cells, it migrates into the nucleus and kills the cell. Cellular localization appears to be crucial. Through a yeast two-hybrid screen, we identified human Peptidyl-prolyl isomerase-like 3 (Ppil3) as one of the Apoptin-associated proteins. Ppil3 could bind Apoptin directly, and held Apoptin in cytoplasm even in tumor cells. We then demonstrated that the nuclearcytoplasmic distribution of Apoptin is related to the expression level of intrinsic Ppil3. Moreover, extrinsic modifying of Ppil3 levels also resulted in nuclearcytoplasmic shuffling of Apoptin. The Apoptin P109A mutant, located between the putative nuclear localization and export signals, could significantly impair the function of Ppil3. Our results suggest a new direction for the localization mechanism study of Apoptin in cells.

  11. Structural, Biochemical, and in Vivo Characterization of the First Virally Encoded Cyclophilin from the Mimivirus

    PubMed Central

    Thai, Vu; Renesto, Patricia; Fowler, C. Andrew; Brown, Darin J.; Davis, Tara; Gu, Wanjun; Pollock, David D.; Kern, Dorothee; Raoult, Didier; Eisenmesser, Elan Z.

    2010-01-01

    Although multiple viruses utilize host cell cyclophilins, including severe acute respiratory syndrome (SARS) and human immunodeficiency virus type-1(HIV-1), their role in infection is poorly understood. To help elucidate these roles, we have characterized the first virally encoded cyclophilin (mimicyp) derived from the largest virus discovered to date (the Mimivirus) that is also a causative agent of pneumonia in humans. Mimicyp adopts a typical cyclophilin-fold, yet it also forms trimers unlike any previously characterized homologue. Strikingly, immunofluorescence assays reveal that mimicyp localizes to the surface of the mature virion, as recently proposed for several viruses that recruit host cell cyclophilins such as SARS and HIV-1. Additionally mimicyp lacks peptidyl-prolyl isomerase activity in contrast to human cyclophilins. Thus, this study suggests that cyclophilins, whether recruited from host cells (i.e. HIV-1 and SARS) or virally encoded (i.e. Mimivirus), are localized on viral surfaces for at least a subset of viruses. PMID:18342330

  12. Chemotactic Activity of Cyclophilin A in the Skin Mucus of Yellow Catfish (Pelteobagrus fulvidraco) and Its Active Site for Chemotaxis.

    PubMed

    Dawar, Farman Ullah; Tu, Jiagang; Xiong, Yang; Lan, Jiangfeng; Dong, Xing Xing; Liu, Xiaoling; Khattak, Muhammad Nasir Khan; Mei, Jie; Lin, Li

    2016-01-01

    Fish skin mucus is a dynamic barrier for invading pathogens with a variety of anti-microbial enzymes, including cyclophilin A (CypA), a multi-functional protein with peptidyl-prolyl cis/trans isomerase (PPIase) activity. Beside various other immunological functions, CypA induces leucocytes migration in vitro in teleost. In the current study, we have discovered several novel immune-relevant proteins in yellow catfish skin mucus by mass spectrometry (MS). The CypA present among them was further detected by Western blot. Moreover, the CypA present in the skin mucus displayed strong chemotactic activity for yellow catfish leucocytes. Interestingly, asparagine (like arginine in mammals) at position 69 was the critical site in yellow catfish CypA involved in leucocyte attraction. These novel efforts do not only highlight the enzymatic texture of skin mucus, but signify CypA to be targeted for anti-inflammatory therapeutics. PMID:27589721

  13. Structural, Biochemical, and in Vivo Characterization of the First Virally Encoded Cyclophilin from the Mimivirus

    SciTech Connect

    Thai,V.; Renesto, P.; Fowler, C.; Brown, D.; Davis, T.; Gu, W.; Pollock, D.; Kern, D.; Raoult, D.; Eisenmesser, E.

    2008-01-01

    Although multiple viruses utilize host cell cyclophilins, including severe acute respiratory syndrome (SARS) and human immunodeficiency virus type-1(HIV-1), their role in infection is poorly understood. To help elucidate these roles, we have characterized the first virally encoded cyclophilin (mimicyp) derived from the largest virus discovered to date (the Mimivirus) that is also a causative agent of pneumonia in humans. Mimicyp adopts a typical cyclophilin-fold, yet it also forms trimers unlike any previously characterized homologue. Strikingly, immunofluorescence assays reveal that mimicyp localizes to the surface of the mature virion, as recently proposed for several viruses that recruit host cell cyclophilins such as SARS and HIV-1. Additionally mimicyp lacks peptidyl-prolyl isomerase activity in contrast to human cyclophilins. Thus, this study suggests that cyclophilins, whether recruited from host cells (ie HIV-1 and SARS) or virally encoded (ie Mimivirus), are localized on viral surfaces for at least a subset of viruses.

  14. Biochemical characterization of recombinant phosphoglucose isomerase of Mycobacterium tuberculosis

    SciTech Connect

    Mathur, Divya; Ahsan, Zaid; Tiwari, Madhulika; Garg, Lalit C. . E-mail: lalitcgarg@yahoo.com

    2005-11-18

    Phosphoglucose isomerase (PGI) is a well-characterized ubiquitous enzyme involved in the glycolytic pathway. It catalyzes the reversible isomerization of D-glucopyranose-6-phosphate and D-fructofuranose-6-phosphate and is present in all living cells. However, there is interspecies variation at the level of the primary structure which sometimes produces heterogeneity at the structural and functional levels. In order to evaluate and characterize the mycobacterial PGI, the gene encoding the PGI from Mycobacterium tuberculosis H37Rv was cloned in pET-22b(+) vector and expressed in Escherichia coli. The target DNA was PCR amplified from the bacterial artificial chromosome using specific primers and cloned under the control of T7 promoter. Upon induction with IPTG, the recombinant PGI (rPGI) expressed partly as soluble protein and partly as inclusion bodies. The rPGI from the soluble fraction was purified to near homogeneity by ion-exchange chromatography. Mass spectrum analysis of the purified rPGI revealed its mass to be 61.45 kDa. The purified rPGI was enzymatically active and the specific activity was 600 U/mg protein. The K {sub m} of rPGI was determined to be 0.318 mM for fructose-6-phosphate and the K {sub i} was 0.8 mM for 6-phosphogluconate. The rPGI exhibited optimal activity at 37 deg C and pH 9.0, and did not require mono- or divalent cations for its activity.

  15. Identification of fibrillogenic regions in human triosephosphate isomerase.

    PubMed

    Carcamo-Noriega, Edson N; Saab-Rincon, Gloria

    2016-01-01

    Background. Amyloid secondary structure relies on the intermolecular assembly of polypeptide chains through main-chain interaction. According to this, all proteins have the potential to form amyloid structure, nevertheless, in nature only few proteins aggregate into toxic or functional amyloids. Structural characteristics differ greatly among amyloid proteins reported, so it has been difficult to link the fibrillogenic propensity with structural topology. However, there are ubiquitous topologies not represented in the amyloidome that could be considered as amyloid-resistant attributable to structural features, such is the case of TIM barrel topology. Methods. This work was aimed to study the fibrillogenic propensity of human triosephosphate isomerase (HsTPI) as a model of TIM barrels. In order to do so, aggregation of HsTPI was evaluated under native-like and destabilizing conditions. Fibrillogenic regions were identified by bioinformatics approaches, protein fragmentation and peptide aggregation. Results. We identified four fibrillogenic regions in the HsTPI corresponding to the β3, β6, β7 y α8 of the TIM barrel. From these, the β3-strand region (residues 59-66) was highly fibrillogenic. In aggregation assays, HsTPI under native-like conditions led to amorphous assemblies while under partially denaturing conditions (urea 3.2 M) formed more structured aggregates. This slightly structured aggregates exhibited residual cross-β structure, as demonstrated by the recognition of the WO1 antibody and ATR-FTIR analysis. Discussion. Despite the fibrillogenic regions present in HsTPI, the enzyme maintained under native-favoring conditions displayed low fibrillogenic propensity. This amyloid-resistance can be attributed to the three-dimensional arrangement of the protein, where β-strands, susceptible to aggregation, are protected in the core of the molecule. Destabilization of the protein structure may expose inner regions promoting β-aggregation, as well as the

  16. Identification of fibrillogenic regions in human triosephosphate isomerase

    PubMed Central

    Carcamo-Noriega, Edson N.

    2016-01-01

    Background. Amyloid secondary structure relies on the intermolecular assembly of polypeptide chains through main-chain interaction. According to this, all proteins have the potential to form amyloid structure, nevertheless, in nature only few proteins aggregate into toxic or functional amyloids. Structural characteristics differ greatly among amyloid proteins reported, so it has been difficult to link the fibrillogenic propensity with structural topology. However, there are ubiquitous topologies not represented in the amyloidome that could be considered as amyloid-resistant attributable to structural features, such is the case of TIM barrel topology. Methods. This work was aimed to study the fibrillogenic propensity of human triosephosphate isomerase (HsTPI) as a model of TIM barrels. In order to do so, aggregation of HsTPI was evaluated under native-like and destabilizing conditions. Fibrillogenic regions were identified by bioinformatics approaches, protein fragmentation and peptide aggregation. Results. We identified four fibrillogenic regions in the HsTPI corresponding to the β3, β6, β7 y α8 of the TIM barrel. From these, the β3-strand region (residues 59–66) was highly fibrillogenic. In aggregation assays, HsTPI under native-like conditions led to amorphous assemblies while under partially denaturing conditions (urea 3.2 M) formed more structured aggregates. This slightly structured aggregates exhibited residual cross-β structure, as demonstrated by the recognition of the WO1 antibody and ATR-FTIR analysis. Discussion. Despite the fibrillogenic regions present in HsTPI, the enzyme maintained under native-favoring conditions displayed low fibrillogenic propensity. This amyloid-resistance can be attributed to the three-dimensional arrangement of the protein, where β-strands, susceptible to aggregation, are protected in the core of the molecule. Destabilization of the protein structure may expose inner regions promoting β-aggregation, as well as the

  17. Purification, characterization and catalytic properties of human sterol 8-isomerase.

    PubMed Central

    Nes, W David; Zhou, Wenxu; Dennis, Allen L; Li, Haoxia; Jia, Zhonghua; Keith, Richard A; Piser, Timothy M; Furlong, Stephen T

    2002-01-01

    CHO 2, encoding human sterol 8-isomerase (hSI), was introduced into plasmids pYX213 or pET23a. The resulting native protein was overexpressed in erg 2 yeast cells and purified to apparent homogeneity. The enzyme exhibited a K (m) of 50 microM and a turnover number of 0.423 s(-1) for zymosterol, an isoelectric point of 7.70, a native molecular mass of 107000 Da and was tetrameric. The structural features of zymosterol provided optimal substrate acceptability. Biomimetic studies of acid-catalysed isomerization of zymosterol resulted in formation of cholest-8(14)-enol, whereas the enzyme-generated product was a Delta(7)-sterol, suggesting absolute stereochemical control of the reaction by hSI. Using (2)H(2)O and either zymosterol or cholesta-7,24-dienol as substrates, the reversibility of the reaction was confirmed by GC-MS of the deuterated products. The positional specific incorporation of deuterium at C-9alpha was established by a combination of (1)H- and (13)C-NMR analyses of the enzyme-generated cholesta-7,24-dienol. Kinetic analyses indicated the reaction equilibrium ( K (eq)=14; DeltaG(o')=-6.5 kJ/mol) for double-bond isomerization favoured the forward direction, Delta(8) to Delta(7). Treatment of hSI with different high-energy intermediate analogues produced the following dissociation constants ( K (i)): emopamil (2 microM)=tamoxifen (1 microM)=tridemorph (1 microM)<25-azacholesterol (21 microM)

  18. Purification, characterization, and amino acid sequencing of a. delta. /sup 5/-3-oxosteroid isomerase from Pseudomonas putida biotype B

    SciTech Connect

    Linden, K.G.

    1986-01-01

    Studies were performed on the ..delta../sup 5/-3-oxosteroid isomerase from Pseudomonas putida biotype B. The studies have involved three broad areas: improvement in the purification of the enzyme, further characterization of the purified enzyme, and completion of the amino acid sequence of the enzyme. For the purification of the enzyme, techniques for removing the isomerase from whole cells were studied, the effects of ionic strength on the binding of the isomerase to steroidal affinity resins was explored, and a new affinity resin was developed. Absorption spectra and the proton NMR spectra of the isomerase were obtained. Amino acid sequencing of the oxosteroid isomerase indicates that the enzyme is a dimeric protein consisting of two identical subunits each consisting of a polypeptide chain of 131 residues and a M/sub r/ = 14,536.

  19. Characterization of the microsomal steroid-8-ene isomerase of cholesterol biosynthesis.

    PubMed

    Yamaga, N; Gaylor, J L

    1978-03-01

    Rat liver microsomes contain an enzyme that catalyzes the isomerization of the nuclear double bond of steroids from the 8(9) position to the 7(8) position. The enzyme is most active with zymosterol, 5alpha-cholesta-8,24-dien-3beta-ol, which is a precursor of cholesterol. Properties of the microsomal isomerase have now been studied, and preliminary data are reported on both regulation of enzymic activity and first steps in the solubilization of the enzyme from membranes. After a brief lag period, the velocity of isomerase is relatively constant for about 5 min of incubation, and then isomerization subsides. The apparent Michaelis constant (52-70 micro M) is difficult to determine accurately, due to these complex kinetic changes. V(max) is 4.0-4.7 nmol/min per mg of microsomal protein. The apparent specific activity is more than ten times that of liver microsomal methyl sterol oxidase. The maximal specific activity of microsomal isomerase is approximately doubled when rats are fed an intestinal bile acid sequestrant, cholestyramine. Changes in specific activity of isomerase parallel changes in activities of other microsomal enzymes of cholesterol biosynthesis, such as 3-hydroxy-3-methylglutaryl-CoA reductase and 4-methyl sterol oxidase. Isomerase activity is destroyed by phospholipase A digestion, high concentration of bile salts, and solvent extraction, all of which are known either to remove phospholipid or to alter microsomal membrane integrity. On the other hand, isomerase remains active in the presence of a mild, nonionic detergent, Triton WR-1339; thus, solubilization with nonionic detergents is under study. PMID:650094

  20. Purification and characterization of thermostable glucose isomerase from Clostridium thermosulfurogenes and Thermoanaerobacter strain B6A.

    PubMed Central

    Lee, C Y; Zeikus, J G

    1991-01-01

    Glucose isomerases produced by Thermoanaerobacter strain B6A and Clostridium thermosulfurogenes strain 4B were purified 10-11-fold to homogeneity and their physicochemical and catalytic properties were determined. Both purified enzymes displayed very similar properties (native Mr 200,000, tetrameric subunit composition, and apparent pH optima 7.0-7.5). The enzymes were stable at pH 5.5-12.0, and maintained more than 90% activity after incubation at high temperature (85 degrees C) for 1 h in the presence of metal ions. The N-terminal amino acid sequences of both thermostable glucose isomerases were Met-Asn-Lys-Tyr-Phe-Glu-Asn and were not similar to that of the thermolabile Bacillus subtilis enzyme. The glucose isomerase from C. thermosulfurogenes and Thermoanaerobacter displayed pI values of 4.9 and 4.8, and their kcat. and Km values for D-glucose at 65 degrees C were 1040 and 1260 min-1 and 140 and 120 mM respectively. Both enzymes displayed higher kcat. and lower Km values for D-xylose than for D-glucose. The C. thermosulfurogenes enzyme required Co2+ or Mg2+ for thermal stability and glucose isomerase activity, and Mn2+ or these metals for xylose isomerase activity. Crystals of C. thermosulfurogenes glucose isomerase were formed at room temperature by the hanging-drop method using 16-18% poly(ethylene glycol) (PEG) 4000 in 0.1 M-citrate buffer. Images Fig. 1. Fig. 5. PMID:1996956

  1. Xylose Isomerase Improves Growth and Ethanol Production Rates from Biomass Sugars for Both Saccharomyces Pastorianus and Saccharomyces Cerevisiae

    PubMed Central

    Miller, Kristen P.; Gowtham, Yogender Kumar; Henson, J. Michael; Harcum, Sarah W.

    2013-01-01

    The demand for biofuel ethanol made from clean, renewable nonfood sources is growing. Cellulosic biomass, such as switch grass (Panicum virgatum L.), is an alternative feedstock for ethanol production; however, cellulosic feedstock hydrolysates contain high levels of xylose, which needs to be converted to ethanol to meet economic feasibility. In this study, the effects of xylose isomerase on cell growth and ethanol production from biomass sugars representative of switch grass were investigated using low cell density cultures. The lager yeast species Saccharomyces pastorianus was grown with immobilized xylose isomerase in the fermentation step to determine the impact of the glucose and xylose concentrations on the ethanol production rates. Ethanol production rates were improved due to xylose isomerase; however, the positive effect was not due solely to the conversion of xylose to xylulose. Xylose isomerase also has glucose isomerase activity, so to better understand the impact of the xylose isomerase on S. pastorianus, growth and ethanol production were examined in cultures provided fructose as the sole carbon. It was observed that growth and ethanol production rates were higher for the fructose cultures with xylose isomerase even in the absence of xylose. To determine whether the positive effects of xylose isomerase extended to other yeast species, a side-by-side comparison of S. pastorianus and Saccharomyces cerevisiae was conducted. These comparisons demonstrated that the xylose isomerase increased ethanol productivity for both the yeast species by increasing the glucose consumption rate. These results suggest that xylose isomerase can contribute to improved ethanol productivity, even without significant xylose conversion. PMID:22866331

  2. Expression of the Escherichia coli xylose isomerase gene in Saccharomyces cerevisiae

    SciTech Connect

    Sarthy, A.V.; McConaughy, B.L.; Lobo, Z.; Sundstrom, J.A.; Furlong, C.E.; Hall, B.D.

    1987-09-01

    Transformation of Saccharomyces cerevisiae by yeast expression plasmids bearing the Escherichia coli xylose isomerase gene leads to production of the protein. Western blotting experiments show that immunoreactive protein chains which comigrate with the E. coli enzyme are made in the transformant strains and that the amount produced parallels the copy number of the plasmid. When comparable amounts of immunologically cross-reactive xylose isomerase protein made in E. coli or S. cerevisiae were assayed for enzymatic activity, however, the yeast protein was at least 10/sup 3/-fold less active.

  3. Amino acid sequences around the cysteine residues of rabbit muscle triose phosphate isomerase

    PubMed Central

    Miller, Janet C.; Waley, S. G.

    1971-01-01

    1. The nature of the subunits in rabbit muscle triose phosphate isomerase has been investigated. 2. Amino acid analyses show that there are five cysteine residues and two methionine residues/subunit. 3. The amino acid sequences around the cysteine residues have been determined; these account for about 75 residues. 4. Cleavage at the methionine residues with cyanogen bromide gave three fragments. 5. These results show that the subunits correspond to polypeptide chains, containing about 230 amino acid residues. The chains in triose phosphate isomerase seem to be shorter than those of other glycolytic enzymes. PMID:5165707

  4. Arabidopsis Phosphomannose Isomerase 1, but Not Phosphomannose Isomerase 2, Is Essential for Ascorbic Acid Biosynthesis*S⃞

    PubMed Central

    Maruta, Takanori; Yonemitsu, Miki; Yabuta, Yukinori; Tamoi, Masahiro; Ishikawa, Takahiro; Shigeoka, Shigeru

    2008-01-01

    We studied molecular and functional properties of Arabidopsis phosphomannose isomerase isoenzymes (PMI1 and PMI2) that catalyze reversible isomerization between d-fructose 6-phosphate and d-mannose 6-phosphate (Man-6P). The apparent Km and Vmax values for Man-6P of purified recombinant PMI1 were 41.3 ± 4.2 μm and 1.89 μmol/min/mg protein, respectively, whereas those of purified recombinant PMI2 were 372 ± 13 μm and 22.5 μmol/min/mg protein, respectively. Both PMI1 and PMI2 were inhibited by incubation with EDTA, Zn2+, Cd2+, and l-ascorbic acid (AsA). Arabidopsis PMI1 protein was constitutively expressed in both vegetative and reproductive organs under normal growth conditions, whereas the PMI2 protein was not expressed in any organs under light. The induction of PMI1 expression and an increase in the AsA level were observed in leaves under continuous light, whereas the induction of PMI2 expression and a decrease in the AsA level were observed under long term darkness. PMI1 showed a diurnal expression pattern in parallel with the total PMI activity and the total AsA content in leaves. Moreover, a reduction of PMI1 expression through RNA interference resulted in a substantial decrease in the total AsA content of leaves of knockdown PMI1 plants, whereas the complete inhibition of PMI2 expression did not affect the total AsA levels in leaves of knock-out PMI2 plants. Consequently, this study improves our understanding of the molecular and functional properties of Arabidopsis PMI isoenzymes and provides genetic evidence of the involvement of PMI1, but not PMI2, in the biosynthesis of AsA in Arabidopsis plants. PMID:18755683

  5. Both the isomerase and chaperone activities of protein disulfide isomerase are required for the reactivation of reduced and denatured acidic phospholipase A2.

    PubMed Central

    Yao, Y; Zhou, Y; Wang, C

    1997-01-01

    The spontaneous reactivation yield of acidic phospholipase A2 (APLA2), a protein containing seven disulfide bonds, after reduction and denaturation in guanidine hydrochloride is very low. Protein disulfide isomerase (PDI) markedly increases the reactivation yield and prevents the aggregation of APLA2 during refolding in a redox buffer containing GSH and GSSG. S-methylated PDI (mPDI), with no isomerase but as nearly full chaperone activity as native PDI, has no effect on either the reactivation or aggregation of APLA2. However, the simultaneous presence of PDI and mPDI in molar ratios to APLA2 of 0.1 and 0.9 respectively fully reactivates the denatured enzyme, as does PDI alone at a ratio of 1. At ratios of 0.1 and 0.15 respectively, they completely suppress APLA2 aggregation, as does PDI alone at a ratio of 0.25. Moreover, delayed addition of PDI to the refolding buffer greatly diminished the reactivation yield of APLA2, but this deteriorating effect can be alleviated markedly by the presence of mPDI in the refolding buffer. Without GSSG, mPDI prevents the aggregation of APLA2 during refolding. It is proposed that the in vitro action of PDI as a foldase consists of both isomerase and chaperone activities, and the latter activity can be fully replaced by mPDI. PMID:9034346

  6. Water in the Active Site of Ketosteroid Isomerase

    PubMed Central

    Hanoian, Philip; Hammes-Schiffer, Sharon

    2011-01-01

    Classical molecular dynamics simulations were utilized to investigate the structural and dynamical properties of water in the active site of ketosteroid isomerase (KSI) to provide insight into the role of these water molecules in the enzyme-catalyzed reaction. This reaction is thought to proceed via a dienolate intermediate that is stabilized by hydrogen bonding with residues Tyr16 and Asp103. A comparative study was performed for the wild-type (WT) KSI and the Y16F, Y16S, and Y16F/Y32F/Y57F (FFF) mutants. These systems were studied with three different bound ligands: equilenin, which is an intermediate analog, and the intermediate states of two steroid substrates. Several distinct water occupation sites were identified in the active site of KSI for the WT and mutant systems. Three additional sites were identified in the Y16S mutant that were not occupied in WT KSI or the other mutants studied. The number of water molecules directly hydrogen bonded to the ligand oxygen was approximately two waters in the Y16S mutant, one water in the Y16F and FFF mutants, and intermittent hydrogen bonding of one water molecule in WT KSI. The molecular dynamics trajectories of the Y16F and FFF mutants reproduced the small conformational changes of residue 16 observed in the crystal structures of these two mutants. Quantum mechanical/molecular mechanical calculations of 1H NMR chemical shifts of the protons in the active site hydrogen-bonding network suggest that the presence of water in the active site does not prevent the formation of short hydrogen bonds with far-downfield chemical shifts. The molecular dynamics simulations indicate that the active site water molecules exchange much more frequently for WT KSI and the FFF mutant than for the Y16F and Y16S mutants. This difference is most likely due to the hydrogen-bonding interaction between Tyr57 and an active site water molecule that is persistent in the Y16F and Y16S mutants but absent in the FFF mutant and significantly less

  7. Production and Purification of Extracellular D-Xylose Isomerase from an Alkaliphilic, Thermophilic Bacillus sp. †

    PubMed Central

    Chauthaiwale, Jyoti; Rao, Mala

    1994-01-01

    An alkaliphilic, thermophilic Bacillus sp. (NCIM 59) produced extracellular xylose isomerase at pH 10 and 50°C by using xylose or wheat bran as the carbon source. The distribution of xylose isomerase as a function of growth in comparison with distributions of extra- and intracellular marker enzymes such as xylanase and β-galactosidase revealed that xylose isomerase was truly secreted as an extracellular enzyme and was not released because of sporulation or lysis. The enzyme was purified to homogeneity by ammonium sulfate precipitation followed by gel filtration, preparative polyacrylamide gel electrophoresis, and ion-exchange chromatography. The molecular weight of xylose isomerase was estimated to be 160,000 by gel filtration and 50,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating the presence of three subunits. The enzyme is most active at pH 8.0 and with incubation at 85°C for 20 min. Divalent metal ions Mg2+, Co2+, and Mn2+ were required for maximum activity of the enzyme. The Km values for D-xylose and D-glucose at 80°C and pH 7.5 were 6.66 and 142 mM, respectively, while Kcat values were 2.3 × 102 s-1 and 0.5 × 102 s-1, respectively. Images PMID:16349464

  8. Genetic engineering in the Precambrian: structure of the chicken triosephosphate isomerase gene.

    PubMed Central

    Straus, D; Gilbert, W

    1985-01-01

    We report the sequence of the single chicken triosephosphate isomerase gene and its flanking regions. The 3-kilobase-long gene is composed of seven similarly sized exons and six introns. By using crystallographic and sequence data, we argue that this ancient gene was originally assembled from the genetic antecedents of exons. Images PMID:3837846

  9. Stereochemistry of 4-carboxymuconolactone decarboxylase and muconolactone isomerase in the. beta. -ketoadipate pathway

    SciTech Connect

    Whitman, C.P.; Chari, R.V.J.; Ngai, K.L.; Kozarich, J.W.

    1986-05-01

    The protocatechuate and catechol pathways, two separate and parallel branches of the ..beta..-ketoadipate pathway in Pseudomonas putida, converge at a common intermediate - ..beta..-ketoadipate enol-lactone. The enol-lactone is generated by 4-carboxymuconolactone decarboxylase in the protocatechuate pathway while muconolactone isomerase produces it in the catechol pathway. The presence of these enzymes as well as ..beta..-carboxymuconate cycloisomerase and its substrate, ..beta..-carboxy-cis,cis-muconate, in a NMR tube, leads to the following sequence of events. Lactonization of ..beta..-carboxy-cis,cis-muconate produces 4-carboxymuconolactone which decarboxylates enzymatically with deuteration by D/sub 2/O to afford 2-(/sup 2/H)-4-ketoadipate enol-lactone - the substrate for muconolactone isomerase. Further conversion of the monodeuterated enol-lactone by muconolactone isomerase affords muconolactone which is nearly completely deuterated at the 4 position. The proton ricochets between the 2 and 4 positions with concurrent washout while in the 2 position. Based on the known absolute stereochemistry of 4-carboxymuconolactone and muconolactone, these results suggest that both the decarboxylase and isomerase proceed by syn mechanisms, but operate on opposite faces of the common enol-lactone substrate.

  10. Comparing the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways in arabinose and xylose fermenting Saccharomyces cerevisiae strains

    PubMed Central

    Bettiga, Maurizio; Hahn-Hägerdal, Bärbel; Gorwa-Grauslund, Marie F

    2008-01-01

    Background Ethanolic fermentation of lignocellulosic biomass is a sustainable option for the production of bioethanol. This process would greatly benefit from recombinant Saccharomyces cerevisiae strains also able to ferment, besides the hexose sugar fraction, the pentose sugars, arabinose and xylose. Different pathways can be introduced in S. cerevisiae to provide arabinose and xylose utilisation. In this study, the bacterial arabinose isomerase pathway was combined with two different xylose utilisation pathways: the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways, respectively, in genetically identical strains. The strains were compared with respect to aerobic growth in arabinose and xylose batch culture and in anaerobic batch fermentation of a mixture of glucose, arabinose and xylose. Results The specific aerobic arabinose growth rate was identical, 0.03 h-1, for the xylose reductase/xylitol dehydrogenase and xylose isomerase strain. The xylose reductase/xylitol dehydrogenase strain displayed higher aerobic growth rate on xylose, 0.14 h-1, and higher specific xylose consumption rate in anaerobic batch fermentation, 0.09 g (g cells)-1 h-1 than the xylose isomerase strain, which only reached 0.03 h-1 and 0.02 g (g cells)-1h-1, respectively. Whereas the xylose reductase/xylitol dehydrogenase strain produced higher ethanol yield on total sugars, 0.23 g g-1 compared with 0.18 g g-1 for the xylose isomerase strain, the xylose isomerase strain achieved higher ethanol yield on consumed sugars, 0.41 g g-1 compared with 0.32 g g-1 for the xylose reductase/xylitol dehydrogenase strain. Anaerobic fermentation of a mixture of glucose, arabinose and xylose resulted in higher final ethanol concentration, 14.7 g l-1 for the xylose reductase/xylitol dehydrogenase strain compared with 11.8 g l-1 for the xylose isomerase strain, and in higher specific ethanol productivity, 0.024 g (g cells)-1 h-1 compared with 0.01 g (g cells)-1 h-1 for the xylose reductase

  11. Analysis of the Arabinose-5-Phosphate Isomerase of Bacteroides fragilis Provides Insight into Regulation of Single-Domain Arabinose Phosphate Isomerases

    PubMed Central

    Cech, David; Wang, Pan Fen; Holler, Tod P.

    2014-01-01

    Arabinose-5-phosphate isomerases (APIs) catalyze the interconversion of d-ribulose-5-phosphate and d-arabinose-5-phosphate, the first step in the biosynthesis of 3-deoxy-d-manno-octulosonic acid (Kdo), an essential component of the lipopolysaccharide in Gram-negative bacteria. Classical APIs, such as Escherichia coli KdsD, contain a sugar isomerase domain and a tandem cystathionine beta-synthase domain. Despite substantial effort, little is known about structure-function relationships in these APIs. We recently reported an API containing only a sugar isomerase domain. This protein, c3406 from E. coli CFT073, has no known physiological function. In this study, we investigated a putative single-domain API from the anaerobic Gram-negative bacterium Bacteroides fragilis. This putative API (UniProt ID Q5LIW1) is the only protein encoded by the B. fragilis genome with significant identity to any known API, suggesting that it is responsible for lipopolysaccharide biosynthesis in B. fragilis. We tested this hypothesis by preparing recombinant Q5LIW1 protein (here referred to by the UniProt ID Q5LIW1), characterizing its API activity in vitro, and demonstrating that the gene encoding Q5LIW1 (GenBank ID YP_209877.1) was able to complement an API-deficient E. coli strain. We demonstrated that Q5LIW1 is inhibited by cytidine 5′-monophospho-3-deoxy-d-manno-2-octulosonic acid, the final product of the Kdo biosynthesis pathway, with a Ki of 1.91 μM. These results support the assertion that Q5LIW1 is the API that supports lipopolysaccharide biosynthesis in B. fragilis and is subject to feedback regulation by CMP-Kdo. The sugar isomerase domain of E. coli KdsD, lacking the two cystathionine beta-synthase domains, demonstrated API activity and was further characterized. These results suggest that Q5LIW1 may be a suitable system to study API structure-function relationships. PMID:24891442

  12. Identification of DES1 as a Vitamin A Isomerase in Müller Glial Cells of the Retina

    PubMed Central

    Kaylor, Joanna J.; Yuan, Quan; Cook, Jeremy; Sarfare, Shanta; Makshanoff, Jacob; Miu, Anh; Kim, Anita; Kim, Paul; Habib, Samer; Roybal, C. Nathaniel; Xu, Tongzhou; Nusinowitz, Steven; Travis, Gabriel H.

    2012-01-01

    Absorption of a light particle by an opsin-pigment causes photoisomerization of its retinaldehyde chromophore. Restoration of light sensitivity to the resulting apo-opsin requires chemical re-isomerization of the photobleached chromophore. This is carried out by a multistep enzyme pathway called the visual cycle. Accumulating evidence suggests the existence of an alternate visual cycle for regenerating opsins in daylight. Here, we identified dihydroceramide desaturase-1 (DES1) as a retinol isomerase and an excellent candidate for isomerase-2 in this alternate pathway. DES1 is expressed in retinal Müller cells where it co-immunoprecipitates with cellular retinaldehyde binding protein (CRALBP). Adenoviral gene therapy with DES1 partially rescued the biochemical and physiological phenotypes in rpe65 −/− mice lacking isomerohydrolase (isomerase-1). Knockdown of DES1 expression by RNA-interference concordantly reduced isomerase-2 activity in cultured Müller cells. Purified DES1 possessed very high isomerase-2 activity in the presence of appropriate cofactors, suggesting that DES1 by itself is sufficient for isomerase activity. PMID:23143414

  13. Mutations in FKBP14 cause a variant of Ehlers-Danlos syndrome with progressive kyphoscoliosis, myopathy, and hearing loss.

    PubMed

    Baumann, Matthias; Giunta, Cecilia; Krabichler, Birgit; Rüschendorf, Franz; Zoppi, Nicoletta; Colombi, Marina; Bittner, Reginald E; Quijano-Roy, Susana; Muntoni, Francesco; Cirak, Sebahattin; Schreiber, Gudrun; Zou, Yaqun; Hu, Ying; Romero, Norma Beatriz; Carlier, Robert Yves; Amberger, Albert; Deutschmann, Andrea; Straub, Volker; Rohrbach, Marianne; Steinmann, Beat; Rostásy, Kevin; Karall, Daniela; Bönnemann, Carsten G; Zschocke, Johannes; Fauth, Christine

    2012-02-10

    We report on an autosomal-recessive variant of Ehlers-Danlos syndrome (EDS) characterized by severe muscle hypotonia at birth, progressive scoliosis, joint hypermobility, hyperelastic skin, myopathy, sensorineural hearing impairment, and normal pyridinoline excretion in urine. Clinically, the disorder shares many features with the kyphoscoliotic type of EDS (EDS VIA) and Ullrich congenital muscular dystrophy. Linkage analysis in a large Tyrolean kindred identified a homozygous frameshift mutation in FKBP14 in two affected individuals. Based on the cardinal clinical characteristics of the disorder, four additional individuals originating from different European countries were identified who carried either homozygous or compound heterozygous mutations in FKBP14. FKBP14 belongs to the family of FK506-binding peptidyl-prolyl cis-trans isomerases (PPIases). ER-resident FKBPs have been suggested to act as folding catalysts by accelerating cis-trans isomerization of peptidyl-prolyl bonds and to act occasionally also as chaperones. We demonstrate that FKBP14 is localized in the endoplasmic reticulum (ER) and that deficiency of FKBP14 leads to enlarged ER cisterns in dermal fibroblasts in vivo. Furthermore, indirect immunofluorescence of FKBP14-deficient fibroblasts indicated an altered assembly of the extracellular matrix in vitro. These findings suggest that a disturbance of protein folding in the ER affecting one or more components of the extracellular matrix might cause the generalized connective tissue involvement in this disorder. FKBP14 mutation analysis should be considered in all individuals with apparent kyphoscoliotic type of EDS and normal urinary pyridinoline excretion, in particular in conjunction with sensorineural hearing impairment. PMID:22265013

  14. A Quasi-Laue Neutron Crystallographic Study of D-Xylose Isomerase

    NASA Technical Reports Server (NTRS)

    Meilleur, Flora; Snell, Edward H.; vanderWoerd, Mark; Judge, Russell A.; Myles, Dean A. A.

    2006-01-01

    Hydrogen atom location and hydrogen bonding interaction determination are often critical to explain enzymatic mechanism. Whilst it is difficult to determine the position of hydrogen atoms using X-ray crystallography even with subatomic (less than 1.0 Angstrom) resolution data available, neutron crystallography provides an experimental tool to directly localise hydrogeddeuteriwn atoms in biological macromolecules at resolution of 1.5-2.0 Angstroms. Linearisation and isomerisation of xylose at the active site of D-xylose isomerase rely upon a complex hydrogen transfer. Neutron quasi-Laue data were collected on Streptomyces rubiginosus D-xylose isomerase crystal using the LADI instrument at ILL with the objective to provide insight into the enzymatic mechanism (Myles et al. 1998). The neutron structure unambiguously reveals the protonation state of His 53 in the active site, identifying the model for the enzymatic pathway.

  15. Utilization of plant hemicellulose for production of xylanase and glucose isomerase

    SciTech Connect

    Han, Y.W.

    1983-01-01

    The hemicellulose fraction of sugarcane bagasse and ryegrass straw was extracted with NaOH and used for production of xylanase and glucose isomerase by Streptomyces flavogriseus. As much as 33% of hemicellulose per dry weight of the plant material could be obtained by treating the plant biomass with 5-10% NaOH for 1 h at 121 degrees or for 24 h at room temperature. The amount of hemicellulose extracted greatly increased as the concentration of NaOH increased up to 10%, thereafter the rate of increase slowed. Hemicellulose was further fractionated into hemicellulose A and hemicellulose B. Growth of the organism on plant hemicellulose yielded glucose isomerase (2.2 units/mL culture) and xylanase (27 units/mL culture). The levels of the enzymes produced on hemicellulose were about the same as on xylose or xylan. The organism, however, grew poorly on sugarcane bagasse and produced little enzymes.

  16. Crystallization and preliminary X-ray characterization of phosphoglucose isomerase from Mycobacterium tuberculosis H37Rv

    PubMed Central

    Mathur, Divya; Anand, Kanchan; Mathur, Deepika; Jagadish, Nirmala; Suri, Anil; Garg, Lalit C.

    2007-01-01

    Phosphoglucose isomerase is a ubiquitous enzyme that catalyzes the isomerization of d-glucopyranose-6-phosphate to d-fructofuranose-6-phosphate. The present investigation reports the expression, purification, crystallization and preliminary crystallographic studies of the phosphoglucose isomerase from Mycobacterium tuberculosis H37Rv, which shares 46% sequence identity with that of its human host. The recombinant protein, which was prepared using an Escherichia coli expression system, was crystallized by the hanging-drop vapour-diffusion method. The crystals diffracted to a resolution of 2.8 Å and belonged to the orthorhombic space group I212121, with unit-cell parameters a = 109.0, b = 119.8, c = 138.9 Å. PMID:17401215

  17. Expression, purification, crystallization and preliminary X-ray diffraction analysis of Bifidobacterium adolescentis xylose isomerase

    PubMed Central

    dos Reis, Caio Vinicius; Bernardes, Amanda; Polikarpov, Igor

    2013-01-01

    Xylose isomerase (EC 5.3.1.5) is a key enzyme in xylose metabolism which is industrially important for the transformation of glucose and xylose into fructose and xylulose, respectively. The Bifidobacterium adolescentis xylA gene (NC_008618.1) encoding xylose isomerase (XI) was cloned and the enzyme was overexpressed in Escherichia coli. Purified recombinant XI was crystallized using the sitting-drop vapour-diffusion method with polyethylene glycol 3350 as the precipitating agent. A complete native data set was collected to 1.7 Å resolution using a synchrotron-radiation source. The crystals belonged to the orthorhombic space group P21212, with unit-cell parameters a = 88.78, b = 123.98, c = 78.63 Å. PMID:23695585

  18. Purification of Δ(5)-3-ketosteroid isomerase from Digitalis lanata.

    PubMed

    Meitinger, Nadine; Geiger, Daniel; Augusto, Thierry W; Maia de Pádua, Rodrigo; Kreis, Wolfgang

    2015-01-01

    The isomerization of 5-pregnene-3,20-dione into 4-pregnene-3,20-dione was investigated to shed further light on cardenolide biosynthesis and to characterize the enzymes involved in cardenolide formation. It was shown that the Δ(5)-3-ketosteroid isomerase of Digitalis lanata, which catalyzes this isomerization, is an individual enzyme and not, as previously thought, associated with Δ(5)-3β-hydroxysteroid dehydrogenase. The enzyme was purified by fractionated ammonium sulfate precipitation, hydrophobic interaction chromatography and gel filtration. The purification protocol resulted in a 68.1-fold enriched specific enzyme activity with a yield of 2.2%. After an additional chromatofocusing step the 3KSI activity appeared as a single protein band at 17kDa in SDS-PAGE. Plant 3KSI displayed similar properties to microbial 3-ketosteroid isomerases. PMID:25468533

  19. Novel roles for protein disulphide isomerase in disease states: a double edged sword?

    PubMed Central

    Parakh, Sonam; Atkin, Julie D.

    2015-01-01

    Protein disulphide isomerase (PDI) is a multifunctional redox chaperone of the endoplasmic reticulum (ER). Since it was first discovered 40 years ago the functions ascribed to PDI have evolved significantly and recent studies have recognized its distinct functions, with adverse as well as protective effects in disease. Furthermore, post translational modifications of PDI abrogate its normal functional roles in specific disease states. This review focusses on recent studies that have identified novel functions for PDI relevant to specific diseases. PMID:26052512

  20. Analysis of the Isomerase and Chaperone-Like Activities of an Amebic PDI (EhPDI)

    PubMed Central

    Meléndez-López, Samuel G.

    2015-01-01

    Protein disulfide isomerases (PDI) are eukaryotic oxidoreductases that catalyze the formation and rearrangement of disulfide bonds during folding of substrate proteins. Structurally, PDI enzymes share as a common feature the presence of at least one active thioredoxin-like domain. PDI enzymes are also involved in holding, refolding, and degradation of unfolded or misfolded proteins during stressful conditions. The EhPDI enzyme (a 38 kDa polypeptide with two active thioredoxin-like domains) has been used as a model to gain insights into protein folding and disulfide bond formation in E. histolytica. Here, we performed a functional complementation assay, using a ΔdsbC mutant of E. coli, to test whether EhPDI exhibits isomerase activity in vivo. Our preliminary results showed that EhPDI exhibits isomerase activity; however, further mutagenic analysis revealed significant differences in the functional role of each thioredoxin-like domain. Additional studies confirmed that EhPDI protects heat-labile enzymes against thermal inactivation, extending our knowledge about its chaperone-like activity. The characterization of EhPDI, as an oxidative folding catalyst with chaperone-like function, represents the initial step to dissect the molecular mechanisms involved in protein folding in E. histolytica. PMID:25695056

  1. Structure of the Noncatalytic Domains and Global Fold of the Protein Disulfide Isomerase ERp72

    SciTech Connect

    Kozlov, G.; Määttänen, P; Schrag, J; Hura, G; Gabrielli, L; Cygler, M; Thomas, D; Gehring, K

    2009-01-01

    Protein disulfide isomerases are a family of proteins that catalyze the oxidation and isomerization of disulfide bonds in newly synthesized proteins in the endoplasmic reticulum. The family includes general enzymes such as PDI that recognize unfolded proteins, and others that are selective for specific classes of proteins. Here, we report the X-ray crystal structure of central non-catalytic domains of a specific isomerase, ERp72 (also called CaBP2 and protein disulfide-isomerase A4) from Rattus norvegicus. The structure reveals strong similarity to ERp57, a PDI-family member that interacts with the lectin-like chaperones calnexin and calreticulin but, unexpectedly, ERp72 does not interact with calnexin as shown by isothermal titration calorimetry and nuclear magnetic resonance (NMR) spectroscopy. Small-angle X-ray scattering (SAXS) of ERp72 was used to develop models of the full-length protein using both rigid body refinement and ab initio simulated annealing of dummy atoms. The two methods show excellent agreement and define the relative positions of the five thioredoxin-like domains of ERp72 and potential substrate or chaperone binding sites.

  2. Triosephosphate isomerase: removal of a putatively electrophilic histidine residue results in a subtle change in catalytic mechanism

    SciTech Connect

    Nickbarg, E.B.; Davenport, R.C.; Petsko, G.A.; Knowles, J.R.

    1988-08-09

    An important active-site residue in the glycolytic enzyme triosephosphate isomerase is His-95, which appears to act as an electrophilic component in catalyzing the enolization of the substrates. With the techniques of site-directed mutagenesis, His-95 has been replaced by Gln in the isomerase from Saccharomyces cerevisiae. The mutant isomerase has been expressed in Escherichia coli strain DF502 and purified to homogeneity. The specific catalytic activity of the mutant enzyme is less than that of wild type by a factor of nearly 400. The mutant enzyme can be resolved from the wild-type isomerase on nondenaturing isoelectric focusing gels, and an isomerase activity stain shows that the observed catalytic activity indeed derives from the mutant protein. The mutant enzyme shows the same stereospecificity of proton transfer as the wild type. Tritium exchange experiments similar to those used to define the free energy profile for the wild-type yeast isomerase, together with a new method of analysis involving /sup 14/C and /sup 3/H doubly labeled substrates, have been used to investigate the energetics of the mutant enzyme catalyzed reaction. The deuterium kinetic isotope effects observed with the mutant isomerase using (1(R)-/sup 2/H)dihydroxyacetone phosphate and (2-/sup 2/H)glyceraldehyde 3-phosphate are 2.15 +/- 0.04 and 2.4 +/- 0.1, respectively. These results lead to the conclusion that substitution of Gln for His-95 so impairs the ability of the enzyme to stabilize the reaction intermediate that there is a change in the pathways of proton transfer mediated by the mutant enzyme.

  3. Xylose isomerase from polycentric fungus Orpinomyces: gene sequencing, cloning, and expression in Saccharomyces cerevisiae for bioconversion of xylose to ethanol.

    PubMed

    Madhavan, Anjali; Tamalampudi, Sriappareddy; Ushida, Kazunari; Kanai, Daisuke; Katahira, Satoshi; Srivastava, Aradhana; Fukuda, Hideki; Bisaria, Virendra S; Kondo, Akihiko

    2009-04-01

    The cDNA sequence of the gene for xylose isomerase from the rumen fungus Orpinomyces was elucidated by rapid amplification of cDNA ends. The 1,314-nucleotide gene was cloned and expressed constitutively in Saccharomyces cerevisiae. The deduced polypeptide sequence encoded a protein of 437 amino acids which showed the highest similarity to the family II xylose isomerases. Further, characterization revealed that the recombinant enzyme was a homodimer with a subunit of molecular mass 49 kDa. Cell extract of the recombinant strain exhibited high specific xylose isomerase activity. The pH optimum of the enzyme was 7.5, while the low temperature optimum at 37 degrees C was the property that differed significantly from the majority of the reported thermophilic xylose isomerases. In addition to the xylose isomerase gene, the overexpression of the S. cerevisiae endogenous xylulokinase gene and the Pichia stipitis SUT1 gene for sugar transporter in the recombinant yeast facilitated the efficient production of ethanol from xylose. PMID:19050860

  4. Crystal structures of two monomeric triosephosphate isomerase variants identified via a directed-evolution protocol selecting for L-arabinose isomerase activity.

    PubMed

    Krause, Mirja; Kiema, Tiila Riikka; Neubauer, Peter; Wierenga, Rik K

    2016-06-01

    The crystal structures are described of two variants of A-TIM: Ma18 (2.7 Å resolution) and Ma21 (1.55 Å resolution). A-TIM is a monomeric loop-deletion variant of triosephosphate isomerase (TIM) which has lost the TIM catalytic properties. Ma18 and Ma21 were identified after extensive directed-evolution selection experiments using an Escherichia coli L-arabinose isomerase knockout strain expressing a randomly mutated A-TIM gene. These variants facilitate better growth of the Escherichia coli selection strain in medium supplemented with 40 mM L-arabinose. Ma18 and Ma21 differ from A-TIM by four and one point mutations, respectively. Ma18 and Ma21 are more stable proteins than A-TIM, as judged from CD melting experiments. Like A-TIM, both proteins are monomeric in solution. In the Ma18 crystal structure loop 6 is open and in the Ma21 crystal structure loop 6 is closed, being stabilized by a bound glycolate molecule. The crystal structures show only small differences in the active site compared with A-TIM. In the case of Ma21 it is observed that the point mutation (Q65L) contributes to small structural rearrangements near Asn11 of loop 1, which correlate with different ligand-binding properties such as a loss of citrate binding in the active site. The Ma21 structure also shows that its Leu65 side chain is involved in van der Waals interactions with neighbouring hydrophobic side-chain moieties, correlating with its increased stability. The experimental data suggest that the increased stability and solubility properties of Ma21 and Ma18 compared with A-TIM cause better growth of the selection strain when coexpressing Ma21 and Ma18 instead of A-TIM. PMID:27303904

  5. Domain swapping in the low-similarity isomerase/hydratase superfamily: the crystal structure of rat mitochondrial Delta3, Delta2-enoyl-CoA isomerase.

    PubMed

    Hubbard, Paul A; Yu, Wenfeng; Schulz, Horst; Kim, Jung-Ja P

    2005-06-01

    Two monofunctional Delta(3), Delta(2)-enoyl-CoA isomerases, one in mitochondria (mECI) and the other in both mitochondria and peroxisomes (pECI), belong to the low-similarity isomerase/hydratase superfamily. Both enzymes catalyze the movement of a double bond from C3 to C2 of an unsaturated acyl-CoA substrate for re-entry into the beta-oxidation pathway. Mutagenesis has shown that Glu165 of rat mECI is involved in catalysis; however, the putative catalytic residue in yeast pECI, Glu158, is not conserved in mECI. To elucidate whether Glu165 of mECI is correctly positioned for catalysis, the crystal structure of rat mECI has been solved. Crystal packing suggests the enzyme is trimeric, in contrast to other members of the superfamily, which appear crystallographically to be dimers of trimers. The polypeptide fold of mECI, like pECI, belongs to a subset of this superfamily in which the C-terminal domain of a given monomer interacts with its own N-terminal domain. This differs from that of crotonase and 1,4-dihydroxy-2-naphtoyl-CoA synthase, whose C-terminal domains are involved in domain swapping with an adjacent monomer. The structure confirms Glu165 as the putative catalytic acid/base, positioned to abstract the pro-R proton from C2 and reprotonate at C4 of the acyl chain. The large tunnel-shaped active site cavity observed in the mECI structure explains the relative substrate promiscuity in acyl-chain length and stereochemistry. Comparison with the crystal structure of pECI suggests the catalytic residues from both enzymes are spatially conserved but not in their primary structures, providing a powerful reminder of how catalytic residues cannot be determined solely by sequence alignments. PMID:15883186

  6. Truncation of a Protein Disulfide Isomerase, PDIL2-1, Delays Embryo Sac Maturation and Disrupts Pollen Tube Guidance in Arabidopsis thaliana

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pollen tubes navigate through different female tissues and deliver the sperm to the embryo sac for fertilization. Protein disulfide isomerases play important roles in the maturation of secreted or plasma membrane proteins. Here we show that truncated versions of a protein disulfide isomerase (PDI), ...

  7. Coimmobilization of glucoamylase and glucose isomerase by molecular deposition technique for one-step conversion of dextrin to fructose.

    PubMed

    Ge, Y; Wang, Y; Zhou, H; Wang, S; Tong, Y; Li, W

    1999-01-01

    Glucose isomerase was immobilized by itself with adsorption and coimmobilized with glucoamylase by molecular deposition technique using macroporous trimethylamine polystyrene beads. Approximately 77.5% of the enzyme added was immobilized. The pH-activity curve of the immobilized glucose isomerase was broadened, resulting in 75% retention of its maximum activity at pH 6.2. The Km of the immobilized glucose isomerase was 1.28-fold that of the soluble one. When the two enzymes were immobilized together, the system was found capable of functioning at pH 6.0 to produce fructose from starch and dextrin. At this pH, the total fructose output of the coimmobilized enzyme system after 24 h was 1.9 times that of the free enzyme system. PMID:9987846

  8. Glucose(xylose) isomerase production by Streptomyces sp. CH7 grown on agricultural residues.

    PubMed

    Chanitnun, Kankiya; Pinphanichakarn, Pairoh

    2012-07-01

    Streptomyces sp. CH7 was found to efficiently produce glucose(xylose) isomerase when grown on either xylan or agricultural residues. This strain produced a glucose(xylose) isomerase activity of roughly 1.8 U/mg of protein when it was grown in medium containing 1% xylose as a carbon source. Maximal enzymatic activities of about 5 and 3 U/mg were obtained when 1% xylan and 2.5% corn husks were used, respectively. The enzyme was purified from a mycelial extract to 16-fold purity with only two consecutive column chromatography steps using Macro-prep DEAE and Sephacryl-300, respectively. The approximate molecular weight of the purified enzyme is 170 kDa, and it has four identical subunits of 43.6 kDa as estimated by SDS-PAGE. Its K m values for glucose and xylose were found to be 258.96 and 82.77 mM, respectively, and its V max values are 32.42 and 63.64 μM/min/mg, respectively. The purified enzyme is optimally active at 85°C and pH 7.0. It is stable at pH 5.5-8.5 and at temperatures up to 60°C after 30 min. These findings indicate that glucose(xylose) isomerase from Streptomyces sp. CH7 has the potential for industrial applications, especially for high-fructose syrup production and bioethanol fermentation from hemicellulosic hydrolysates by Saccharomyces cerevisiae. PMID:24031932

  9. Triosephosphate isomerase: energetics of the reaction catalyzed by the yeast enzyme expressed in Escherichia coli

    SciTech Connect

    Nickbarg, E.B.; Knowles, J.R.

    1988-08-09

    Triosephosphate isomerase from bakers' yeast, expressed in Escherichia coli strain DF502(p12), has been purified to homogeneity. The kinetics of the reaction in each direction have been determined at pH 7.5 and 30 degrees C. Deuterium substitution at the C-2 position of substrate (R)-glyceraldehyde phosphate and at the 1-pro-R position of substrate dihydroxyacetone phosphate results in kinetic isotope effects on kcat of 1.6 and 3.4, respectively. The extent of transfer of tritium from (1(R)-TH)dihydroxyacetone phosphate to product (R)-glyceraldehyde phosphate during the catalyzed reaction is only 3% after 66% conversion to product, indicating that the enzymic base that mediates proton transfer is in rapid exchange with solvent protons. When the isomerase-catalyzed reaction is run in tritiated water in each direction, radioactivity is incorporated both into the remaining substrate and into the product. In the exchange-conversion experiment with dihydroxyacetone phosphate as substrate, the specific radioactivity of remaining dihydroxyacetone phosphate rises as a function of the extent of reaction with a slope of about 0.3, while the specific radioactivity of the products is 54% that of the solvent. In the reverse direction with (R)-glyceraldehyde phosphate as substrate, the specific radioactivity of the product formed is only 11% that of the solvent, while the radioactivity incorporated into the remaining substrate (R)-glyceraldehyde phosphate also rises as a function of the extent of reaction with a slope of 0.3. These results have been analyzed according to the protocol described earlier to yield the free energy profile of the reaction catalyzed by the yeast isomerase.

  10. Affinity labeling and characterization of the active site histidine of glucosephosphate isomerase

    SciTech Connect

    Gibson, D.R.; Gracy, R.W.; Hartman, F.C.

    1980-10-10

    N-bromoacetylethanolamine phosphate was found to act as a specific affinity label for the active center of glucosephosphate isomerase. The inactivation process followed pseudo-first order kinetics, was irreversible, and exhibited rate saturation kinetics with minimal half-lives of inactivation of 4.5 and 6.3 min for the enzyme isolated from human placenta and rabbit muscle, respectively. The pH dependence of the inactivation process closely paralleled the pH dependence of the overall catalytic process with pK/sub a/ values at pH 6.4 and 9.0. The stoichiometry of labeling of either enzyme, as determined with N-bromo(/sup 14/C/sub 2/)acetylethanolamine phosphate, was 1 eq of the affinity label/subunit of enzyme. After acid hydrolysis and amino acid analysis of the radioactive affinity-labeled human enzyme, only radioactive 3-carboxymethyl histidine was found. In the case of the rabbit enzyme, the only radioactive derivative obtained was 1-carboxymethyl histidine. Active site tryptic peptides were isolated by solvent extraction, thin layer peptide fingerprinting, and ion exchange chromatography before and after removal of the phosphate from the active site peptide. Amino acid analysis of the labeled peptides from the two species were very similar. Using high sensitivity methods for sequence analysis, the primary structure of the active site was established as Val-Leu-His-Ala-Glu-Asn-Val-Asp (Gly,Thr,Ser) Glu-Ile (Thr-Gly-His-Lys-Glx)-Tyr-Phe. Apparent sequence homology between the catalytic center of glucosephosphate isomerase and triosephosphate isomerase suggest that the two enzymes may have evolved from a common ancestral gene.

  11. Ribose-5-phosphate isomerase from Saccharomyces cerevisiae: purification and molecular analysis of the enzyme.

    PubMed

    Reuter, R; Naumann, M; Bär, J; Miosga, T; Kopperschläger, G

    1998-01-01

    Purification and molecular analysis of ribose-5-phosphate isomerase (EC 5.3.1.6) from Saccharomyces cerevisiae is described first time. The enzyme was enriched from a haploid deletion mutant containing the wild-type gene on a multicopy plasmid elaborating the following steps: ammonium sulphate precipitation, interfacial salting out on Sepharose 6B, high performance liquid chromatography on Fractogel EMD DEAE and on Resource Phenyl. The enzyme activity was found to be rather unstable possibly caused by removal of stabilizing cofactors or proteins during the purification procedure. The purified enzyme showed a hyperbolic dependence on the substrate ribose-5-phosphate with a K(m)-value of 1.6 +/- 0.3 mmol/l. For the native enzyme a molecular mass of 115 +/- 10 kDa was determined as found by saccharose density gradient centrifugation, sedimentation equilibrium analysis, size exclusion chromatography and polyacrylamide gel electrophoresis. Sodium dodecyl sulphate polyacrylamide gel electrophoresis and Western blotting revealed one band with a molecular mass of 31 +/- 2 kDa. Thus, the native enzyme is composed of four subunits of identical size. The molecular mass of the subunit and the identified N-terminal sequence of 33 amino acids fits well the 258 amino acid protein encoded by the S. cerevisiae RKI open reading frame, which was characterized previously only by increasing specific activities of ribose-5-phosphate isomerase in cells after cloning the gene. On the basis of the conserved amino acids an alignment of the amino acid sequence of ribose-5-phosphate isomerase from yeast with those of the enzyme from mouse, spinach and Escherichia coli is presented. PMID:9763699

  12. Trigonal crystals of glucose isomerase require thymol for their growth and stability

    NASA Astrophysics Data System (ADS)

    Chayen, N. E.; Lloyd, L. F.; Collyer, C. A.; Blow, D. M.

    1989-09-01

    The growth of trigonal bypyramidal crystals of Arthrobacter glucose isomerase is only possible in the presence of thymol at a concentration exceeding 2.5mM; at lower thymol concentrations rectangular and rhombic crystals are formed. When thymol is removed from the supernatant of trigonal crystals, they fall apart and recrystallise as rectangles or rhombs. The disordering can be reversed if thymol is reintroduced before the trigonal crystals have dispersed, but the other crystal forms are stable in thymol. During the disintegration of trigonal crystals the supernatant concentration of protein is increased.

  13. Arabinose 5-phosphate isomerase as a target for antibacterial design: studies with substrate analogues and inhibitors.

    PubMed

    Gabrielli, Luca; Merlo, Silvia; Airoldi, Cristina; Sperandeo, Paola; Gianera, Serena; Polissi, Alessandra; Nicotra, Francesco; Holler, Tod P; Woodard, Ronald W; Cipolla, Laura

    2014-04-15

    Structural requirements of D-arabinose 5-phosphate isomerase (KdsD, E.C. 5.3.1.13) from Pseudomonas aeruginosa were analysed in detail using advanced NMR techniques. We performed epitope mapping studies of the binding between the enzyme and the most potent KdsD inhibitors found to date, together with studies of a set of newly synthesised arabinose 5-phosphate (A5P) mimetics. We report here the first experimental evidence that KdsD may bind the furanose form of A5P, suggesting that catalysis of ring opening may be an important part of KdsD catalysis. PMID:24680056

  14. TrbB from Conjugative Plasmid F Is a Structurally Distinct Disulfide Isomerase That Requires DsbD for Redox State Maintenance ▿ †

    PubMed Central

    Hemmis, Casey W.; Berkmen, Mehmet; Eser, Markus; Schildbach, Joel F.

    2011-01-01

    TrbB, a periplasmic protein encoded by the conjugative plasmid F, has a predicted thioredoxin-like fold and possesses a C-X-X-C redox active site motif. TrbB may function in the conjugative process by serving as a disulfide bond isomerase, facilitating proper folding of a subset of F-plasmid-encoded proteins in the periplasm. Previous studies have demonstrated that a ΔtrbB F plasmid in Escherichia coli lacking DsbCE.coli, its native disulfide bond isomerase, experiences a 10-fold decrease in mating efficiency but have not provided direct evidence for disulfide bond isomerase activity. Here we demonstrate that trbB can partially restore transfer of a variant of the distantly related R27 plasmid when both chromosomal and plasmid genes encoding disulfide bond isomerases have been disrupted. In addition, we show that TrbB displays both disulfide bond isomerase and reductase activities on substrates not involved in the conjugative process. Unlike canonical members of the disulfide bond isomerase family, secondary structure predictions suggest that TrbB lacks both an N-terminal dimerization domain and an α-helical domain found in other disulfide bond isomerases. Phylogenetic analyses support the conclusion that TrbB belongs to a unique family of plasmid-based disulfide isomerases. Interestingly, although TrbB diverges structurally from other disulfide bond isomerases, we show that like those isomerases, TrbB relies on DsbD from E. coli for maintenance of its C-X-X-C redox active site motif. PMID:21742866

  15. Steroidomimetic aminomethyl spiroacetals as novel inhibitors of the enzyme Δ8,7-sterol isomerase in cholesterol biosynthesis.

    PubMed

    Krojer, Melanie; Müller, Christoph; Bracher, Franz

    2014-02-01

    Grundmann's ketone is converted to a spiroacetal containing a 5-hydroxymethyl-5-nitro-1,3-dioxane moiety whose hydroxymethyl group can be esterified or directly substituted with primary and secondary amines. Among the resulting aminomethyl spiroacetals, several ones bearing diamino residues were found to be inhibitors of the enzyme Δ8,7-isomerase in cholesterol biosynthesis. The complex bicyclic building block derived from Grundmann's ketone could be replaced by a properly substituted tetraline scaffold, without noteworthy loss in activity. This opens the opportunity to perform further structural modifications for the design of new steroidomimetic inhibitors of human Δ8,7-isomerase. PMID:24493593

  16. The prolyl-isomerase Pin1 activates the mitochondrial death program of p53

    PubMed Central

    Sorrentino, G; Mioni, M; Giorgi, C; Ruggeri, N; Pinton, P; Moll, U; Mantovani, F; Del Sal, G

    2013-01-01

    In response to intense stress, the tumor protein p53 (p53) tumor suppressor rapidly mounts a direct mitochondrial death program that precedes transcription-mediated apoptosis. By eliminating severely damaged cells, this pathway contributes to tumor suppression as well as to cancer cell killing induced by both genotoxic drugs and non-genotoxic p53-reactivating molecules. Here we have explored the role had in this pathway by the prolyl-isomerase Pin1 (peptidylprolyl cis/trans isomerase, NIMA-interacting 1), a crucial transducer of p53's phosphorylation into conformational changes unleashing its pro-apoptotic activity. We show that Pin1 promotes stress-induced localization of p53 to mitochondria both in vitro and in vivo. In particular, we demonstrate that upon stress-induced phosphorylation of p53 on Ser46 by homeodomain interacting protein kinase 2, Pin1 stimulates its mitochondrial trafficking signal, that is, monoubiquitination. This pathway is induced also by the p53-activating molecule RITA, and we demonstrate the strong requirement of Pin1 for the induction of mitochondrial apoptosis by this compound. These findings have significant implications for treatment of p53-expressing tumors and for prospective use of p53-activating compounds in clinics. PMID:22935610

  17. Control of carotenoid biosynthesis through a heme-based cis-trans isomerase.

    PubMed

    Beltrán, Jesús; Kloss, Brian; Hosler, Jonathan P; Geng, Jiafeng; Liu, Aimin; Modi, Anuja; Dawson, John H; Sono, Masanori; Shumskaya, Maria; Ampomah-Dwamena, Charles; Love, James D; Wurtzel, Eleanore T

    2015-08-01

    Plants synthesize carotenoids, which are essential for plant development and survival. These metabolites also serve as essential nutrients for human health. The biosynthetic pathway for all plant carotenoids occurs in chloroplasts and other plastids and requires 15-cis-ζ-carotene isomerase (Z-ISO). It was not known whether Z-ISO catalyzes isomerization alone or in combination with other enzymes. Here we show that Z-ISO is a bona fide enzyme and integral membrane protein. Z-ISO independently catalyzes the cis-trans isomerization of the 15-15' carbon-carbon double bond in 9,15,9'-cis-ζ-carotene to produce the substrate required by the subsequent biosynthetic-pathway enzyme. We discovered that isomerization depends upon a ferrous heme b cofactor that undergoes redox-regulated ligand switching between the heme iron and alternate Z-ISO amino acid residues. Heme b-dependent isomerization of a large hydrophobic compound in a membrane was previously undescribed. As an isomerase, Z-ISO represents a new prototype for heme b proteins and potentially uses a new chemical mechanism. PMID:26075523

  18. A mutation in the rice chalcone isomerase gene causes the golden hull and internode 1 phenotype.

    PubMed

    Hong, Lilan; Qian, Qian; Tang, Ding; Wang, Kejian; Li, Ming; Cheng, Zhukuan

    2012-07-01

    The biosynthesis of flavonoids, important secondary plant metabolites, has been investigated extensively, but few mutants of genes in this pathway have been identified in rice (Oryza sativa). The rice gold hull and internode (gh) mutants exhibit a reddish-brown pigmentation in the hull and internode and their phenotype has long been used as a morphological marker trait for breeding and genetic study. Here, we characterized that the gh1 mutant was a mutant of the rice chalcone isomerase gene (OsCHI). The result showed that gh1 had a Dasheng retrotransposon inserted in the 5′ UTR of the OsCHI gene, which resulted in the complete loss of OsCHI expression. gh1 exhibited golden pigmentation in hulls and internodes once the panicles were exposed to light. The total flavonoid content in gh1 hulls was increased threefold compared to wild type. Consistent with the gh1 phenotype, OsCHI transcripts were expressed in most tissues of rice and most abundantly in internodes. It was also expressed at high levels in panicles before heading, distributed mainly in lemmas and paleae, but its expression decreased substantially after the panicles emerged from the sheath. OsCHI encodes a protein functionally and structurally conserved to chalcone isomerases in other species. Our findings demonstrated that the OsCHI gene was indispensable for flux of the flavonoid pathway in rice. PMID:22286805

  19. Disclosing the essentiality of ribose-5-phosphate isomerase B in Trypanosomatids.

    PubMed

    Faria, Joana; Loureiro, Inês; Santarém, Nuno; Cecílio, Pedro; Macedo-Ribeiro, Sandra; Tavares, Joana; Cordeiro-da-Silva, Anabela

    2016-01-01

    Ribose-5-phosphate isomerase (RPI) belongs to the non-oxidative branch of the pentose phosphate pathway, catalysing the inter-conversion of D-ribose-5-phosphate and D-ribulose-5-phosphate. Trypanosomatids encode a type B RPI, whereas humans have a structurally unrelated type A, making RPIB worthy of exploration as a potential drug target. Null mutant generation in Leishmania infantum was only possible when an episomal copy of RPIB gene was provided, and the latter was retained both in vitro and in vivo in the absence of drug pressure. This suggests the gene is essential for parasite survival. Importantly, the inability to remove the second allele of RPIB gene in sKO mutants complemented with an episomal copy of RPIB carrying a mutation that abolishes isomerase activity suggests the essentiality is due to its metabolic function. In vitro, sKO promastigotes exhibited no defect in growth, metacyclogenesis or macrophage infection, however, an impairment in intracellular amastigotes' replication was observed. Additionally, mice infected with sKO mutants rescued by RPIB complementation had a reduced parasite burden in the liver. Likewise, Trypanosoma brucei is resistant to complete RPIB gene removal and mice infected with sKO mutants showed prolonged survival upon infection. Taken together our results genetically validate RPIB as a potential drug target in trypanosomatids. PMID:27230471

  20. Role of a chalcone isomerase-like protein in flavonoid biosynthesis in Arabidopsis thaliana

    PubMed Central

    Jiang, Wenbo; Yin, Qinggang; Wu, Ranran; Zheng, Guangshun; Liu, Jinyue; Dixon, Richard A.; Pang, Yongzhen

    2015-01-01

    Flavonoids are important natural products for plant defence and human health. Although almost all the flavonoid pathway genes have been well-documented by biochemical and/or genetic approaches, the role of the Arabidopsis chalcone isomerase-like (CHIL) gene remains unclear. Two chil mutants with a seed colour similar to that of wild-type Arabidopsis have been identified here, but in sharp contrast to the characteristic transparent testa seed phenotype associated with other known flavonoid pathway genes. CHIL loss-of-function mutations led to a strong reduction in the proanthocyanidin and flavonol levels in seeds, but not in the anthocyanin levels in leaves. CHIL over-expression could partially recover the mutant phenotype of the chil mutant and increased both proanthocyanidin and flavonol accumulation in wild-type Arabidopsis. However, the CHIL gene could not rescue the mutant phenotype of TT5 that encodes the intrinsic chalcone isomerase in Arabidopsis. Parallel phenotypical and metabolic analyses of the chil, tt5, chs, and f3h mutants revealed that, genetically, CHIL functions at the same step as TT5. Moreover, it is demonstrated that CHIL co-expresses, co-localizes, and interacts with TT5 in Arabidopsis for flavonoid production. Based on these genetic and metabolic studies, it is concluded that CHIL functions with TT5 to promote flavonoid production, which is a unique enhancer in the flavonoid pathway. PMID:26347569

  1. Protein disulfide isomerases are antibody targets during immune-mediated tumor destruction

    PubMed Central

    Fonseca, Catia; Soiffer, Robert; Ho, Vincent; Vanneman, Matthew; Jinushi, Masahisa; Ritz, Jerome; Neuberg, Donna; Stone, Richard; DeAngelo, Dan

    2009-01-01

    The identification of cancer antigens that contribute to transformation and are linked with immune-mediated tumor destruction is an important goal for immunotherapy. Toward this end, we screened a murine renal cell carcinoma cDNA expression library with sera from mice vaccinated with irradiated tumor cells engineered to secrete granulocyte macrophage colony-stimulating factor (GM-CSF). Multiple nonmutated, overexpressed proteins that function in tumor cell migration, protein/nucleic acid homeostasis, metabolism, and stress responses were detected. Among these, the most frequently recognized clone was protein disulfide isomerase (PDI). High titer antibodies to human PDI were similarly induced in an acute myeloid leukemia patient who achieved a complete response after vac-cination with irradiated, autologous GM-CSF–secreting tumor cells in the setting of nonmyeloablative allogeneic bone marrow transplantation. Moreover, ERp5, a closely related disulfide isomerase involved in major histocompatibility complex (MHC) class I chain-related protein A (MICA) shedding, also evoked potent humoral reactions in diverse solid and hematologic malignancy patients who responded to GM-CSF–secreting tumor cell vaccines or antibody blockade of cytotoxic T lymphocyte–associated antigen 4 (CTLA-4). Together, these findings reveal the unexpected immunogenicity of PDIs and raise the possibility that these gene products might serve as targets for therapeutic monoclonal antibodies. PMID:19008459

  2. In-house SIRAS phasing of the polyunsaturated fatty-acid isomerase from Propionibacterium acnes

    SciTech Connect

    Liavonchanka, Alena; Hornung, Ellen; Feussner, Ivo; Rudolph, Markus

    2006-02-01

    Low iodide concentrations were sufficient to allow SAD and SIRAS phasing of cubic crystals of a novel fatty acid isomerase using Cu Kα radiation. The polyenoic fatty-acid isomerase from Propionibacterium acnes (PAI) catalyzes the double-bond isomerization of linoleic acid to conjugated linoleic acid, which is a dairy- or meat-derived fatty acid in the human diet. PAI was overproduced in Escherichia coli and purified to homogeneity as a yellow-coloured protein. The nature of the bound cofactor was analyzed by absorption and fluorescence spectroscopy. Single crystals of PAI were obtained in two crystal forms. Cubic shaped crystals belong to space group I2{sub 1}3, with a unit-cell parameter of 160.4 Å, and plate-like crystals belong to the monoclinic space group C2, with unit-cell parameters a = 133.7, b = 60.8, c = 72.2 Å, β = 115.8°. Both crystal forms contain one molecule per asymmetric unit and diffract to a resolution of better than 2.0 Å. Initial phases were obtained by SIRAS from in-house data from a cubic crystal that was soaked with an unusually low KI concentration of 0.25 M.

  3. The protein disulphide-isomerase family: unravelling a string of folds.

    PubMed Central

    Ferrari, D M; Söling, H D

    1999-01-01

    The mammalian protein disulphide-isomerase (PDI) family encompasses several highly divergent proteins that are involved in the processing and maturation of secretory proteins in the endoplasmic reticulum. These proteins are characterized by the presence of one or more domains of roughly 95-110 amino acids related to the cytoplasmic protein thioredoxin. All but the PDI-D subfamily are composed entirely of repeats of such domains, with at least one domain containing and one domain lacking a redox-active -Cys-Xaa-Xaa-Cys- tetrapeptide. In addition to their known roles as redox catalysts and isomerases, the last few years have revealed additional functions of the PDI proteins, including peptide binding, cell adhesion and perhaps chaperone activities. Attention is now turning to the non-redox-active domains of the PDIs, which may play an important role in all of the known activities of these proteins. Thus the presence of both redox-active and -inactive domains within these proteins portends a complexity of functions differentially accommodated by the various family members. PMID:10085220

  4. Protein disulfide isomerase homolog TrPDI2 contributing to cellobiohydrolase production in Trichoderma reesei.

    PubMed

    Wang, Guokun; Lv, Pin; He, Ronglin; Wang, Haijun; Wang, Lixian; Zhang, Dongyuan; Chen, Shulin

    2015-09-01

    The majority of the cysteine residues in the secreted proteins form disulfide bonds via protein disulfide isomerase (PDI)-mediated catalysis, stabilizing the enzyme activity. The role of PDI in cellulase production is speculative, as well as the possibility of PDI as a target for improving enzyme production efficiency of Trichoderma reesei, a widely used producer of enzyme for the production of lignocellulose-based biofuels and biochemicals. Here, we report that a PDI homolog, TrPDI2 in T. reesei exhibited a 36.94% and an 11.81% similarity to Aspergillus niger TIGA and T. reesei PDI1, respectively. The capability of TrPDI2 to recover the activity of reduced and denatured RNase by promoting refolding verified its protein disulfide isomerase activity. The overexpression of Trpdi2 increased the secretion and the activity of CBH1 at the early stage of cellulase induction. In addition, both the expression level and redox state of TrPDI2 responded to cellulase induction in T. reesei, providing sustainable oxidative power to ensure cellobiohydrolase maturation and production. The results suggest that TrPDI2 may contribute to cellobiohydrolase secretion by enhancing the capability of disulfide bond formation, which is essential for protein folding and maturation. PMID:26138396

  5. The prolyl-isomerase Pin1 activates the mitochondrial death program of p53.

    PubMed

    Sorrentino, G; Mioni, M; Giorgi, C; Ruggeri, N; Pinton, P; Moll, U; Mantovani, F; Del Sal, G

    2013-02-01

    In response to intense stress, the tumor protein p53 (p53) tumor suppressor rapidly mounts a direct mitochondrial death program that precedes transcription-mediated apoptosis. By eliminating severely damaged cells, this pathway contributes to tumor suppression as well as to cancer cell killing induced by both genotoxic drugs and non-genotoxic p53-reactivating molecules. Here we have explored the role had in this pathway by the prolyl-isomerase Pin1 (peptidylprolyl cis/trans isomerase, NIMA-interacting 1), a crucial transducer of p53's phosphorylation into conformational changes unleashing its pro-apoptotic activity. We show that Pin1 promotes stress-induced localization of p53 to mitochondria both in vitro and in vivo. In particular, we demonstrate that upon stress-induced phosphorylation of p53 on Ser46 by homeodomain interacting protein kinase 2, Pin1 stimulates its mitochondrial trafficking signal, that is, monoubiquitination. This pathway is induced also by the p53-activating molecule RITA, and we demonstrate the strong requirement of Pin1 for the induction of mitochondrial apoptosis by this compound. These findings have significant implications for treatment of p53-expressing tumors and for prospective use of p53-activating compounds in clinics. PMID:22935610

  6. Control of carotenoid biosynthesis through a heme-based cis-trans isomerase

    PubMed Central

    Beltrán, Jesús; Kloss, Brian; Hosler, Jonathan P.; Geng, Jiafeng; Liu, Aimin; Modi, Anuja; Dawson, John H.; Sono, Masanori; Shumskaya, Maria; Ampomah-Dwamena, Charles; Love, James D.; Wurtzel, Eleanore T.

    2015-01-01

    Plants synthesize carotenoids essential for plant development and survival. These metabolites also serve as essential nutrients for human health. The biosynthetic pathway leading to all plant carotenoids occurs in chloroplasts and other plastids and requires 15-cis-ζ-carotene isomerase (Z-ISO). It was not certain whether isomerization was achieved by Z-ISO alone or in combination with other enzymes. Here we show that Z-ISO is a bona fide enzyme and integral membrane protein. Z-ISO independently catalyzes the cis-to-trans isomerization of the 15–15′ C=C bond in 9,15,9′-cis-ζ-carotene to produce the substrate required by the following biosynthetic pathway enzyme. We discovered that isomerization depends upon a ferrous heme b cofactor that undergoes redox-regulated ligand-switching between the heme iron and alternate Z-ISO amino acid residues. Heme b-dependent isomerization of a large, hydrophobic compound in a membrane is unprecedented. As an isomerase, Z-ISO represents a new prototype for heme b proteins and potentially utilizes a novel chemical mechanism. PMID:26075523

  7. Crystallization and preliminary X-ray diffraction studies of l-rhamnose isomerase from Pseudomonas stutzeri

    SciTech Connect

    Yoshida, Hiromi; Wayoon, Poonperm; Takada, Goro; Izumori, Ken; Kamitori, Shigehiro

    2006-06-01

    Recombinant l-rhamnose isomerase from P. stutzeri has been crystallized. Diffraction data have been collected to 2.0 Å resolution. l-Rhamnose isomerase from Pseudomonas stutzeri (P. stutzeril-RhI) catalyzes not only the reversible isomerization of l-rhamnose to l-rhamnulose, but also isomerization between various rare aldoses and ketoses. Purified His-tagged P. stutzeril-RhI was crystallized by the hanging-drop vapour-diffusion method. The crystals belong to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 74.3, b = 104.0, c = 107.0 Å, β = 106.8°. Diffraction data have been collected to 2.0 Å resolution. The molecular weight of the purified P. stutzeril-RhI with a His tag at the C-terminus was confirmed to be 47.7 kDa by MALDI–TOF mass-spectrometric analysis and the asymmetric unit is expected to contain four molecules.

  8. Ethanol production from lignocellulosic hydrolysates using engineered Saccharomyces cerevisiae harboring xylose isomerase-based pathway.

    PubMed

    Ko, Ja Kyong; Um, Youngsoon; Woo, Han Min; Kim, Kyoung Heon; Lee, Sun-Mi

    2016-06-01

    The efficient co-fermentation of glucose and xylose is necessary for the economically feasible bioethanol production from lignocellulosic biomass. Even with xylose utilizing Saccharomyces cerevisiae, the efficiency of the lignocellulosic ethanol production remains suboptimal mainly due to the low conversion yield of xylose to ethanol. In this study, we evaluated the co-fermentation performances of SXA-R2P-E, a recently engineered isomerase-based xylose utilizing strain, in mixed sugars and in lignocellulosic hydrolysates. In a high-sugar fermentation with 70g/L of glucose and 40g/L of xylose, SXA-R2P-E produced 50g/L of ethanol with an yield of 0.43gethanol/gsugars at 72h. From dilute acid-pretreated hydrolysates of rice straw and hardwood (oak), the strain produced 18-21g/L of ethanol with among the highest yield of 0.43-0.46gethanol/gsugars ever reported. This study shows a highly promising potential of a xylose isomerase-expressing strain as an industrially relevant ethanol producer from lignocellulosic hydrolysates. PMID:26990396

  9. Disclosing the essentiality of ribose-5-phosphate isomerase B in Trypanosomatids

    PubMed Central

    Faria, Joana; Loureiro, Inês; Santarém, Nuno; Cecílio, Pedro; Macedo-Ribeiro, Sandra; Tavares, Joana; Cordeiro-da-Silva, Anabela

    2016-01-01

    Ribose-5-phosphate isomerase (RPI) belongs to the non-oxidative branch of the pentose phosphate pathway, catalysing the inter-conversion of D-ribose-5-phosphate and D-ribulose-5-phosphate. Trypanosomatids encode a type B RPI, whereas humans have a structurally unrelated type A, making RPIB worthy of exploration as a potential drug target. Null mutant generation in Leishmania infantum was only possible when an episomal copy of RPIB gene was provided, and the latter was retained both in vitro and in vivo in the absence of drug pressure. This suggests the gene is essential for parasite survival. Importantly, the inability to remove the second allele of RPIB gene in sKO mutants complemented with an episomal copy of RPIB carrying a mutation that abolishes isomerase activity suggests the essentiality is due to its metabolic function. In vitro, sKO promastigotes exhibited no defect in growth, metacyclogenesis or macrophage infection, however, an impairment in intracellular amastigotes’ replication was observed. Additionally, mice infected with sKO mutants rescued by RPIB complementation had a reduced parasite burden in the liver. Likewise, Trypanosoma brucei is resistant to complete RPIB gene removal and mice infected with sKO mutants showed prolonged survival upon infection. Taken together our results genetically validate RPIB as a potential drug target in trypanosomatids. PMID:27230471

  10. Overexpression, purification, crystallization and preliminary diffraction studies of the Protaminobacter rubrum sucrose isomerase SmuA

    SciTech Connect

    Ravaud, Stéphanie; Watzlawick, Hildegard; Haser, Richard; Mattes, Ralf; Aghajari, Nushin

    2006-01-01

    The P. rubrum sucrose isomerase SmuA, a key enzyme in the industrial production of isomaltulose, was crystallized and diffraction data were collected to 1.95 Å resolution. Palatinose (isomaltulose, α-d-glucosylpyranosyl-1,6-d-fructofuranose), a nutritional and acariogenic reducing sugar, is industrially obtained from sucrose by using immobilized cells of Protaminobacter rubrum that produce the sucrose isomerase SmuA. The isomerization of sucrose catalyzed by this enzyme also results in the formation of trehalulose (α-d-glucosylpyranosyl-1,1-d-fructofuranose) in smaller amounts and glucose, fructose and eventually isomaltose as by-products, which lower the yield of the reaction and complicate the recovery of palatinose. The determination of the three-dimensional structure of SmuA will provide a basis for rational protein-engineering studies in order to optimize the industrial production of palatinose. A recombinant form of the 67.3 kDa SmuA enzyme has been crystallized in the native state by the vapour-diffusion method. Crystals belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 61.6, b = 81.4, c = 135.6 Å, and diffract to 1.95 Å resolution on a synchrotron-radiation source.

  11. Human cellular retinaldehyde-binding protein has secondary thermal 9-cis-retinal isomerase activity.

    PubMed

    Bolze, Christin S; Helbling, Rachel E; Owen, Robin L; Pearson, Arwen R; Pompidor, Guillaume; Dworkowski, Florian; Fuchs, Martin R; Furrer, Julien; Golczak, Marcin; Palczewski, Krzysztof; Cascella, Michele; Stocker, Achim

    2014-01-01

    Cellular retinaldehyde-binding protein (CRALBP) chaperones 11-cis-retinal to convert opsin receptor molecules into photosensitive retinoid pigments of the eye. We report a thermal secondary isomerase activity of CRALBP when bound to 9-cis-retinal. UV/vis and (1)H NMR spectroscopy were used to characterize the product as 9,13-dicis-retinal. The X-ray structure of the CRALBP mutant R234W:9-cis-retinal complex at 1.9 Å resolution revealed a niche in the binding pocket for 9-cis-aldehyde different from that reported for 11-cis-retinal. Combined computational, kinetic, and structural data lead us to propose an isomerization mechanism catalyzed by a network of buried waters. Our findings highlight a specific role of water molecules in both CRALBP-assisted specificity toward 9-cis-retinal and its thermal isomerase activity yielding 9,13-dicis-retinal. Kinetic data from two point mutants of CRALBP support an essential role of Glu202 as the initial proton donor in this isomerization reaction. PMID:24328211

  12. Kinase-dead ATM protein is highly oncogenic and can be preferentially targeted by Topo-isomerase I inhibitors

    PubMed Central

    Yamamoto, Kenta; Wang, Jiguang; Sprinzen, Lisa; Xu, Jun; Haddock, Christopher J; Li, Chen; Lee, Brian J; Loredan, Denis G; Jiang, Wenxia; Vindigni, Alessandro; Wang, Dong; Rabadan, Raul; Zha, Shan

    2016-01-01

    Missense mutations in ATM kinase, a master regulator of DNA damage responses, are found in many cancers, but their impact on ATM function and implications for cancer therapy are largely unknown. Here we report that 72% of cancer-associated ATM mutations are missense mutations that are enriched around the kinase domain. Expression of kinase-dead ATM (AtmKD/-) is more oncogenic than loss of ATM (Atm-/-) in mouse models, leading to earlier and more frequent lymphomas with Pten deletions. Kinase-dead ATM protein (Atm-KD), but not loss of ATM (Atm-null), prevents replication-dependent removal of Topo-isomerase I-DNA adducts at the step of strand cleavage, leading to severe genomic instability and hypersensitivity to Topo-isomerase I inhibitors. Correspondingly, Topo-isomerase I inhibitors effectively and preferentially eliminate AtmKD/-, but not Atm-proficientor Atm-/- leukemia in animal models. These findings identify ATM kinase-domain missense mutations as a potent oncogenic event and a biomarker for Topo-isomerase I inhibitor based therapy. DOI: http://dx.doi.org/10.7554/eLife.14709.001 PMID:27304073

  13. Physiological and Pathogenic Roles of Prolyl Isomerase Pin1 in Metabolic Regulations via Multiple Signal Transduction Pathway Modulations.

    PubMed

    Nakatsu, Yusuke; Matsunaga, Yasuka; Yamamotoya, Takeshi; Ueda, Koji; Inoue, Yuki; Mori, Keiichi; Sakoda, Hideyuki; Fujishiro, Midori; Ono, Hiraku; Kushiyama, Akifumi; Asano, Tomoichiro

    2016-01-01

    Prolyl isomerases are divided into three groups, the FKBP family, Cyclophilin and the Parvulin family (Pin1 and Par14). Among these isomerases, Pin1 is a unique prolyl isomerase binding to the motif including pSer/pThr-Pro that is phosphorylated by kinases. Once bound, Pin1 modulates the enzymatic activity, protein stability or subcellular localization of target proteins by changing the cis- and trans-formations of proline. Several studies have examined the roles of Pin1 in the pathogenesis of cancers and Alzheimer's disease. On the other hand, recent studies have newly demonstrated Pin1 to be involved in regulating glucose and lipid metabolism. Interestingly, while Pin1 expression is markedly increased by high-fat diet feeding, Pin1 KO mice are resistant to diet-induced obesity, non-alcoholic steatohepatitis and diabetic vascular dysfunction. These phenomena result from the binding of Pin1 to several key factors regulating metabolic functions, which include insulin receptor substrate-1, AMPK, Crtc2 and NF-κB p65. In this review, we focus on recent advances in elucidating the physiological roles of Pin1 as well as the pathogenesis of disorders involving this isomerase, from the viewpoint of the relationships between signal transductions and metabolic functions. PMID:27618008

  14. 40 CFR 174.527 - Phosphomannose isomerase in all plants; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS PROCEDURES AND REQUIREMENTS FOR PLANT...; exemption from the requirement of a tolerance. Residues of the phosphomannose isomerase (PMI) enzyme in plants are exempt from the requirement of a tolerance when used as plant-incorporated protectant...

  15. 40 CFR 174.527 - Phosphomannose isomerase in all plants; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS PROCEDURES AND REQUIREMENTS FOR PLANT...; exemption from the requirement of a tolerance. Residues of the phosphomannose isomerase (PMI) enzyme in plants are exempt from the requirement of a tolerance when used as plant-incorporated protectant...

  16. 40 CFR 174.527 - Phosphomannose isomerase in all plants; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS PROCEDURES AND REQUIREMENTS FOR PLANT...; exemption from the requirement of a tolerance. Residues of the phosphomannose isomerase (PMI) enzyme in plants are exempt from the requirement of a tolerance when used as plant-incorporated protectant...

  17. 40 CFR 174.527 - Phosphomannose isomerase in all plants; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS PROCEDURES AND REQUIREMENTS FOR PLANT...; exemption from the requirement of a tolerance. Residues of the phosphomannose isomerase (PMI) enzyme in plants are exempt from the requirement of a tolerance when used as plant-incorporated protectant...

  18. 40 CFR 174.527 - Phosphomannose isomerase in all plants; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS PROCEDURES AND REQUIREMENTS FOR PLANT...; exemption from the requirement of a tolerance. Residues of the phosphomannose isomerase (PMI) enzyme in plants are exempt from the requirement of a tolerance when used as plant-incorporated protectant...

  19. Studies on the combined action of amylases and glucose isomerase on starch and its hydrolyzate. Part I. Production, extraction, purification and kinetic behavior of glucose isomerase.

    PubMed

    Attia, R M; Ghali, Y; Roushdi, M; Eldin, M A

    1980-06-01

    Glucose isomerase was produced from Streptomyces phaeochromogenes by aerobic fermentation at 28 degrees C for 24 hrs. The crude enzyme was obtained by disintegrating the harvested cells. It was found that ammonium sulphate at a saturation of 0.3-0.5 gave the maximum enzyme recovery (88.8%) from the crude extract, while acetone gave 66.2% at a concentration of 3/1 (V/V). On this basis the crude enzyme extract was purified following several steps as concentration, dialysis, precipitation with (NH4)2 SO4, then passing through column of Amberlite CG-50, and the eluate was treated with acetone to precipitate the enzyme. The kinetics behavior was studied and it was found that: optimum D-glucose concentration was 0.8 M, Km was 0.25 M, optimum pH was 7.0 and temperature was 70 degrees C. Magnesium at concentration of 0.07 M gave the maximum activity and its Km was 0.024 M. Antagonistic effects of Na+, Ca++ and Fe+++ in presence of 0.07 M of Mg++ were studied. Km and Vmax at different levels of Mg++ concentration were determined and no change in Km value was observed, while Vmax was affected. These findings indicate that the Mg++ combined with enzyme independently of the substrate. PMID:7424065

  20. De novo design and evolution of artificial disulfide isomerase enzymes analogous to the bacterial DsbC.

    PubMed

    Arredondo, Silvia; Segatori, Laura; Gilbert, Hiram F; Georgiou, George

    2008-11-14

    The Escherichia coli disulfide isomerase, DsbC is a V-shaped homodimer with each monomer comprising a dimerization region that forms part of a putative peptide-binding pocket and a thioredoxin catalytic domain. Disulfide isomerases from prokaryotes and eukaryotes exhibit little sequence homology but display very similar structural organization with two thioredoxin domains facing each other on top of the dimerization/peptide-binding region. To aid the understanding of the mechanistic significance of thioredoxin domain dimerization and of the peptide-binding cleft of DsbC, we constructed a series of protein chimeras comprising unrelated protein dimerization domains fused to thioredoxin superfamily enzymes. Chimeras consisting of the dimerization domain and the alpha-helical linker of the bacterial proline cis/trans isomerase FkpA and the periplasmic oxidase DsbA gave rise to enzymes that catalyzed the folding of multidisulfide substrate proteins in vivo with comparable efficiency to E. coli DsbC. In addition, expression of FkpA-DsbAs conferred modest resistance to CuCl2, a phenotype that depends on disulfide bond isomerization. Selection for resistance to elevated CuCl2 concentrations led to the isolation of FkpA-DsbA mutants containing a single amino acid substitution that changed the active site of the DsbA domain from CPHC into CPYC, increasing the similarity to the DsbC active site (CGYC). Unlike DsbC, which is resistant to oxidation by DsbB-DsbA and does not normally catalyze disulfide bond formation under physiological conditions, the FkpA-DsbA chimeras functioned both as oxidases and isomerases. The engineering of these efficient artificial isomerases delineates the key features of catalysis of disulfide bond isomerization and enhances our understanding of its evolution. PMID:18782764

  1. De Novo Design and Evolution of Artificial Disulfide Isomerase Enzymes Analogous to the Bacterial DsbC*S⃞

    PubMed Central

    Arredondo, Silvia; Segatori, Laura; Gilbert, Hiram F.; Georgiou, George

    2008-01-01

    The Escherichia coli disulfide isomerase, DsbC is a V-shaped homodimer with each monomer comprising a dimerization region that forms part of a putative peptide-binding pocket and a thioredoxin catalytic domain. Disulfide isomerases from prokaryotes and eukaryotes exhibit little sequence homology but display very similar structural organization with two thioredoxin domains facing each other on top of the dimerization/peptide-binding region. To aid the understanding of the mechanistic significance of thioredoxin domain dimerization and of the peptide-binding cleft of DsbC, we constructed a series of protein chimeras comprising unrelated protein dimerization domains fused to thioredoxin superfamily enzymes. Chimeras consisting of the dimerization domain and the α-helical linker of the bacterial proline cis/trans isomerase FkpA and the periplasmic oxidase DsbA gave rise to enzymes that catalyzed the folding of multidisulfide substrate proteins in vivo with comparable efficiency to E. coli DsbC. In addition, expression of FkpA-DsbAs conferred modest resistance to CuCl2, a phenotype that depends on disulfide bond isomerization. Selection for resistance to elevated CuCl2 concentrations led to the isolation of FkpA-DsbA mutants containing a single amino acid substitution that changed the active site of the DsbA domain from CPHC into CPYC, increasing the similarity to the DsbC active site (CGYC). Unlike DsbC, which is resistant to oxidation by DsbB-DsbA and does not normally catalyze disulfide bond formation under physiological conditions, the FkpA-DsbA chimeras functioned both as oxidases and isomerases. The engineering of these efficient artificial isomerases delineates the key features of catalysis of disulfide bond isomerization and enhances our understanding of its evolution. PMID:18782764

  2. Bioproduction of D-Tagatose from D-Galactose Using Phosphoglucose Isomerase from Pseudomonas aeruginosa PAO1.

    PubMed

    Patel, Manisha J; Patel, Arti T; Akhani, Rekha; Dedania, Samir; Patel, Darshan H

    2016-07-01

    Pseudomonas aeruginosa PAO1 phosphoglucose isomerase was purified as an active soluble form by a single-step purification using Ni-NTA chromatography that showed homogeneity on SDS-PAGE with molecular mass ∼62 kDa. The optimum temperature and pH for the maximum isomerization activity with D-galactose were 60 °C and 7.0, respectively. Generally, sugar phosphate isomerases show metal-independent activity but PA-PGI exhibited metal-dependent isomerization activity with aldosugars and optimally catalyzed the D-galactose isomerization in the presence of 1.0 mM MnCl2. The apparent Km and Vmax for D-galactose under standardized conditions were calculated to be 1029 mM (±31.30 with S.E.) and 5.95 U/mg (±0.9 with S.E.), respectively. Equilibrium reached after 180 min with production of 567.51 μM D-tagatose from 1000 mM of D-galactose. Though, the bioconversion ratio is low but it can be increased by immobilization and enzyme engineering. Although various L-arabinose isomerases have been characterized for bioproduction of D-tagatose, P. aeruginosa glucose phosphate isomerase is distinguished from the other L-arabinose isomerases by its optimal temperature (60 °C) for D-tagatose production being mesophilic bacteria, making it an alternate choice for bulk production. PMID:26922727

  3. X-ray structure of linalool dehydratase/isomerase from Castellaniella defragrans reveals enzymatic alkene synthesis.

    PubMed

    Weidenweber, Sina; Marmulla, Robert; Ermler, Ulrich; Harder, Jens

    2016-05-01

    Linalool dehydratase/isomerase (Ldi), an enzyme of terpene degradation in Castellaniella defragrans, isomerizes the primary monoterpene alcohol geraniol into the tertiary alcohol (S)-linalool and dehydrates (S)-linalool to the alkene β-myrcene. Here we report on the crystal structures of Ldi with and without terpene substrates, revealing a cofactor-free homopentameric enzyme. The substrates were embedded inside a hydrophobic channel between two monomers of the (α,α)6 barrel fold class and flanked by three clusters of polar residues involved in acid-base catalysis. The detailed view into the active site will guide future biotechnological applications of Ldi, in particular, for industrial butadiene and isoprene production from renewable sources. PMID:27062179

  4. Structural Elucidation of a Small Molecule Inhibitor of Protein Disulfide Isomerase

    PubMed Central

    2015-01-01

    Compound libraries provide a starting point for multiple biological investigations, but the structural integrity of compounds is rarely assessed experimentally until a late stage in the research process. Here, we describe the discovery of a neuroprotective small molecule that was originally incorrectly annotated with a chemical structure. We elucidated the correct structure of the active compound using analytical chemistry, revealing it to be the natural product securinine. We show that securinine is protective in a cell model of Huntington disease and identify the binding site of securinine to its target, protein disulfide isomerase using NMR chemical shift perturbation studies. We show that securinine displays favorable pharmaceutical properties, making it a promising compound for in vivo studies in neurodegenerative disease models. In addition to finding this unexpected activity of securinine, this study provides a systematic roadmap to those who encounter compounds with incorrect structural annotation in the course of screening campaigns. PMID:26500720

  5. Production of ethanol from D-xylose by using D-xylose isomerase and yeasts

    SciTech Connect

    Gong, C.S.; Chem, L.F.; Flickinger, M.C.; Chiang, L.C.; Tsao, G.T.

    1981-02-01

    D-xylulose, an intermediate of D-xylose catabolism, was observed to be fermentable to ethanol and carbon dioxide in a yield of greater than 80% by yeasts (including industrial bakers' yeast) under fermentative conditions. This conversion appears to be carried out by many yeasts known for D-glucose fermentation. In some yeasts, xylitol, in addition to ethanol, was produced from D-xylulose. Fermenting yeasts are also able to produce ethanol from D-xylose when D-xylose isomerizing enzyme is present. The results indicate that ethanol could be produced from D-xylose in a yield of greater than 80% by a two-step process. First. D-xylose is converted to D-xylulose by xylose isomerase. D-xylulose is then fermented to ethanol by yeasts.

  6. Rapid expansion of the protein disulfide isomerase gene family facilitates the folding of venom peptides.

    PubMed

    Safavi-Hemami, Helena; Li, Qing; Jackson, Ronneshia L; Song, Albert S; Boomsma, Wouter; Bandyopadhyay, Pradip K; Gruber, Christian W; Purcell, Anthony W; Yandell, Mark; Olivera, Baldomero M; Ellgaard, Lars

    2016-03-22

    Formation of correct disulfide bonds in the endoplasmic reticulum is a crucial step for folding proteins destined for secretion. Protein disulfide isomerases (PDIs) play a central role in this process. We report a previously unidentified, hypervariable family of PDIs that represents the most diverse gene family of oxidoreductases described in a single genus to date. These enzymes are highly expressed specifically in the venom glands of predatory cone snails, animals that synthesize a remarkably diverse set of cysteine-rich peptide toxins (conotoxins). Enzymes in this PDI family, termed conotoxin-specific PDIs, significantly and differentially accelerate the kinetics of disulfide-bond formation of several conotoxins. Our results are consistent with a unique biological scenario associated with protein folding: The diversification of a family of foldases can be correlated with the rapid evolution of an unprecedented diversity of disulfide-rich structural domains expressed by venomous marine snails in the superfamily Conoidea. PMID:26957604

  7. Myb-binding site regulates the expression of glucosamine-6-phosphate isomerase in Dictyostelium discoideum.

    PubMed

    Tabata, K; Matsuda, Y; Viller, E; Masamune, Y; Katayama, T; Yasukawa, H

    2001-10-01

    A homolog of the glucosamine-6-phosphate isomerase in the cellular slime mold Dictyostelium discoideum has been analyzed. The gene disruption mutant was arrested at the mound stage, demonstrating that the gene is important for development. The gene was expressed in vegetatively growing cells, silenced on starvation and expressed again in prestalk cells during the multicellular stages. The upstream region of the gene (1376 bp relative to ATG) was cloned and sequenced to study the transcription control mechanisms. Analysis of deletion mutants and a site-directed mutant indicated that the Myb-binding sequence (5'-AACTG-3') localized in the upstream region is important for gene expression. The results of gel-shift assays showed the presence of an Myb-related protein binding to the sequence at the growing phase and another protein binding to the sequence at developmental stages. PMID:11576175

  8. Extreme electric fields power catalysis in the active site of ketosteroid isomerase

    PubMed Central

    Fried, Stephen D.; Bagchi, Sayan; Boxer, Steven G.

    2015-01-01

    Enzymes use protein architecture to impose specific electrostatic fields onto their bound substrates, but the magnitude and catalytic effect of these electric fields have proven difficult to quantify with standard experimental approaches. Using vibrational Stark effect spectroscopy, we found that the active site of the enzyme ketosteroid isomerase (KSI) exerts an extremely large electric field onto the C=O chemical bond that undergoes a charge rearrangement in KSI’s rate-determining step. Moreover, we found that the magnitude of the electric field exerted by the active site strongly correlates with the enzyme’s catalytic rate enhancement, enabling us to quantify the fraction of the catalytic effect that is electrostatic in origin. The measurements described here may help explain the role of electrostatics in many other enzymes and biomolecular systems. PMID:25525245

  9. Ribose 5-phosphate isomerase inhibits LC3 processing and basal autophagy.

    PubMed

    Heintze, Jacob; Costa, Joana R; Weber, Melanie; Ketteler, Robin

    2016-09-01

    Autophagy and cellular metabolism are tightly linked processes, but how individual metabolic enzymes regulate the process of autophagy is not well understood. This study implicates ribose-5-phosphate isomerase (RPIA), a key regulator of the pentose phosphate pathway, in the control of autophagy. We used a dual gene deletion strategy, combining shRNA-mediated knockdown studies with CRISPR/Cas9 genome editing. Knockdown of RPIA by shRNA or genomic deletion by CRISPR/Cas9 genome editing, results in an increase of ATG4B-mediated LC3 processing and in the appearance of LC3-positive autophagosomes in cells. Increased LC3 processing upon knockdown of RPIA can be reversed by treatment with the antioxidant N-acetyl cysteine. The results are consistent with a model in which RPIA suppresses autophagy and LC3 processing by modulation of redox signaling. PMID:27328773

  10. Agrobacterium and biolistic transformation of onion using non-antibiotic selection marker phosphomannose isomerase.

    PubMed

    Aswath, Chenna Reddy; Mo, Sung Youn; Kim, Doo Hwan; Park, S Won

    2006-03-01

    A new selection system for onion transformation that does not require the use of antibiotics or herbicides was developed. The selection system used the Escherichia coli gene that encodes phosphomannose isomerase (pmi). Transgenic plants carrying the manA gene that codes for pmi can detoxify mannose-6-phosphate by conversion to fructose-6-phosphate, an intermediate of glycolysis, via the pmi activity. Six-week-old embryogenic callus initiated from seedling radicle was used for transformation. Transgenic plants were produced efficiently with transformation rates of 27 and 23% using Agrobacterium and biolistic system, respectively. Untransformed shoots were eliminated by a stepwise increase from 10 g l(-1) sucrose with 10 g l(-1) mannose in the first selection to only 10 g l(-1) mannose in the second selection. Integrative transformation was confirmed by PCR, RT-PCR and Southern hybridization. PMID:16211408

  11. Concerted Proton Transfer Mechanism of Clostridium thermocellum Ribose-5-phosphate Isomerase

    PubMed Central

    Wang, Jun; Yang, Weitao

    2013-01-01

    Ribose-5-phosphate isomerase (Rpi) catalyzes the interconversion of D-ribose-5-phosphate and D-ribulose-5-phosphate and plays an essential role in the pentose phosphate pathway and the Calvin cycle of photosynthesis. RpiB, one of the two isoforms of Rpi, is also a potential drug target for some pathogenic bacteria. Clostridium thermocellum ribose-5-phosphate isomerase (CtRpi), belonging to RpiB family, has recently been employed to the industrial production of rare sugars because of it fast reactions kinetics and narrow substrate specificity. It is known this enzyme adopts proton transfer mechanism. It was suggested that the deprotonated Cys65 attracts the proton at C2 of substrate to initiate the isomerization reaction and this step is the rate-limiting step. However the elaborate catalytic mechanism is still unclear. We have performed quantum mechanical/molecular mechanical simulations of this rate-limiting step of the reaction catalyzed by CtRpi with the substrate D-ribose. Our results demonstrate that the deprotonated Cys65 is not a stable reactant. Instead, our calculations revealed a concerted proton-transfer mechanism: Asp8, a highly conserved residue in the RpiB family performs as the base to abstract the proton at Cys65 and Cys65 in turn abstracts the proton of the D-ribose simultaneously. Moreover, we found Thr67 cannot catalyze the proton transfer from O2 to O1 of the D-ribose alone. Water molecule(s) may assist this proton transfer with Thr67. Our findings lead to a clear understanding of the catalysis mechanism of RpiB family and should guide the experiments to increase the catalysis efficiency. This study also highlights the importance of initial protonation states of cysteines. PMID:23875675

  12. Analysis of bacterial xylose isomerase gene diversity using gene-targeted metagenomics.

    PubMed

    Nurdiani, Dini; Ito, Michihiro; Maruyama, Toru; Terahara, Takeshi; Mori, Tetsushi; Ugawa, Shin; Takeyama, Haruko

    2015-08-01

    Bacterial xylose isomerases (XI) are promising resources for efficient biofuel production from xylose in lignocellulosic biomass. Here, we investigated xylose isomerase gene (xylA) diversity in three soil metagenomes differing in plant vegetation and geographical location, using an amplicon pyrosequencing approach and two newly-designed primer sets. A total of 158,555 reads from three metagenomic DNA replicates for each soil sample were classified into 1127 phylotypes, detected in triplicate and defined by 90% amino acid identity. The phylotype coverage was estimated to be within the range of 84.0-92.7%. The xylA gene phylotypes obtained were phylogenetically distributed across the two known xylA groups. They shared 49-100% identities with their closest-related XI sequences in GenBank. Phylotypes demonstrating <90% identity with known XIs in the database accounted for 89% of the total xylA phylotypes. The differences among xylA members and compositions within each soil sample were significantly smaller than they were between different soils based on a UniFrac distance analysis, suggesting soil-specific xylA genotypes and taxonomic compositions. The differences among xylA members and their compositions in the soil were strongly correlated with 16S rRNA variation between soil samples, also assessed by amplicon pyrosequencing. This is the first report of xylA diversity in environmental samples assessed by amplicon pyrosequencing. Our data provide information regarding xylA diversity in nature, and can be a basis for the screening of novel xylA genotypes for practical applications. PMID:25656071

  13. Mannose supplements induce embryonic lethality and blindness in phosphomannose isomerase hypomorphic mice

    PubMed Central

    Sharma, Vandana; Nayak, Jonamani; DeRossi, Charles; Charbono, Adriana; Ichikawa, Mie; Ng, Bobby G.; Grajales-Esquivel, Erika; Srivastava, Anand; Wang, Ling; He, Ping; Scott, David A.; Russell, Joseph; Contreras, Emily; Guess, Cherise M.; Krajewski, Stan; Del Rio-Tsonis, Katia; Freeze, Hudson H.

    2014-01-01

    Patients with congenital disorder of glycosylation (CDG), type Ib (MPI-CDG or CDG-Ib) have mutations in phosphomannose isomerase (MPI) that impair glycosylation and lead to stunted growth, liver dysfunction, coagulopathy, hypoglycemia, and intestinal abnormalities. Mannose supplements correct hypoglycosylation and most symptoms by providing mannose-6-P (Man-6-P) via hexokinase. We generated viable Mpi hypomorphic mice with residual enzymatic activity comparable to that of patients, but surprisingly, these mice appeared completely normal except for modest (∼15%) embryonic lethality. To overcome this lethality, pregnant dams were provided 1–2% mannose in their drinking water. However, mannose further reduced litter size and survival to weaning by 40 and 66%, respectively. Moreover, ∼50% of survivors developed eye defects beginning around midgestation. Mannose started at birth also led to eye defects but had no effect when started after eye development was complete. Man-6-P and related metabolites accumulated in the affected adult eye and in developing embryos and placentas. Our results demonstrate that disturbing mannose metabolic flux in mice, especially during embryonic development, induces a highly specific, unanticipated pathological state. It is unknown whether mannose is harmful to human fetuses during gestation; however, mothers who are at risk for having MPI-CDG children and who consume mannose during pregnancy hoping to benefit an affected fetus in utero should be cautious.—Sharma, V., Nayak, J., DeRossi, C., Charbono, A., Ichikawa, M., Ng, B. G., Grajales-Esquivel, E., Srivastava, A., Wang, L., He, P., Scott, D. A., Russell, J., Contreras, E., Guess, C. M., Krajewski, S., Del Rio-Tsonis, K., Freeze, H. H. Mannose supplements induce embryonic lethality and blindness in phosphomannose isomerase hypomorphic mice. PMID:24421398

  14. Role of conserved Met112 residue in the catalytic activity and stability of ketosteroid isomerase.

    PubMed

    Cha, Hyung Jin; Jang, Do Soo; Jeong, Jae-Hee; Hong, Bee Hak; Yun, Young Sung; Shin, Eun Ju; Choi, Kwan Yong

    2016-10-01

    Ketosteroid isomerase (3-oxosteroid Δ(5)-Δ(4)-isomerase, KSI) from Pseudomonas putida catalyzes allylic rearrangement of the 5,6-double bond of Δ(5)-3-ketosteroid to 4,5-position by stereospecific intramolecular transfer of a proton. The active site of KSI is formed by several hydrophobic residues and three catalytic residues (Tyr14, Asp38, and Asp99). In this study, we investigated the role of a hydrophobic Met112 residue near the active site in the catalysis, steroid binding, and stability of KSI. Replacing Met112 with alanine (yields M112A) or leucine (M112L) decreased the kcat by 20- and 4-fold, respectively. Compared with the wild type (WT), M112A and M112L KSIs showed increased KD values for equilenin, an intermediate analogue; these changes suggest that loss of packing at position 112 might lead to unfavorable steroid binding, thereby resulting in decreased catalytic activity. Furthermore, M112A and M112L mutations reduced melting temperature (Tm) by 6.4°C and 2.5°C, respectively. These changes suggest that favorable packing in the core is important for the maintenance of stability in KSI. The M112K mutation decreased kcat by 2000-fold, compared with the WT. In M112K KSI structure, a new salt bridge was formed between Asp38 and Lys112. This bridge could change the electrostatic potential of Asp38, and thereby contribute to the decreased catalytic activity. The M112K mutation also decreased the stability by reducing Tm by 4.1°C. Our data suggest that the Met112 residue may contribute to the catalytic activity and stability of KSI by providing favorable hydrophobic environments and compact packing in the catalytic core. PMID:27375051

  15. Adverse Outcomes Associated with Cigarette Smoke Radicals Related to Damage to Protein-disulfide Isomerase.

    PubMed

    Kenche, Harshavardhan; Ye, Zhi-Wei; Vedagiri, Kokilavani; Richards, Dylan M; Gao, Xing-Huang; Tew, Kenneth D; Townsend, Danyelle M; Blumental-Perry, Anna

    2016-02-26

    Identification of factors contributing to the development of chronic obstructive pulmonary disease (COPD) is crucial for developing new treatments. An increase in the levels of protein-disulfide isomerase (PDI), a multifaceted endoplasmic reticulum resident chaperone, has been demonstrated in human smokers, presumably as a protective adaptation to cigarette smoke (CS) exposure. We found a similar increase in the levels of PDI in the murine model of COPD. We also found abnormally high levels (4-6 times) of oxidized and sulfenilated forms of PDI in the lungs of murine smokers compared with non-smokers. PDI oxidation progressively increases with age. We begin to delineate the possible role of an increased ratio of oxidized PDI in the age-related onset of COPD by investigating the impact of exposure to CS radicals, such as acrolein (AC), hydroxyquinones (HQ), peroxynitrites (PN), and hydrogen peroxide, on their ability to induce unfolded protein response (UPR) and their effects on the structure and function of PDIs. Exposure to AC, HQ, PN, and CS resulted in cysteine and tyrosine nitrosylation leading to an altered three-dimensional structure of the PDI due to a decrease in helical content and formation of a more random coil structure, resulting in protein unfolding, inhibition of PDI reductase and isomerase activity in vitro and in vivo, and subsequent induction of endoplasmic reticulum stress response. Addition of glutathione prevented the induction of UPR, and AC and HQ induced structural changes in PDI. Exposure to PN and glutathione resulted in conjugation of PDI possibly at active site tyrosine residues. The findings presented here propose a new role of PDI in the pathogenesis of COPD and its age-dependent onset. PMID:26728460

  16. Structure and catalytic mechanism of the evolutionarily unique bacterial chalcone isomerase.

    PubMed

    Thomsen, Maren; Tuukkanen, Anne; Dickerhoff, Jonathan; Palm, Gottfried J; Kratzat, Hanna; Svergun, Dmitri I; Weisz, Klaus; Bornscheuer, Uwe T; Hinrichs, Winfried

    2015-04-01

    Flavonoids represent a large class of secondary metabolites produced by plants. These polyphenolic compounds are well known for their antioxidative abilities, are antimicrobial phytoalexins responsible for flower pigmentation to attract pollinators and, in addition to other properties, are also specific bacterial regulators governing the expression of Rhizobium genes involved in root nodulation (Firmin et al., 1986). The bacterial chalcone isomerase (CHI) from Eubacterium ramulus catalyses the first step in a flavanone-degradation pathway by ring opening of (2S)-naringenin to form naringenin chalcone. The structural biology and enzymology of plant CHIs have been well documented, whereas the existence of bacterial CHIs has only recently been elucidated. This first determination of the structure of a bacterial CHI provides detailed structural insights into the key step of the flavonoid-degradation pathway. The active site could be confirmed by co-crystallization with the substrate (2S)-naringenin. The stereochemistry of the proposed mechanism of the isomerase reaction was verified by specific (1)H/(2)H isotope exchange observed by (1)H NMR experiments and was further supported by mutagenesis studies. The active site is shielded by a flexible lid, the varying structure of which could be modelled in different states of the catalytic cycle using small-angle X-ray scattering data together with the crystallographic structures. Comparison of bacterial CHI with the plant enzyme from Medicago sativa reveals that they have unrelated folds, suggesting that the enzyme activity evolved convergently from different ancestor proteins. Despite the lack of any functional relationship, the tertiary structure of the bacterial CHI shows similarities to the ferredoxin-like fold of a chlorite dismutase and the stress-related protein SP1. PMID:25849401

  17. Protein disulfide isomerase A3-specific Th1 effector cells infiltrate colon cancer tissue of patients with circulating anti-protein disulfide isomerase A3 autoantibodies.

    PubMed

    Caorsi, Cristiana; Niccolai, Elena; Capello, Michela; Vallone, Rosario; Chattaragada, Michelle S; Alushi, Brunilda; Castiglione, Anna; Ciccone, Gianni; Mautino, Alessandro; Cassoni, Paola; De Monte, Lucia; Álvarez-Fernández, Sheila M; Amedei, Amedeo; Alessio, Massimo; Novelli, Francesco

    2016-05-01

    To investigate novel colorectal cancer (CRC)-associated antigens that could be targets of humoral or cellular responses, we analyzed the reactivity of serum from a long-surviving CRC patient (for more than 100 months of follow-up) in clinical remission, by serologic proteome analysis. Two-dimensional Western blotting (2D-WB) and mass spectrometry analysis revealed a strong reactivity of this serum against protein disulfide isomerase A3 (PDIA3). Anti-PDIA3 antibodies are not a diagnostic marker of CRC, 2D-WB and Luminex analysis revealed that they were equally present in about 10% of sera from healthy subjects and CRC patients. Kaplan-Meier analysis of survival in CRC patient cohort, after 48 months of follow-up, showed a trend of higher survival in patients with increased levels of autoantibodies to PDIA3. Therefore, the interplay between the presence of these antibodies and T-cell response was investigated. Peripheral blood T cells from CRC patients with high immunoglobulin G (IgG) reactivity to PDIA3 also secreted interferon gamma (IFN-γ) when stimulated in vitro with recombinant PDIA3, whereas those from CRC with low IgG reactivity to PDIA3 did not. PDIA3-pulsed dendritic cells efficiently induced proliferation and IFN-γ production of autologous CD4(+) and CD8(+) T cells. Finally, ex vivo analysis of tumor-infiltrating T lymphocytes from CRC patients with autoantibodies to PDIA3 revealed that PDIA3-specific Th1 effector cells accumulated in tumor tissue. These data indicate that the presence of autoantibodies to PDIA3 favors the development of an efficient and specific T-cell response against PDIA3 in CRC patients. These results may be relevant for the design of novel immunotherapeutic strategies in CRC patients. PMID:26772958

  18. Structural Analysis of the Pin1-CPEB1 interaction and its potential role in CPEB1 degradation

    PubMed Central

    Schelhorn, Constanze; Martín-Malpartida, Pau; Suñol, David; Macias, Maria J.

    2015-01-01

    The Cytoplasmic Polyadenylation Element Binding proteins are RNA binding proteins involved in the translational regulation of mRNA. During cell cycle progression, CPEB1 is labeled for degradation by phosphorylation-dependent ubiquitination by the SCFβ−TrCP ligase. The peptidyl-prolyl isomerase Pin1 plays a key role in CPEB1 degradation. Conditioned by the cell cycle stage, CPEB1 and Pin1 interactions occur in a phosphorylation-independent or -dependent manner. CPEB1 contains six potential phosphorylatable Pin1 binding sites. Using a set of biophysical techniques, we discovered that the pS210 site is unique, since it displays binding activity not only to the WW domain but also to the prolyl-isomerase domain of Pin1. The NMR structure of the Pin1 WW-CPEB1 pS210 (PDB ID: 2n1o) reveals that the pSerPro motif is bound in trans configuration through contacts with amino acids located in the first turn of the WW domain and the conserved tryptophan in the β3-strand. NMR relaxation analyses of Pin1 suggest that inter-domain flexibility is conferred by the modulation of the interaction with peptides containing the pS210 site, which is essential for degradation. PMID:26456073

  19. Pin1 down-regulates transforming growth factor-beta (TGF-beta) signaling by inducing degradation of Smad proteins.

    PubMed

    Nakano, Ayako; Koinuma, Daizo; Miyazawa, Keiji; Uchida, Takafumi; Saitoh, Masao; Kawabata, Masahiro; Hanai, Jun-ichi; Akiyama, Hirotada; Abe, Masahiro; Miyazono, Kohei; Matsumoto, Toshio; Imamura, Takeshi

    2009-03-01

    Transforming growth factor-beta (TGF-beta) is crucial in numerous cellular processes, such as proliferation, differentiation, migration, and apoptosis. TGF-beta signaling is transduced by intracellular Smad proteins that are regulated by the ubiquitin-proteasome system. Smad ubiquitin regulatory factor 2 (Smurf2) prevents TGF-beta and bone morphogenetic protein signaling by interacting with Smads and inducing their ubiquitin-mediated degradation. Here we identified Pin1, a peptidylprolyl cis-trans isomerase, as a novel protein binding Smads. Pin1 interacted with Smad2 and Smad3 but not Smad4; this interaction was enhanced by the phosphorylation of (S/T)P motifs in the Smad linker region. (S/T)P motif phosphorylation also enhanced the interaction of Smad2/3 with Smurf2. Pin1 reduced Smad2/3 protein levels in a manner dependent on its peptidyl-prolyl cis-trans isomerase activity. Knockdown of Pin1 increased the protein levels of endogenous Smad2/3. In addition, Pin1 both enhanced the interaction of Smurf2 with Smads and enhanced Smad ubiquitination. Pin1 inhibited TGF-beta-induced transcription and gene expression, suggesting that Pin1 negatively regulates TGF-beta signaling by down-regulating Smad2/3 protein levels via induction of Smurf2-mediated ubiquitin-proteasomal degradation. PMID:19122240

  20. Pin1 regulates TGF-β1 production by activated human and murine eosinophils and contributes to allergic lung fibrosis

    PubMed Central

    Shen, Zhong-Jian; Esnault, Stephane; Rosenthal, Louis A.; Szakaly, Renee J.; Sorkness, Ronald L.; Westmark, Pamela R.; Sandor, Matyas; Malter, James S.

    2008-01-01

    Eosinophilic inflammation is a cornerstone of chronic asthma that often culminates in subepithelial fibrosis with variable airway obstruction. Pulmonary eosinophils (Eos) are a predominant source of TGF-β1, which drives fibroblast proliferation and extracellular matrix deposition. We investigated the regulation of TGF-β1 and show here that the peptidyl-prolyl isomerase (PPIase) Pin1 promoted the stability of TGF-β1 mRNA in human Eos. In addition, Pin1 regulated cytokine production by both in vitro and in vivo activated human Eos. We found that Pin1 interacted with both PKC-α and protein phosphatase 2A, which together control Pin1 isomerase activity. Pharmacologic blockade of Pin1 in a rat asthma model selectively reduced eosinophilic pulmonary inflammation, TGF-β1 and collagen expression, and airway remodeling. Furthermore, chronically challenged Pin1–/– mice showed reduced peribronchiolar collagen deposition compared with wild-type controls. These data suggest that pharmacologic suppression of Pin1 may be a novel therapeutic option to prevent airway fibrosis in individuals with chronic asthma. PMID:18188456

  1. Optimization of Fermentation Medium for the Production of Glucose Isomerase Using Streptomyces sp. SB-P1

    PubMed Central

    Bhasin, Sheetal; Modi, H. A.

    2012-01-01

    The combination of medium ingredients has a profound influence on the metabolic pathways running in the microorganism which regulates the production of numerous metabolites. Glucose isomerase (GI), an enzyme with huge potential in the market, can isomerise glucose into fructose. GI is used widely for the production of High-Fructose Corn Syrup (HFCS). HFCS is used as a sweetener in food and pharmaceutical industries. Streptomyces are well-known producers of numerous enzymes including glucose isomerase. An array of 75 isolates was screened for the production of glucose isomerase. The isolate Streptomyces sp. SB-P1 was found to produce maximum amount of extracellular GI. Sucrose and raffinose among pure carbon sources and corn cob and wheat husk among crude agro residues were found to yield high enzyme titers. Potassium nitrate among pure nitrogen sources and soy residues among crude sources gave maximum production. Quantitative effect of carbon, nitrogen, and inducer on GI was also determined. Plackett-Burman design was used to study the effect of different medium ingredients. Sucrose and xylose as carbon sources and peptone and soy residues as nitrogen sources proved to be beneficial for GI production. PMID:22900192

  2. FKBP51 employs both scaffold and isomerase functions to promote NF-κB activation in melanoma

    PubMed Central

    Romano, Simona; Xiao, Yichuan; Nakaya, Mako; D'Angelillo, Anna; Chang, Mikyoung; Jin, Jin; Hausch, Felix; Masullo, Mariorosario; Feng, Xixi; Romano, Maria Fiammetta; Sun, Shao-Cong

    2015-01-01

    Melanoma is the most aggressive skin cancer; its prognosis, particularly in advanced stages, is disappointing largely due to the resistance to conventional anticancer treatments and high metastatic potential. NF-κB constitutive activation is a major factor for the apoptosis resistance of melanoma. Several studies suggest a role for the immunophilin FKBP51 in NF-κB activation, but the underlying mechanism is still unknown. In the present study, we demonstrate that FKBP51 physically interacts with IKK subunits, and facilitates IKK complex assembly. FKBP51-knockdown inhibits the binding of IKKγ to the IKK catalytic subunits, IKK-α and -β, and attenuates the IKK catalytic activity. Using FK506, an inhibitor of the FKBP51 isomerase activity, we found that the IKK-regulatory role of FKBP51 involves both its scaffold function and its isomerase activity. Moreover, FKBP51 also interacts with TRAF2, an upstream mediator of IKK activation. Interestingly, both FKBP51 TPR and PPIase domains are required for its interaction with TRAF2 and IKKγ, whereas only the TPR domain is involved in interactions with IKKα and β. Collectively, these results suggest that FKBP51 promotes NF-κB activation by serving as an IKK scaffold as well as an isomerase. Our findings have profound implications for designing novel melanoma therapies based on modulation of FKBP51. PMID:26101251

  3. The Mechanism of the Reaction Catalyzed by Uronate Isomerase Illustrates How an Isomerase May Have Evolved from a Hydrolase within the Amidohydrolase Superfamily

    SciTech Connect

    Nguyen, T.; Fedorov, A; Williams, L; Fedorov, E; Li, Y; Xu, C; Almo, S; Raushel, F

    2009-01-01

    Uronate isomerase (URI) catalyzes the reversible isomerization of d-glucuronate to d-fructuronate and of d-galacturonate to d-tagaturonate. URI is a member of the amidohydrolase superfamily (AHS), a highly divergent group of enzymes that catalyze primarily hydrolytic reactions. The chemical mechanism and active site structure of URI were investigated in an attempt to improve our understanding of how an active site template that apparently evolved to catalyze hydrolytic reactions has been reforged to catalyze an isomerization reaction. The pH-rate profiles for kcat and kcat/Km for URI from Escherichia coli are bell-shaped and indicate that one group must be unprotonated and another residue must be protonated for catalytic activity. Primary isotope effects on the kinetic constants with [2-2H]-d-glucuronate and the effects of changes in solvent viscosity are consistent with product release being the rate-limiting step. The X-ray structure of Bh0493, a URI from Bacillus halodurans, was determined in the presence of the substrate d-glucuronate. The bound complex showed that the mononuclear metal center in the active site is ligated to the C-6 carboxylate and the C-5 hydroxyl group of the substrate. This hydroxyl group is also hydrogen bonded to Asp-355 in the same orientation as the hydroxide or water is bound in those members of the AHS that catalyze hydrolytic reactions. In addition, the C-2 and C-3 hydroxyl groups of the substrate are hydrogen bonded to Arg-357 and the carbonyl group at C-1 is hydrogen bonded to Tyr-50. A chemical mechanism is proposed that utilizes a proton transfer from C-2 of d-glucuronate to C-1 that is initiated by the combined actions of Asp-355 from the end of ?-strand 8 and the C-5 hydroxyl of the substrate that is bound to the metal ion. The formation of the proposed cis-enediol intermediate is further facilitated by the shuttling of the proton between the C-2 and C-1 oxygens by the conserved Tyr-50 and/or Arg-355.

  4. Properties of d-Arabinose Isomerase Purified from Two Strains of Escherichia coli

    PubMed Central

    Boulter, James R.; Gielow, William O.

    1973-01-01

    d-Arabinose isomerase (EC 5.3.1.3) has been isolated from l-fucose-induced cultures of Escherichia coli K-12 and d-arabinose-induced cultures of E. coli B/r. Both enzymes were homogeneous in an ultracentrifuge and migrated as single bands upon disc electrophoresis in acrylamide gels. The s20,w was 14.5 × 10−13 sec for the E. coli K-12 enzyme and 14.3 × 10−13 sec for the E. coli B/r enzyme. The molecular weight, determined by high-speed sedimentation equilibrium, was 3.55 ± 0.06 × 105 for the E. coli K-12 enzyme and 3.42 ± 0.04 × 105 for the enzyme isolated from E. coli B/r. Both enzyme preparations were active wth l-fucose or d-arabinose as substrates and showed no activity on any of the other aldopentoses or aldohexoses tested. With the E. coli K-12 enzyme, the Km was 2.8 × 10−1m for d-arabinose and 4.5 × 10−2m for l-fucose; with the E. coli B/r enzyme, the Km was 1.7 × 10−1m for d-arabinose and 4.2 × 10−2m for l-fucose. Both enzymes were inhibited by several of the polyalcohols tested, ribitol, l-arabitol, and dulcitol being the strongest. Both enzymes exhibited a broad plateau of optimal catalytic activity in the alkaline range. Both enzymes were stimulated by the presence of Mn2+ or Co2+ ions, but were strongly inhibited by the presence of Cd2+ ions. Both enzymes were precipitated by antisera prepared against either enzyme preparation. The amino acid composition for both proteins has been determined; a striking similarity has been detected. Both enzymes could be dissociated, by protonation at pH 2 or by dialysis against buffer containing 8 m urea, into subunits that were homogeneous in an ultracentrifuge and migrated as single bands on disc electrophoresis in acrylamide gels containing urea. The molecular weight of the subunit, determined by high-speed sedimentation equilibrium, was 9.09 ± 0.2 × 104 for the enzyme from E. coli K-12 and 8.46 ± 0.1 × 104 for the enzyme from E. coli B/r. On the basis of biophysical studies, both

  5. Erythritol feeds the pentose phosphate pathway via three new isomerases leading to D-erythrose-4-phosphate in Brucella

    PubMed Central

    Barbier, Thibault; Collard, François; Zúñiga-Ripa, Amaia; Moriyón, Ignacio; Godard, Thibault; Becker, Judith; Wittmann, Christoph; Van Schaftingen, Emile; Letesson, Jean-Jacques

    2014-01-01

    Erythritol is an important nutrient for several α-2 Proteobacteria, including N2-fixing plant endosymbionts and Brucella, a worldwide pathogen that finds this four-carbon polyol in genital tissues. Erythritol metabolism involves phosphorylation to l-erythritol-4-phosphate by the kinase EryA and oxidation of the latter to l-3-tetrulose 4-phosphate by the dehydrogenase EryB. It is accepted that further steps involve oxidation by the putative dehydrogenase EryC and subsequent decarboxylation to yield triose-phosphates. Accordingly, growth on erythritol as the sole C source should require aldolase and fructose-1,6-bisphosphatase to produce essential hexose-6-monophosphate. However, we observed that a mutant devoid of fructose-1,6-bisphosphatases grew normally on erythritol and that EryC, which was assumed to be a dehydrogenase, actually belongs to the xylose isomerase superfamily. Moreover, we found that TpiA2 and RpiB, distant homologs of triose phosphate isomerase and ribose 5-phosphate isomerase B, were necessary, as previously shown for Rhizobium. By using purified recombinant enzymes, we demonstrated that l-3-tetrulose-4-phosphate was converted to d-erythrose 4-phosphate through three previously unknown isomerization reactions catalyzed by EryC (tetrulose-4-phosphate racemase), TpiA2 (d-3-tetrulose-4-phosphate isomerase; renamed EryH), and RpiB (d-erythrose-4-phosphate isomerase; renamed EryI), a pathway fully consistent with the isotopomer distribution of the erythrose-4-phosphate-derived amino acids phenylalanine and tyrosine obtained from bacteria grown on 13C-labeled erythritol. d-Erythrose-4-phosphate is then converted by enzymes of the pentose phosphate pathway to glyceraldehyde 3-phosphate and fructose 6-phosphate, thus bypassing fructose-1,6-bisphosphatase. This is the first description to our knowledge of a route feeding carbohydrate metabolism exclusively via d-erythrose 4-phosphate, a pathway that may provide clues to the preferential metabolism of

  6. Erythritol feeds the pentose phosphate pathway via three new isomerases leading to D-erythrose-4-phosphate in Brucella.

    PubMed

    Barbier, Thibault; Collard, François; Zúñiga-Ripa, Amaia; Moriyón, Ignacio; Godard, Thibault; Becker, Judith; Wittmann, Christoph; Van Schaftingen, Emile; Letesson, Jean-Jacques

    2014-12-16

    Erythritol is an important nutrient for several α-2 Proteobacteria, including N2-fixing plant endosymbionts and Brucella, a worldwide pathogen that finds this four-carbon polyol in genital tissues. Erythritol metabolism involves phosphorylation to L-erythritol-4-phosphate by the kinase EryA and oxidation of the latter to L-3-tetrulose 4-phosphate by the dehydrogenase EryB. It is accepted that further steps involve oxidation by the putative dehydrogenase EryC and subsequent decarboxylation to yield triose-phosphates. Accordingly, growth on erythritol as the sole C source should require aldolase and fructose-1,6-bisphosphatase to produce essential hexose-6-monophosphate. However, we observed that a mutant devoid of fructose-1,6-bisphosphatases grew normally on erythritol and that EryC, which was assumed to be a dehydrogenase, actually belongs to the xylose isomerase superfamily. Moreover, we found that TpiA2 and RpiB, distant homologs of triose phosphate isomerase and ribose 5-phosphate isomerase B, were necessary, as previously shown for Rhizobium. By using purified recombinant enzymes, we demonstrated that L-3-tetrulose-4-phosphate was converted to D-erythrose 4-phosphate through three previously unknown isomerization reactions catalyzed by EryC (tetrulose-4-phosphate racemase), TpiA2 (D-3-tetrulose-4-phosphate isomerase; renamed EryH), and RpiB (D-erythrose-4-phosphate isomerase; renamed EryI), a pathway fully consistent with the isotopomer distribution of the erythrose-4-phosphate-derived amino acids phenylalanine and tyrosine obtained from bacteria grown on (13)C-labeled erythritol. D-erythrose-4-phosphate is then converted by enzymes of the pentose phosphate pathway to glyceraldehyde 3-phosphate and fructose 6-phosphate, thus bypassing fructose-1,6-bisphosphatase. This is the first description to our knowledge of a route feeding carbohydrate metabolism exclusively via D-erythrose 4-phosphate, a pathway that may provide clues to the preferential metabolism of

  7. Heterologous expression and biochemical characterization of glucose isomerase from Thermobifida fusca.

    PubMed

    Deng, Hui; Chen, Sheng; Wu, Dan; Chen, Jian; Wu, Jing

    2014-06-01

    Glucose isomerase (GIase) catalyzes the isomerization of D-glucose to D-fructose. The GIase from Thermobifida fusca WSH03-11 was expressed in Escherichia coli BL21(DE3), and the purified enzyme took the form of a tetramer in solution and displayed a pI value of 5.05. The temperature optimum of GIase was 80 °C and its half life was about 2 h at 80 °C or 15 h at 70 °C. The pH optimum of GIase was 10 and the enzyme retained 95 % activity over the pH range of 5-10 after incubating at 4 °C for 24 h. Kinetic studies showed that the K m and K cat values of the enzyme are 197 mM and 1,688 min(-1), respectively. The maximum conversion yield of glucose (45 %, w/v) to fructose of the enzyme was 53 % at pH 7.5 and 70 °C. The present study provides the basis for the industrial application of recombinant T. fusca GIase in the production of high fructose syrup. PMID:24317483

  8. The role of phosphomannose isomerase in Leishmania mexicana glycoconjugate synthesis and virulence.

    PubMed

    Garami, A; Ilg, T

    2001-03-01

    Phosphomannose isomerase (PMI) catalyzes the reversible interconversion of fructose 6-phosphate and mannose 6-phosphate, which is the first step in the biosynthesis of activated mannose donors required for the biosynthesis of various glycoconjugates. Leishmania species synthesize copious amounts of mannose-containing glycolipids and glycoproteins, which are involved in virulence of these parasitic protozoa. To investigate the role of PMI for parasite glycoconjugate synthesis, we have cloned the PMI gene (lmexpmi) from Leishmania mexicana, generated gene deletion mutants (Delta lmexpmi), and analyzed their phenotype. Delta lmexpmi mutants lack completely the high PMI activity found in wild type parasites, but are, in contrast to fungi, able to grow in media deficient for free mannose. The mutants are unable to synthesize phosphoglycan repeats [-6-Gal beta 1-4Man alpha 1-PO(4)-] and mannose-containing glycoinositolphospholipids, and the surface expression of the glycosylphosphatidylinositol-anchored dominant surface glycoprotein leishmanolysin is strongly decreased, unless the parasite growth medium is supplemented with mannose. The Delta lmexpmi mutant is attenuated in infections of macrophages in vitro and of mice, suggesting that PMI may be a target for anti-Leishmania drug development. L. mexicana Delta lmexpmi provides the first conditional mannose-controlled system for parasite glycoconjugate assembly with potential applications for the investigation of their biosynthesis, intracellular sorting, and function. PMID:11084042

  9. Cellular and biochemical characterization of two closely related triosephosphate isomerases from Trichomonas vaginalis.

    PubMed

    Figueroa-Angulo, Elisa E; Estrella-Hernández, Priscila; Salgado-Lugo, Holjes; Ochoa-Leyva, Adrián; Gómez Puyou, Armando; Campos, Silvia S; Montero-Moran, Gabriela; Ortega-López, Jaime; Saab-Rincón, Gloria; Arroyo, Rossana; Benítez-Cardoza, Claudia G; Brieba, Luis G

    2012-11-01

    The glycolytic enzyme triosephosphate isomerase catalyses the isomerization between glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. Here we report that Trichomonas vaginalis contains 2 fully functional tpi genes. Both genes are located in separated chromosomal context with different promoter regulatory elements and encode ORFs of 254 amino acids; the only differences between them are the character of 4 amino acids located in α-helices 1, 2 and 8. Semi-quantitative RT-PCR assays showed that tpi2 transcript is approximately 3·3-fold more abundant than tpi1. Using an anti-TvTIM2 polyclonal antibody it was demonstrated that TIM proteins have a cytoplasmic localization and both enzymes are able to complement an Escherichia coli strain carrying a deletion of its endogenous tpi gene. Both TIM proteins assemble as dimers and their secondary structure assessment is essentially identical to TIM from Saccharomyces cerevisiae. The kinetic catalytic constants of the recombinant enzymes using glyceraldehyde-3-phosphate as substrate are similar to the catalytic constants of TIMs from other organisms including parasitic protozoa. As T. vaginalis depends on glycolysis for ATP production, we speculate 2 possible reasons to maintain a duplicated tpi copy on its genome: an increase in gene dosage or an early event of neofunctionalization of TIM as a moonlighting protein. PMID:22931930

  10. The Ess1 prolyl isomerase is required for growth and morphogenetic switching in Candida albicans.

    PubMed Central

    Devasahayam, Gina; Chaturvedi, Vishnu; Hanes, Steven D

    2002-01-01

    Prolyl-isomerases (PPIases) are found in all organisms and are important for the folding and activity of many proteins. Of the 13 PPIases in Saccharomyces cerevisiae only Ess1, a parvulin-class PPIase, is essential for growth. Ess1 is required to complete mitosis, and Ess1 and its mammalian homolog, Pin1, interact directly with RNA polymerase II. Here, we isolate the ESS1 gene from the pathogenic fungus Candida albicans and show that it is functionally homologous to the S. cerevisiae ESS1. We generate conditional-lethal (ts) alleles of C. albicans ESS1 and use these mutations to demonstrate that ESS1 is essential for growth in C. albicans. We also show that reducing the dosage or activity of ESS1 blocks morphogenetic switching from the yeast to the hyphal and pseudohyphal forms under certain conditions. Analysis of double mutants of ESS1 and TUP1 or CPH1, two genes known to be involved in morphogenetic switching, suggests that ESS1 functions in the same pathway as CPH1 and upstream of or in parallel to TUP1. Given that switching is important for virulence of C. albicans, inhibitors of Ess1 might be useful as antifungal agents. PMID:11805043

  11. Enhancement of protein secretion in Pichia pastoris by overexpression of protein disulfide isomerase.

    PubMed

    Inan, Mehmet; Aryasomayajula, Dinesh; Sinha, Jayanta; Meagher, Michael M

    2006-03-01

    A potential vaccine candidate, Necator americanus secretory protein (Na-ASP1), against hookworm infections, has been expressed in Pichia pastoris. Na-ASP1, a 45 kDa protein containing 20 cysteines, was directed outside the cell by fusing the protein to the preprosequence of the alpha-mating factor of Saccharomyces cerevisiae. Most of the protein produced by single copy clones was secreted outside the cell. However, increasing gene copy number of Na-ASP1 protein in P. pastoris saturated secretory capacity and therefore, decreased the amount of secreted protein in clones harboring multiple copies of Na-ASP1 gene. Overexpression of the endoplasmic reticulum (ER) resident, homologous chaperone protein, protein disulfide isomerase (PDI) was able to increase the secretion of (Na-ASP1) protein in high copy clones. The effect of PDI levels on secretion of Na-ASP1 protein was examined in clones with varying copy number of PDI gene. Increase in secreted Na-ASP1 secretion is correlated well with the PDI copy number. Increasing levels of PDI also increased overall Na-ASP1 protein production in all the clones. Nevertheless, there was still accumulation of intracellular Na-ASP1 protein in P. pastoris clones over-expressing Na-ASP1 and PDI proteins. PMID:16255058

  12. The Unfolded Protein Response and the Role of Protein Disulfide Isomerase in Neurodegeneration

    PubMed Central

    Perri, Emma R.; Thomas, Colleen J.; Parakh, Sonam; Spencer, Damian M.; Atkin, Julie D.

    2016-01-01

    The maintenance and regulation of proteostasis is a critical function for post-mitotic neurons and its dysregulation is increasingly implicated in neurodegenerative diseases. Despite having different clinical manifestations, these disorders share similar pathology; an accumulation of misfolded proteins in neurons and subsequent disruption to cellular proteostasis. The endoplasmic reticulum (ER) is an important component of proteostasis, and when the accumulation of misfolded proteins occurs within the ER, this disturbs ER homeostasis, giving rise to ER stress. This triggers the unfolded protein response (UPR), distinct signaling pathways that whilst initially protective, are pro-apoptotic if ER stress is prolonged. ER stress is increasingly implicated in neurodegenerative diseases, and emerging evidence highlights the complexity of the UPR in these disorders, with both protective and detrimental components being described. Protein Disulfide Isomerase (PDI) is an ER chaperone induced during ER stress that is responsible for the formation of disulfide bonds in proteins. Whilst initially considered to be protective, recent studies have revealed unconventional roles for PDI in neurodegenerative diseases, distinct from its normal function in the UPR and the ER, although these mechanisms remain poorly defined. However, specific aspects of PDI function may offer the potential to be exploited therapeutically in the future. This review will focus on the evidence linking ER stress and the UPR to neurodegenerative diseases, with particular emphasis on the emerging functions ascribed to PDI in these conditions. PMID:26779479

  13. Extracellular protein disulfide isomerase regulates coagulation on endothelial cells through modulation of phosphatidylserine exposure

    PubMed Central

    Popescu, Narcis I.; Lupu, Cristina

    2010-01-01

    Tissue factor (TF) is the cellular receptor for plasma protease factor VIIa (FVIIa), and the TF-FVIIa complex initiates coagulation in both hemostasis and thrombosis. Cell surface-exposed TF is mainly cryptic and requires activation to fully exhibit the procoagulant potential. Recently, the protein disulfide isomerase (PDI) has been hypothesized to regulate TF decryption through the redox switch of an exposed disulfide in TF extracellular domain. In this study, we analyzed PDI contribution to coagulation using an in vitro endothelial cell model. In this model, extracellular PDI is detected by imaging and flow cytometry. Inhibition of cell surface PDI induces a marked increase in TF procoagulant function, whereas exogenous addition of PDI inhibits TF decryption. The coagulant effects of PDI inhibition were sensitive to annexin V treatment, suggesting exposure of phosphatidylserine (PS), which was confirmed by prothrombinase assays and direct labeling. In contrast, exogenous PDI addition enhanced PS internalization. Analysis of fluorescent PS revealed that PDI affects both the apparent flippase and floppase activities on endothelial cells. In conclusion, we identified a new mechanism for PDI contribution to coagulation on endothelial cells, namely, the regulation of PS exposure, where PDI acts as a negative regulator of coagulation. PMID:20448108

  14. Protein disulfide isomerase-immunopositive inclusions in patients with amyotrophic lateral sclerosis.

    PubMed

    Honjo, Yasuyuki; Kaneko, Satoshi; Ito, Hidefumi; Horibe, Tomohisa; Nagashima, Masato; Nakamura, Masataka; Fujita, Kengo; Takahashi, Ryosuke; Kusaka, Hirofumi; Kawakami, Koji

    2011-11-01

    The major pathological hallmarks of amyotrophic lateral sclerosis (ALS) are neuronal cytoplasmic inclusions (NCIs) and swollen neurites. Superoxide dismutase (SOD)-1-immunopositive NCIs are observed in patients with familial ALS (FALS), and TAR DNA-binding protein 43kDa (TDP-43)-immunopositive NCIs are found in patients with sporadic ALS (SALS). Protein disulfide isomerase (PDI) is a member of the thioredoxin superfamily and is believed to accelerate the folding of disulfide-bonded proteins by catalyzing the disulfide interchange reaction, which is the rate-limiting step during protein folding in the luminal space of the endoplasmic reticulum. Post mortem spinal cord specimens from five patients with SALS and one with FALS (I113T), and five normal controls were utilized in this immunohistochemical study. We found PDI-immunopositive swollen neurites and NCIs in the patients with ALS. Furthermore, PDI was colocalized with TDP-43 and SOD1 in NCIs. The accumulation of misfolding proteins may disturb axon transport and make swollen neurites. As the motor neuron is the longest cell in the nervous system, the motor system may selectively be disturbed. In conclusion, we assume that PDI is S-nitrosylated in the affected neurons, which inhibits its enzymatic activity and thus allows protein misfolding to occur in ALS. PMID:21745122

  15. Understanding protein lids: structural analysis of active hinge mutants in triosephosphate isomerase.

    PubMed

    Kursula, I; Salin, M; Sun, J; Norledge, B V; Haapalainen, A M; Sampson, N S; Wierenga, R K

    2004-04-01

    The conformational switch from open to closed of the flexible loop 6 of triosephosphate isomerase (TIM) is essential for the catalytic properties of TIM. Using a directed evolution approach, active variants of chicken TIM with a mutated C-terminal hinge tripeptide of loop 6 have been generated (Sun,J. and Sampson,N.S., Biochemistry, 1999, 38, 11474-11481). In chicken TIM, the wild-type C-terminal hinge tripeptide is KTA. Detailed enzymological characterization of six variants showed that some of these (LWA, NPN, YSL, KTK) have decreased catalytic efficiency, whereas others (KVA, NSS) are essentially identical with wild-type. The structural characterization of these six variants is reported. No significant structural differences compared with the wild-type are found for KVA, NSS and LWA, but substantial structural adaptations are seen for NPN, YSL and KTK. These structural differences can be understood from the buried position of the alanine side chain in the C-hinge position 3 in the open conformation of wild-type loop 6. Replacement of this alanine with a bulky side chain causes the closed conformation to be favored, which correlates with the decreased catalytic efficiency of these variants. The structural context of loop 6 and loop 7 and their sequence conservation in 133 wild-type sequences is also discussed. PMID:15166315

  16. Reactivation of triosephosphate isomerase from three trypanosomatids and human: effect of suramin.

    PubMed Central

    Gao, X G; Garza-Ramos, G; Saavedra-Lira, E; Cabrera, N; De Gómez-Puyou, M T; Perez-Montfort, R; Gómez-Puyou, A

    1998-01-01

    The reactivation of the homodimeric triosephosphate isomerases (TIMs) from Trypanosoma brucei, T. cruzi, Leishmania mexicana and humans was determined after their denaturation with guanidine hydrochloride. In the range of 2-32 microg of T. brucei TIM per ml and 0.2-5 microg of the other enzymes per ml, the rate and extent of TIM reactivation depended on protein concentration, indicating that at these protein concentrations, the rate-limiting step of reactivation is monomer association and not monomer folding. The rate of monomer association was more than one order of magnitude lower in the T. brucei enzyme than in the other three enzymes. Suramin is a drug of choice in the treatment of sleeping sickness, but its mechanism of action is not known. At micromolar concentrations, Suramin inhibited the reactivation of the four enzymes, but the extent of inhibition by Suramin decreased with increasing protein concentration as consequence of a diminution of the life time of the folded monomer. Since the life time of the monomer of T. brucei TIM is longer than that of the other enzymes, Suramin is a more effective inhibitor of the reactivation of TIM from T. brucei, particularly at monomer concentrations above 1 microg of protein per ml (monomer concentration approx. 37 nM). Compounds that are structurally related to Suramin also inhibit TIM reactivation; their effect was about five times more pronounced in the enzyme from T. brucei than in human TIM. PMID:9576855

  17. Lung cancer chemotherapy agents increase procoagulant activity via protein disulfide isomerase-dependent tissue factor decryption.

    PubMed

    Lysov, Zakhar; Swystun, Laura L; Kuruvilla, Sara; Arnold, Andrew; Liaw, Patricia C

    2015-01-01

    Lung cancer patients undergoing chemotherapy have an elevated risk for thrombosis. However, the mechanisms by which chemotherapy agents increase the risk for thrombosis remains unclear. The aim of this study was to determine the mechanism(s) by which lung cancer chemotherapy agents cisplatin, carboplatin, gemcitabine, and paclitaxel elicit increased tissue factor activity on endothelial cells, A549 cells, and monocytes. Tissue factor activity, tissue factor antigen, and phosphatidylserine exposure were measured on chemotherapy-treated human umbilical vein endothelial cells (HUVEC), A549 cells, and monocytes. Cell surface protein disulfide isomerase (PDI) and cell surface free thiol levels were measured on HUVEC and A549 non-small cell lung carcinoma cells. Treatment of HUVECs, A549 cells, and monocytes with lung cancer chemotherapy significantly increased cell surface tissue factor activity. However, elevated tissue factor antigen levels were observed only on cisplatin-treated and gemcitabine-treated monocytes. Cell surface levels of phosphatidylserine were increased on HUVEC and monocytes treated with cisplatin/gemcitabine combination therapy. Chemotherapy also resulted in increased cell surface levels of PDI and reduced cell surface free thiol levels. Glutathione treatment and PDI inhibition, but not phosphatidylserine inhibition, attenuated tissue factor activity. Furthermore, increased tissue factor activity was reversed by reducing cysteines with dithiothreitol. These studies are the first to demonstrate that lung cancer chemotherapy agents increase procoagulant activity on endothelial cells and A549 cells by tissue factor decryption through a disulfide bond formation in a PDI-dependent mechanism. PMID:24911456

  18. Gene sequencing, modelling and immunolocalization of the protein disulfide isomerase from Plasmodium chabaudi.

    PubMed

    Novo, Carlos; Martins, Tiago M; Prata, Sofia; Lopes, Angela; Armada, Ana

    2009-11-01

    Malaria remains one of the major human parasitic diseases, particularly in subtropical regions. Most of the fatal cases are caused by Plasmodium falciparum. The rodent parasite Plasmodium chabaudi has been the model of choice in research due to its similarities to human malaria, including developmental cycle, preferential invasion of mature erythrocytes, synchrony of asexual development, antigenic variation, gene sinteny as well as similar resistance mechanisms. Protein disulfide isomerase (PDI) is an essential catalyst of the endoplasmic reticulum in different biological systems with folding and chaperone activities. Most of the proteins exported by parasites have to pass through the endoplasmic reticulum before reaching their final destination and their correct folding is critical for parasite survival. PDI constitutes a potential target for the development of alternative therapy strategies based on the inhibition of folding and chaperoning of exported proteins. We here describe the sequencing of the gene coding for the PDI from P. chabaudi and analyse the relationship to its counterpart enzymes, particularly with the PDI from other Plasmodium species. The model constructed, based on the recent model deduced from the crystallographic structure 2B5E, was compared with the previous theoretical model for the whole PDI molecule constructed by threading. A recombinant PDI from P. chabaudi was also produced and used as an antigen for monoclonal antibody production for application in PDI immunolocalization. PMID:19615402

  19. GnRH immunization alters the expression and distribution of protein disulfide isomerases in the epididymis.

    PubMed

    Schorr-Lenz, A M; Alves, J; Henckes, N A C; Seibel, P M; Benham, A M; Bustamante-Filho, I C

    2016-09-01

    Hypogonadism is defined as the inadequate gonadal production of testosterone. Low serum testosterone leads to infertility by impairing spermatogenesis and reducing sperm count, however, the impact of hypogonadism in epididymal sperm maturation is poorly understood. From the testis, spermatozoa are transported into the epididymis where they find a specific microenvironment composed of a complex mixture of proteins that facilitate sperm storage and maturation. Inside the epididymal ductule, spermatozoa undergo several changes, resulting in their becoming capable of fertilizing eggs. Protein disulfide isomerases (PDIs) are known to participate in the folding and assembly of secreted proteins in the endoplasmic reticulum. However, little is known about the control and function of PDIs in the testis and epididymis, particularly during male development. The aim of this work was to compare the expression and distribution of PDI and PDIA3 (ERp57) in the testis and epididymis of healthy and GnRH-immunized boars. We detected higher amounts of PDIA3 and PDI in sperm preparations and fluid from the proximal regions of the epididymis of healthy boars. However, we observed an increase in PDIA3 expression in the testis and cauda epididymis in the immunocastrated group. GnRH-immunized boars showed a marked increase in PDI content in cauda spermatozoa and fluid, indicating a possible endocrine dysregulation of PDI. The results of our study suggest that PDIs are associated with epididymal sperm maturation and may be attractive candidates for monitoring male fertility. PMID:27323298

  20. Cryptococcal phosphoglucose isomerase is required for virulence factor production, cell wall integrity and stress resistance.

    PubMed

    Zhang, Ping; Wei, Dongsheng; Li, Zhongming; Sun, Zhixiong; Pan, Jiao; Zhu, Xudong

    2015-11-01

    Regulation of virulence factor production in the pathogen Cryptococcus neoformans remains to be fully illustrated. We present here a finding that a gene, encoding the glycolysis enzyme phosphoglucose isomerase (Pgi1), is critical for the biosynthesis of melanin and capsule, cell wall integrity and resistance to stress conditions. A leaky mutant of the yeast, LZM19, resulted from an insertion of T-DNA in the PGI1 promoter region, expressed PGI1 at a level only 1.9% of the wild type. LZM19 could synthesize the pigment melanin in the presence of 2% glucose, suggesting a status of LAC1 derepression. Phenotypically, capsule biosynthesis in LZM19 was remarkably reduced. Integrity of the cell wall and plasma membrane of LZM19 were impaired based on its sensitivity to Congo red and SDS. Also, LZM19 exhibited hypersensitivity to osmotic stress generated by 2 M NaCl or 1 M KCl, indicating possible impairment in the HOG signaling pathway. Furthermore, LZM19 failed to utilize mannose and fructose, suggesting a possible involvement of Pgi1 in the breakdown of these two sugars. Our results revealed a crucial role of PGI1 in coordination of the production of virulence factors, cell wall integrity and stress response in C. neoformans. PMID:26271120

  1. Protein disulphide isomerase as a target for nanoparticle-mediated sensitisation of cancer cells to radiation.

    PubMed

    Taggart, L E; McMahon, S J; Butterworth, K T; Currell, F J; Schettino, G; Prise, K M

    2016-05-27

    Radiation resistance and toxicity in normal tissues are limiting factors in the efficacy of radiotherapy. Gold nanoparticles (GNPs) have been shown to be effective at enhancing radiation-induced cell death, and were initially proposed to physically enhance the radiation dose deposited. However, biological responses of GNP radiosensitization based on physical assumptions alone are not predictive of radiosensitisation and therefore there is a fundamental research need to determine biological mechanisms of response to GNPs alone and in combination with ionising radiation. This study aimed to identify novel mechanisms of cancer cell radiosensitisation through the use of GNPs, focusing on their ability to induce cellular oxidative stress and disrupt mitochondrial function. Using N-acetyl-cysteine, we found mitochondrial oxidation to be a key event prior to radiation for the radiosensitisation of cancer cells and suggests the overall cellular effects of GNP radiosensitisation are a result of their interaction with protein disulphide isomerase (PDI). This investigation identifies PDI and mitochondrial oxidation as novel targets for radiosensitisation. PMID:27080849

  2. Development of a recombinant d-mannose isomerase and its characterizations for d-mannose synthesis.

    PubMed

    Hu, Xing; Zhang, Peng; Miao, Ming; Zhang, Tao; Jiang, Bo

    2016-08-01

    d-Mannose isomerase (MIase) catalyzes the conversion of d-fructose to d-mannose. In this study, the MIase encoding gene (yihS) from Escherichia coli BL21 contains an ORF of 1242bp, was cloned and expressed in Bacillus subtilis WB800. This heterologous expression resulted in a hexamer with a molecular weight of 274.5kDa and Tm of 61.4°C. Efficient MIase secretory expression by the robust recombinant B. subtilis was achieved with activity of 51.2U/ml (d-mannose forming). Its optimal temperature and pH were 45.0°C and 7.0, respectively. Using d-fructose as the substrate, Km, kcat and catalytic efficiency value of kinetic reaction were 203.7±6.7mM, 27.7±0.7s(-1) and 136.0±2.9M(-1)s(-1), respectively. The production of d-mannose reached about 150g/l with approximately 25% turnover yield under the optimum conditions. These results demonstrated that B. subtilis was a promising candidate of MIase expression system for d-mannose production. PMID:27138861

  3. Functional Role of the Disulfide Isomerase ERp57 in Axonal Regeneration.

    PubMed

    Castillo, Valentina; Oñate, Maritza; Woehlbier, Ute; Rozas, Pablo; Andreu, Catherine; Medinas, Danilo; Valdés, Pamela; Osorio, Fabiola; Mercado, Gabriela; Vidal, René L; Kerr, Bredford; Court, Felipe A; Hetz, Claudio

    2015-01-01

    ERp57 (also known as grp58 and PDIA3) is a protein disulfide isomerase that catalyzes disulfide bonds formation of glycoproteins as part of the calnexin and calreticulin cycle. ERp57 is markedly upregulated in most common neurodegenerative diseases downstream of the endoplasmic reticulum (ER) stress response. Despite accumulating correlative evidence supporting a neuroprotective role of ERp57, the contribution of this foldase to the physiology of the nervous system remains unknown. Here we developed a transgenic mouse model that overexpresses ERp57 in the nervous system under the control of the prion promoter. We analyzed the susceptibility of ERp57 transgenic mice to undergo neurodegeneration. Unexpectedly, ERp57 overexpression did not affect dopaminergic neuron loss and striatal denervation after injection of a Parkinson's disease-inducing neurotoxin. In sharp contrast, ERp57 transgenic animals presented enhanced locomotor recovery after mechanical injury to the sciatic nerve. These protective effects were associated with enhanced myelin removal, macrophage infiltration and axonal regeneration. Our results suggest that ERp57 specifically contributes to peripheral nerve regeneration, whereas its activity is dispensable for the survival of a specific neuronal population of the central nervous system. These results demonstrate for the first time a functional role of a component of the ER proteostasis network in peripheral nerve regeneration. PMID:26361352

  4. Phosphomannose Isomerase Inhibitors Improve N-Glycosylation in Selected Phosphomannomutase-deficient Fibroblasts*

    PubMed Central

    Sharma, Vandana; Ichikawa, Mie; He, Ping; Bravo, Yalda; Dahl, Russell; Ng, Bobby G.; Cosford, Nicholas D. P.; Freeze, Hudson H.

    2011-01-01

    Congenital disorders of glycosylation (CDG) are rare genetic disorders due to impaired glycosylation. The patients with subtypes CDG-Ia and CDG-Ib have mutations in the genes encoding phosphomannomutase 2 (PMM2) and phosphomannose isomerase (MPI or PMI), respectively. PMM2 (mannose 6-phosphate → mannose 1-phosphate) and MPI (mannose 6-phosphate ⇔ fructose 6-phosphate) deficiencies reduce the metabolic flux of mannose 6-phosphate (Man-6-P) into glycosylation, resulting in unoccupied N-glycosylation sites. Both PMM2 and MPI compete for the same substrate, Man-6-P. Daily mannose doses reverse most of the symptoms of MPI-deficient CDG-Ib patients. However, CDG-Ia patients do not benefit from mannose supplementation because >95% Man-6-P is catabolized by MPI. We hypothesized that inhibiting MPI enzymatic activity would provide more Man-6-P for glycosylation and possibly benefit CDG-Ia patients with residual PMM2 activity. Here we show that MLS0315771, a potent MPI inhibitor from the benzoisothiazolone series, diverts Man-6-P toward glycosylation in various cell lines including fibroblasts from CDG-Ia patients and improves N-glycosylation. Finally, we show that MLS0315771 increases mannose metabolic flux toward glycosylation in zebrafish embryos. PMID:21949237

  5. Arabidopsis cotyledon-specific chloroplast biogenesis factor CYO1 is a protein disulfide isomerase.

    PubMed

    Shimada, Hiroshi; Mochizuki, Mariko; Ogura, Kan; Froehlich, John E; Osteryoung, Katherine W; Shirano, Yumiko; Shibata, Daisuke; Masuda, Shinji; Mori, Kazuki; Takamiya, Ken-Ichiro

    2007-10-01

    Chloroplast development in cotyledons differs in a number of ways from that in true leaves, but the cotyledon-specific program of chloroplast biogenesis has not been clarified. The cyo1 mutant in Arabidopsis thaliana has albino cotyledons but normal green true leaves. Chloroplasts develop abnormally in cyo1 mutant plants grown in the light, but etioplasts are normal in mutants grown in the dark. We isolated CYO1 by T-DNA tagging and verified that the mutant allele was responsible for the albino cotyledon phenotype by complementation. CYO1 has a C(4)-type zinc finger domain similar to that of Escherichia coli DnaJ. CYO1 is expressed mainly in young plants under light conditions, and the CYO1 protein localizes to the thylakoid membrane in chloroplasts. Transcription of nuclear photosynthetic genes is generally unaffected by the cyo1 mutation, but the level of photosynthetic proteins is decreased in cyo1 mutants. Recombinant CYO1 accelerates disulfide bond reduction in the model substrate insulin and renatures RNase A, indicating that CYO1 has protein disulfide isomerase activity. These results suggest that CYO1 has a chaperone-like activity required for thylakoid biogenesis in cotyledons. PMID:17921316

  6. Mass spectrometric analysis of dimer-disrupting mutations in Plasmodium triosephosphate isomerase.

    PubMed

    Bandyopadhyay, Debarati; Prakash, Sunita; Gupta, Kallol; Balaram, Padmanabhan

    2016-05-01

    Electrospray ionization mass spectrometry (ESI MS) under nanospray conditions has been used to examine the effects of mutation at two key dimer interface residues, Gln (Q) 64 and Thr (T) 75, in Plasmodium falciparum triosephosphate isomerase. Both residues participate in an intricate network of intra- and intersubunit hydrogen bonds. The gas phase distributions of dimeric and monomeric protein species have been examined for the wild type enzyme (TWT) and three mutants, Q64N, Q64E, and T75S, under a wide range of collision energies (40-160 eV). The results established the order of dimer stability as TWT > T75S > Q64E ∼ Q64N. The mutational effects on dimer stability are in good agreement with the previously reported estimates, based on the concentration dependence of enzyme activity. Additional experiments in solution, using inhibition of activity by a synthetic dimer interface peptide, further support the broad agreement between gas phase and solution studies. PMID:26919806

  7. Hyphal tip extension in Aspergillus nidulans requires the manA gene, which encodes phosphomannose isomerase.

    PubMed Central

    Smith, D J; Payton, M A

    1994-01-01

    A strain of Aspergillus nidulans carrying a temperature-sensitive mutation in the manA gene produces cell walls depleted of D-mannose and forms hyphal tip balloons at the restrictive temperature (B.P. Valentine and B.W. Bainbridge, J. Gen. Microbiol. 109:155-168, 1978). We have isolated and characterized the manA gene and physically located it between 3.5 and 5.5 kb centromere distal of the riboB locus on chromosome VIII. The manA gene contains four introns and encodes a 50.6-kDa protein which has significant sequence identity to type I phosphomannose isomerase proteins from other eukaryotes. We have constructed by integrative transformation a null mutation in the manA gene which can only be maintained in a heterokaryotic strain with wild-type manA+ nuclei. Thus, a manA null mutation is lethal in A. nidulans. The phenotype of the mutation was analyzed in germinating conidia. Such conidia are able to commence germination but swell abnormally, sometimes producing a misshapen germ tube, before growth ceases. The reason for the lethality is probably the lack of synthesis of mannose-containing cell wall polymers that must be required for normal cell wall development in growing hyphae. Images PMID:8065336

  8. Quantitative dissection of hydrogen bond-mediated proton transfer in the ketosteroid isomerase active site

    PubMed Central

    Sigala, Paul A.; Fafarman, Aaron T.; Schwans, Jason P.; Fried, Stephen D.; Fenn, Timothy D.; Caaveiro, Jose M. M.; Pybus, Brandon; Ringe, Dagmar; Petsko, Gregory A.; Boxer, Steven G.; Herschlag, Daniel

    2013-01-01

    Hydrogen bond networks are key elements of protein structure and function but have been challenging to study within the complex protein environment. We have carried out in-depth interrogations of the proton transfer equilibrium within a hydrogen bond network formed to bound phenols in the active site of ketosteroid isomerase. We systematically varied the proton affinity of the phenol using differing electron-withdrawing substituents and incorporated site-specific NMR and IR probes to quantitatively map the proton and charge rearrangements within the network that accompany incremental increases in phenol proton affinity. The observed ionization changes were accurately described by a simple equilibrium proton transfer model that strongly suggests the intrinsic proton affinity of one of the Tyr residues in the network, Tyr16, does not remain constant but rather systematically increases due to weakening of the phenol–Tyr16 anion hydrogen bond with increasing phenol proton affinity. Using vibrational Stark spectroscopy, we quantified the electrostatic field changes within the surrounding active site that accompany these rearrangements within the network. We were able to model these changes accurately using continuum electrostatic calculations, suggesting a high degree of conformational restriction within the protein matrix. Our study affords direct insight into the physical and energetic properties of a hydrogen bond network within a protein interior and provides an example of a highly controlled system with minimal conformational rearrangements in which the observed physical changes can be accurately modeled by theoretical calculations. PMID:23798390

  9. The Catalytic Activity of Protein-Disulfide Isomerase Requires a Conformationally Flexible Molecule

    SciTech Connect

    Tian, G.; Kober, F; Lewandrowski, U; Sickmann, A; Lennarz, W; Schindelin, H

    2008-01-01

    Protein-disulfide isomerase (PDI) catalyzes the formation of the correct pattern of disulfide bonds in secretory proteins. A low resolution crystal structure of yeast PDI described here reveals large scale conformational changes compared with the initially reported structure, indicating that PDI is a highly flexible molecule with its catalytic domains, a and a?, representing two mobile arms connected to a more rigid core composed of the b and b? domains. Limited proteolysis revealed that the linker between the a domain and the core is more susceptible to degradation than that connecting the a? domain to the core. By restricting the two arms with inter-domain disulfide bonds, the molecular flexibility of PDI, especially that of its a domain, was demonstrated to be essential for the enzymatic activity in vitro and in vivo. The crystal structure also featured a PDI dimer, and a propensity to dimerize in solution and in the ER was confirmed by cross-linking experiments and the split green fluorescent protein system. Although sedimentation studies suggested that the self-association of PDI is weak, we hypothesize that PDI exists as an interconvertible mixture of monomers and dimers in the endoplasmic reticulum due to its high abundance in this compartment.

  10. Protein disulphide isomerase as a target for nanoparticle-mediated sensitisation of cancer cells to radiation

    NASA Astrophysics Data System (ADS)

    Taggart, L. E.; McMahon, S. J.; Butterworth, K. T.; Currell, F. J.; Schettino, G.; Prise, K. M.

    2016-05-01

    Radiation resistance and toxicity in normal tissues are limiting factors in the efficacy of radiotherapy. Gold nanoparticles (GNPs) have been shown to be effective at enhancing radiation-induced cell death, and were initially proposed to physically enhance the radiation dose deposited. However, biological responses of GNP radiosensitization based on physical assumptions alone are not predictive of radiosensitisation and therefore there is a fundamental research need to determine biological mechanisms of response to GNPs alone and in combination with ionising radiation. This study aimed to identify novel mechanisms of cancer cell radiosensitisation through the use of GNPs, focusing on their ability to induce cellular oxidative stress and disrupt mitochondrial function. Using N-acetyl-cysteine, we found mitochondrial oxidation to be a key event prior to radiation for the radiosensitisation of cancer cells and suggests the overall cellular effects of GNP radiosensitisation are a result of their interaction with protein disulphide isomerase (PDI). This investigation identifies PDI and mitochondrial oxidation as novel targets for radiosensitisation.