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Sample records for cis-trans isomerase involved

  1. Peptidyl-prolyl cis-trans isomerases: structure and functions.

    PubMed

    Pliyev, B K; Gurvits, B Y

    1999-07-01

    Peptidyl-prolyl cis-trans isomerases (PPI) catalyze cis-trans isomerization of imide bonds in peptides and proteins. This review summarizes the literature on the structure and functions of PPIs, their involvement in protein folding, and organization of PPI-containing receptors and membrane channels. A possible role of several PPIs in distant interactions between cells is discussed.

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

    PubMed Central

    Moro, A; Ruiz-Cabello, F; Fernández-Cano, A; Stock, R P; González, A

    1995-01-01

    Macrophage infectivity potentiators are membrane proteins described as virulence factors in bacterial intracellular parasites, such as Legionella and Chlamydia. These factors share amino acid homology to eukaryotic peptidyl-prolyl cis-trans isomerases that are inhibited by FK506, an inhibitor of signal transduction in mammalian cells with potent immunosuppressor activity. We report here the characterization of a protein released into the culture medium by the infective stage of the protozoan intracellular parasite Trypanosoma cruzi. The protein possesses a peptidyl-prolyl cis-trans isomerase activity that is inhibited by FK506 and its non-immunosuppressing derivative L-685,818. The corresponding gene presents sequence homology with bacterial macrophage infectivity potentiators. The addition of the protein, produced heterologously in Escherichia coli, to cultures of trypomastigotes and simian epithelial or HeLa cells enhances invasion of the mammalian cells by the parasites. Antibodies raised in mice against the T.cruzi isomerase greatly reduce infectivity. A similar reduction of infectivity is obtained by addition to the cultures of FK506 and L-685,818. We concluded that the T.cruzi isomerase is involved in cell invasion. Images PMID:7540135

  3. Peptidyl-prolyl cis-trans isomerase of Bacillus subtilis: identification of residues involved in cyclosporin A affinity and catalytic efficiency.

    PubMed

    Göthel, S F; Herrler, M; Marahiel, M A

    1996-03-19

    The 17-kDa peptidyl-prolyl cis-trans-isomerase from Bacillus subtilis (PPiB) is a member of the cyclophilin family and shows strong homology to PPIases of eukaryotic origin (40%) and less identify to PPIase sequences of Gram-negative bacteria (27-32%). Although the majority of residues that form the PPIase active site are highly conserved, three residues, V52, H90, and H109 in the sequence of the B.subtilis PPIase, were found to differ from the sequences found in human (hCyP) and Escherichia coli (eCyP). Also, the binding affinity of cyclosporin A (CsA) to the different PPIases varies in IC(50) values from 6 nM for human PPIase hCyPA and 84 nM for the human hCyPB to over 120 nM for B. subtilis and 3000 nM for E. coli. In addition, a variety of k(cat)/K(m) values, ranging from 1.1 mM(-1) s(-1) for the B. subtilis PPIase to over 10 mM(-1) s(-1) for human and 13 mM(-1) s(-1) for E. coli, were detected using the common substrate suc-Ala-Ala-Pro-Phe-pNA. Through site-specific mutagenesis we demonstrate that the differences in the three mentioned residues are mainly responsible for the variations in IC(50) and k(cat)/K(m) values. Replacement of H90 to N90, or H109 to W109, resembling the amino acid sequence of human hCyPA, resulted in more efficient CsA binding (IC(50) value for H90N, 60 nM, and for H109W, 95 nm), whereas replacement of H90 to R90, or H109 to F109, resembling the amino acid sequence of E. coli eCyP, resulted in less efficient CsA binding (IC(50) value for H90R, 2000nM, and for H109F, 5000 nM). In addition to lower CsA affinity, mutant protein H109F shows a k(cat)/K(m) value of 10.5 mM(-1) s(-1), comparably high to that of the wild-type E. coli protein. In contrast, other mutants like C57F, H90N, H90R, and H109W do not differ significantly in k(cat)/K(m) values from wild-type PPiB. Replacement of V52 to M52, which is conserved in E. coli and all known eukaryotic PPIases, does not show any effect in CsA binding affinity (IC(50) value for V52M, 120 nM), but

  4. 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

  5. Cyclophilin A (CyPA) Induces Chemotaxis Independent of Its Peptidylprolyl Cis-Trans Isomerase Activity

    PubMed Central

    Song, Fei; Zhang, Xin; Ren, Xiao-Bai; Zhu, Ping; Xu, Jing; Wang, Li; Li, Yi-Fei; Zhong, Nan; Ru, Qiang; Zhang, Da-Wei; Jiang, Jian-Li; Xia, Bin; Chen, Zhi-Nan

    2011-01-01

    Cyclophilin A (CyPA) is a ubiquitously distributed peptidylprolyl cis-trans isomerase (PPIase) that possesses diverse biological functions. Extracellular CyPA is a potent chemokine, which can directly induce leukocyte chemotaxis and contribute to the pathogenesis of inflammation-mediated diseases. Although it has been identified that the chemotaxis activity of CyPA is mediated through its cell surface signaling receptor CD147, the role of CyPA PPIase activity in this process is disputable, and the underlying molecular mechanism is still poorly understood. In this study, we present the first evidence that CyPA induces leukocyte chemotaxis through a direct binding with the ectodomain of CD147 (CD147ECT), independent of its PPIase activity. Although NMR study indicates that the CD147ECT binding site on CyPA overlaps with the PPIase active site, the PPIase inactive mutant CyPAR55A exhibits similar CD147ECT binding ability and chemotaxis activity to those of CyPAWT. Furthermore, we have identified three key residues of CyPA involved in CD147ECT binding and found that mutations H70A, T107A, and R69A result in similar levels of reduction in CD147ECT binding ability and chemotaxis activity for CyPA, without affecting the PPIase activity. Our findings indicate that there exists a novel mechanism for CyPA to regulate cellular signaling processes, shedding new light on its applications in drug development and providing a new targeting site for drug design. PMID:21245143

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

    PubMed Central

    Zarnt, T; Lang, K; Burtscher, H; Fischer, G

    1995-01-01

    Free in solution, the immunosuppressive compounds cyclosporin A (CsA), FK506, ascomycin and rapamycin are present in many solvents in various slowly interconverting conformations. Together with their cellular receptor proteins, cyclophilin (CyP) and FK506-binding protein (FKBP), however, these inhibitors have been shown to have a homogeneous conformation. The existence of a slow cis-trans interconversion of an imidic bond in the inhibitor molecule during the course of the formation of the CsA-CyP18cy complex (where CyP18cy is human 18 kDa cytosolic CyP) prompted us to investigate the reaction of the peptidomacrolides FK506, ascomycin and rapamycin with two specific binding-proteins in more detail. Since formation of the FK506-FKBP complex results in the inhibition of the peptidylprolyl cis-trans-isomerase activity of the binding protein, we used the enzyme's decrease in enzymic activity to monitor binding of the inhibitors to their enzyme targets. For FK506, the kinetics of inhibition of human 12 kDa cytosolic FKBP (FKBP12cy) were clearly dependent on time. Subsequent to a rapid inactivation reaction, not resolved in its kinetics due to manual mixing, a slow dominant first-order inactivation process with a relaxation time of 1163 s at 10 degrees C was observed. Concomitantly the Ki value of the slow phase dropped 2.6-fold within the first 60 min of incubation. Using the FKBP12cy homologue 25 kDa membrane FKBP (FKBP25mem), a bacterial peptidylprolyl cis-trans-isomerase, the rate and amplitudes of the inhibition reactions were very similar to FKBP12cy. On the other hand, the kinetics and amplitudes of the inhibition of FKBP12cy varied significantly if rapamycin was used as an inhibitor instead of FK 506. Owing to reduced conformation transition in rapamycin upon binding to FKBP12cy, the slow phase during inhibition was significantly decreased in amplitude. A likely reason for this became apparent when the activation-enthalpy and the pH-dependence of the rate

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

    PubMed

    Zarnt, T; Lang, K; Burtscher, H; Fischer, G

    1995-01-01

    Free in solution, the immunosuppressive compounds cyclosporin A (CsA), FK506, ascomycin and rapamycin are present in many solvents in various slowly interconverting conformations. Together with their cellular receptor proteins, cyclophilin (CyP) and FK506-binding protein (FKBP), however, these inhibitors have been shown to have a homogeneous conformation. The existence of a slow cis-trans interconversion of an imidic bond in the inhibitor molecule during the course of the formation of the CsA-CyP18cy complex (where CyP18cy is human 18 kDa cytosolic CyP) prompted us to investigate the reaction of the peptidomacrolides FK506, ascomycin and rapamycin with two specific binding-proteins in more detail. Since formation of the FK506-FKBP complex results in the inhibition of the peptidylprolyl cis-trans-isomerase activity of the binding protein, we used the enzyme's decrease in enzymic activity to monitor binding of the inhibitors to their enzyme targets. For FK506, the kinetics of inhibition of human 12 kDa cytosolic FKBP (FKBP12cy) were clearly dependent on time. Subsequent to a rapid inactivation reaction, not resolved in its kinetics due to manual mixing, a slow dominant first-order inactivation process with a relaxation time of 1163 s at 10 degrees C was observed. Concomitantly the Ki value of the slow phase dropped 2.6-fold within the first 60 min of incubation. Using the FKBP12cy homologue 25 kDa membrane FKBP (FKBP25mem), a bacterial peptidylprolyl cis-trans-isomerase, the rate and amplitudes of the inhibition reactions were very similar to FKBP12cy. On the other hand, the kinetics and amplitudes of the inhibition of FKBP12cy varied significantly if rapamycin was used as an inhibitor instead of FK 506. Owing to reduced conformation transition in rapamycin upon binding to FKBP12cy, the slow phase during inhibition was significantly decreased in amplitude. A likely reason for this became apparent when the activation-enthalpy and the pH-dependence of the rate

  8. Activation of Colicin M by the FkpA Prolyl Cis-Trans Isomerase/Chaperone*

    PubMed Central

    Helbig, Stephanie; Patzer, Silke I.; Schiene-Fischer, Cordelia; Zeth, Kornelius; Braun, Volkmar

    2011-01-01

    Colicin M (Cma) is specifically imported into the periplasm of Escherichia coli and kills the cells. Killing depends on the periplasmic peptidyl prolyl cis-trans isomerase/chaperone FkpA. To identify the Cma prolyl bonds targeted by FkpA, we replaced the 15 proline residues individually with alanine. Seven mutant proteins were fully active; Cma(P129A), Cma(P176A), and Cma(P260A) displayed 1%, and Cma(P107A) displayed 10% of the wild-type activity. Cma(P107A), Cma(P129A), and Cma(P260A), but not Cma(P176A), killed cells after entering the periplasm via osmotic shock, indicating that the former mutants were translocation-deficient; Cma(P129A) did not bind to the FhuA outer membrane receptor. The crystal structures of Cma and Cma(P176A) were identical, excluding inactivation of the activity domain located far from Pro-176. In a new peptidyl prolyl cis-trans isomerase assay, FkpA isomerized the Cma prolyl bond in peptide Phe-Pro-176 at a high rate, but Lys-Pro-107 and Leu-Pro-260 isomerized at only <10% of that rate. The four mutant proteins secreted into the periplasm via a fused signal sequence were toxic but much less than wild-type Cma. Wild-type and mutant Cma proteins secreted or translocated across the outer membrane by energy-coupled import or unspecific osmotic shock were only active in the presence of FkpA. We propose that Cma unfolds during transfer across the outer or cytoplasmic membrane and refolds to the active form in the periplasm assisted by FkpA. Weak refolding of Cma(P176A) would explain its low activity in all assays. Of the four proline residues identified as being important for Cma activity, Phe-Pro-176 is most likely targeted by FkpA. PMID:21149455

  9. 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. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Peptidyl-prolyl cis-trans isomerase Pin1 in ageing, cancer and Alzheimer disease.

    PubMed

    Lee, Tae Ho; Pastorino, Lucia; Lu, Kun Ping

    2011-06-20

    Phosphorylation of proteins on serine or threonine residues preceding proline is a key signalling mechanism in diverse physiological and pathological processes. Pin1 (peptidyl-prolyl cis-trans isomerase) is the only enzyme known that can isomerise specific Ser/Thr-Pro peptide bonds after phosphorylation and regulate their conformational changes with high efficiency. These Pin1-catalysed conformational changes can have profound effects on phosphorylation signalling by regulating a spectrum of target activities. Interestingly, Pin1 deregulation is implicated in a number of diseases, notably ageing and age-related diseases, including cancer and Alzheimer disease. Pin1 is overexpressed in most human cancers; it activates numerous oncogenes or growth enhancers and also inactivates a large number of tumour suppressors or growth inhibitors. By contrast, ablation of Pin1 prevents cancer, but eventually leads to premature ageing and neurodegeneration. Consistent with its neuroprotective role, Pin1 has been shown to be inactivated in neurons of patients with Alzheimer disease. Therefore, Pin1-mediated phosphorylation-dependent prolyl isomerisation represents a unique signalling mechanism that has a pivotal role in the development of human diseases, and might offer an attractive new diagnostic and therapeutic target.

  11. Identification and comparative analysis of sixteen fungal peptidyl-prolyl cis/trans isomerase repertoires

    PubMed Central

    Pemberton, Trevor J

    2006-01-01

    Background The peptidyl-prolyl cis/trans isomerase (PPIase) class of proteins is present in all known eukaryotes, prokaryotes, and archaea, and it is comprised of three member families that share the ability to catalyze the cis/trans isomerisation of a prolyl bond. Some fungi have been used as model systems to investigate the role of PPIases within the cell, however how representative these repertoires are of other fungi or humans has not been fully investigated. Results PPIase numbers within these fungal repertoires appears associated with genome size and orthology between repertoires was found to be low. Phylogenetic analysis showed the single-domain FKBPs to evolve prior to the multi-domain FKBPs, whereas the multi-domain cyclophilins appear to evolve throughout cyclophilin evolution. A comparison of their known functions has identified, besides a common role within protein folding, multiple roles for the cyclophilins within pre-mRNA splicing and cellular signalling, and within transcription and cell cycle regulation for the parvulins. However, no such commonality was found with the FKBPs. Twelve of the 17 human cyclophilins and both human parvulins, but only one of the 13 human FKBPs, identified orthologues within these fungi. hPar14 orthologues were restricted to the Pezizomycotina fungi, and R. oryzae is unique in the known fungi in possessing an hCyp33 orthologue and a TPR-containing FKBP. The repertoires of Cryptococcus neoformans, Aspergillus fumigatus, and Aspergillus nidulans were found to exhibit the highest orthology to the human repertoire, and Saccharomyces cerevisiae one of the lowest. Conclusion Given this data, we would hypothesize that: (i) the evolution of the fungal PPIases is driven, at least in part, by the size of the proteome, (ii) evolutionary pressures differ both between the different PPIase families and the different fungi, and (iii) whilst the cyclophilins and parvulins have evolved to perform conserved functions, the FKBPs have

  12. Selective inactivation of parvulin-like peptidyl-prolyl cis/trans isomerases by juglone.

    PubMed

    Hennig, L; Christner, C; Kipping, M; Schelbert, B; Rücknagel, K P; Grabley, S; Küllertz, G; Fischer, G

    1998-04-28

    In contrast to FK506 binding proteins and cyclophilins, the parvulin family of peptidyl-prolyl cis/trans isomerases (PPIases; E.C. 5.2.1.8) cannot be inhibited by either FK506 or cyclosporin A. We have found that juglone, 5-hydroxy-1,4-naphthoquinone, irreversibly inhibits the enzymatic activity of several parvulins, like the E. coli parvulin, the yeast Ess1/Ptf1, and human Pin1, in a specific manner, thus allowing selective inactivation of these enzymes in the presence of other PPIases. The mode of action was studied by analyzing the inactivation kinetics and the nature of products of the reaction of E. coli parvulin and its Cys69Ala variant with juglone. For all parvulins investigated, complete inactivation was obtained by a slow process that is characterized by pseudo-first-order rate constants in the range of 5.3 x 10(-)4 to 4. 5 x 10(-)3 s-1. The inactivated parvulin contains two juglone molecules that are covalently bound to the side chains of Cys41 and Cys69 because of a Michael addition of the thiol groups to juglone. Redox reactions did not contribute to the inactivation process. Because thiol group modification was shown to proceed 5-fold faster than the rate of enzyme inactivation, it was considered as a necessary but not sufficient condition for inactivation. When measured by far-UV circular dichroism (CD), the rate of structural alterations following thiol group modification parallels exactly the rate of inactivation. Thus, partial unfolding of the active site of the parvulins was thought to be the cause of the deterioration of PPIase activity.

  13. Identification of Conus Peptidylprolyl Cis-Trans Isomerases (PPIases) and Assessment of Their Role in the Oxidative Folding of Conotoxins

    PubMed Central

    Safavi-Hemami, Helena; Bulaj, Grzegorz; Olivera, Baldomero M.; Williamson, Nicholas A.; Purcell, Anthony W.

    2010-01-01

    Peptidylprolyl cis-trans isomerases (PPIases) are ubiquitous proteins that catalyze the cis-trans isomerization of prolines. A number of proteins, such as Drosophila rhodopsin and the human immunodeficiency viral protein HIV-1 Gag, have been identified as endogenous substrates for PPIases. However, very little is known about the interaction of PPIases with small, disulfide-rich peptides. Marine cone snails synthesize a wide array of cysteine-rich peptides, called conotoxins, many of which contain one or more prolines or hydroxyprolines. To identify whether PPIase-associated cis-trans isomerization of these residues affects the oxidative folding of conotoxins, we identified, sequenced, and expressed three functionally active isoforms of PPIase from the venom gland of Conus novaehollandiae, and we characterized their ability to facilitate oxidative folding of conotoxins in vitro. Three conotoxins, namely μ-GIIIA, μ-SIIIA, and ω-MVIIC, derived from two distinct toxin gene families were assayed. Conus PPIase significantly increased the rate of appearance of the native form of μ-GIIIA, a peptide containing three hydroxyprolines. In contrast, the presence of PPIase had no effect on the folding of μ-SIIIA and ω-MVIIC, peptides containing no or one proline residue, respectively. We further showed that an endoplasmic reticulum-resident PPIase isoform facilitated folding of μ-GIIIA more efficiently than two cytosolic isoforms. This is the first study to demonstrate PPIase-assisted folding of conotoxins, small disulfide-rich peptides with unique structural properties. PMID:20147296

  14. Presence of autoantibodies to peptidyl-prolyl cis-trans isomerase (cyclosporin A-binding protein) in systemic lupus erythematosus.

    PubMed

    Harigai, M; Hara, M; Takahashi, N; Kitani, A; Hirose, T; Suzuki, K; Kawakami, M; Hidaka, T; Kawaguchi, Y; Ishizuka, T

    1992-04-01

    Several autoantibodies against cytoplasmic or nuclear components of cells have been reported in autoimmune diseases. We report here a previously unrecognized autoantibody to peptidyl-prolyl cis-trans isomerase (PPIase) in patients with systemic lupus erythematosus (SLE). PPIase, which catalyzes the cis-trans isomerization of proline imidic peptide bonds in oligopeptides, has recently been found to be identical to cyclophilin, a specific binding protein of a potent immunosuppressant, cyclosporin A. IgG and IgM anti-PPIase antibodies were detected in 40 and 20% of unselected patients with SLE, respectively, by ELISA. The reactivity of these sera was confirmed by immunoblotting experiments. Sera from rheumatoid arthritis patients showed no reactivity and 1 of 8 sera from systemic sclerosis patients and 1 of 25 sera from normal controls showed only weak reactivity. Unexpectedly, the anti-PPIase antibody was unable to inhibit PPIase activity, indicating that the autoantibody recognizes an epitope of PPIase which is different from the active site of PPIase. The levels of the anti-PPIase antibody in SLE patients correlated with remissions and flares of the disease. The anti-PPIase antibody was higher in patients with active SLE than those with inactive disease. The prevalence of the active stage of the disease was significantly higher in IgG anti-PPIase antibody-positive SLE patients as compared to antibody-negative SLE patients. These data define the presence of a new autoantibody against PPIase and its association with the activity and certain clinical manifestations in SLE.

  15. Semiautomated microtiter plate assay for monitoring peptidylprolyl cis/trans isomerase activity in normal and pathological human sera.

    PubMed

    Küllertz, G; Lüthe, S; Fischer, G

    1998-03-01

    An UV/VIS spectrophotometric assay technique was developed that was able to routinely monitor peptidylprolyl cis/trans isomerase (PPIase) activity of biological fluids in 96-well microtiter plates. The assay, based on monitoring the cis-to-trans isomerization of succinyl-Phe-cisPro-Phe-4-nitroanilide as substrate in a chymotrypsin-coupled reaction, yields a throughput of 96 samples per 30 min. The assay's capacity was exemplified by dealing with the PPIase activity in several normal and pathological human sera. Reference values of 151 healthy subjects (83 females, 69 males, 17 to 60 years old) were found to possess significant sex-specific differences. PPIase activity factor K of the sera was significantly greater in males (5th, 50th, 95th percentiles: 17, 36, 55 K) than females (14, 30, 48 K). PPIase activities of sera from healthy donors (n = 151) were significantly higher (Mann-Whitney rank-sum test P < 0.0001) than those of patients (n = 47). PPIase activity in serum samples stored at 4 degrees C was stable for at least 20 h.

  16. 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

  17. 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.

  18. A cyclophilin-like peptidyl-prolyl cis/trans isomerase from Legionella pneumophila--characterization, molecular cloning and overexpression.

    PubMed

    Schmidt, B; Tradler, T; Rahfeld, J U; Ludwig, B; Jain, B; Mann, K; Rücknagel, K P; Janowski, B; Schierhorn, A; Küllertz, G; Hacker, J; Fischer, G

    1996-09-01

    Legionella pneumophila is the causative agent of a severe form of pneumonia in humans (Legionnaires' disease). A major virulence factor, the Mip protein (FK506-binding protein, FKBP25mem), belongs to the enzyme family of peptidyl-prolyl cis/trans isomerases (PPlases). Here we show that L. pneumophila Philadelphia I possesses an additional cytoplasmic PPlase at a level of enzyme activity comparable to that of FKBP25mem. The N-terminal amino acid sequence of the purified protein was obtained by Edman degradation and showed that the protein is a member of the cyclophilin family of PPlases. The Icy gene (Legionella cyclophilin) was cloned and sequenced. It encodes a putative 164-amino-acid protein with a molecular mass of 17968 Da called L. pneumophila cyclophilin 18 (L.p.Cyp18). Amino acid sequence comparison displays considerable similarity to the cytoplasmic and the periplasmic cyclophilins of Escherichia coli with 60.5% and 51.5% identity, respectively. The substrate specificity and inhibition by cyclosporin A revealed a pattern that is typically found for other bacterial cyclophilins. An L. pneumophila Cyp18 derivative with a 19-amino-acid polypeptide extension including a 6-histidine tag and an enterokinase cleavage site exhibits PPlase activity when produced at high levels in E. coli K-12. After removal of the extension by enterokinase, the properties of the recombinant Cyp18 were indistinguishable from those of the authentic enzyme. In order to investigate the influence of Cyp18 on intracellular survival of L. pneumophila an Icy-negative L. pneumophila strain was constructed. Compared with the wild-type strain, the mutant did not exhibit a significant phenotype but was 10-fold less invasive for Acanthamoeba castellanii. Like human cyclophilin, the L. p. Cyp18 exhibits nuclease activity, but this enzymatic activity does not appear to be linked with the native structure of the protein.

  19. 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. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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

    PubMed Central

    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

  1. Cyclophilin A (CyPA) induces chemotaxis independent of its peptidylprolyl cis-trans isomerase activity: direct binding between CyPA and the ectodomain of CD147.

    PubMed

    Song, Fei; Zhang, Xin; Ren, Xiao-Bai; Zhu, Ping; Xu, Jing; Wang, Li; Li, Yi-Fei; Zhong, Nan; Ru, Qiang; Zhang, Da-Wei; Jiang, Jian-Li; Xia, Bin; Chen, Zhi-Nan

    2011-03-11

    Cyclophilin A (CyPA) is a ubiquitously distributed peptidylprolyl cis-trans isomerase (PPIase) that possesses diverse biological functions. Extracellular CyPA is a potent chemokine, which can directly induce leukocyte chemotaxis and contribute to the pathogenesis of inflammation-mediated diseases. Although it has been identified that the chemotaxis activity of CyPA is mediated through its cell surface signaling receptor CD147, the role of CyPA PPIase activity in this process is disputable, and the underlying molecular mechanism is still poorly understood. In this study, we present the first evidence that CyPA induces leukocyte chemotaxis through a direct binding with the ectodomain of CD147 (CD147(ECT)), independent of its PPIase activity. Although NMR study indicates that the CD147(ECT) binding site on CyPA overlaps with the PPIase active site, the PPIase inactive mutant CyPA(R55A) exhibits similar CD147(ECT) binding ability and chemotaxis activity to those of CyPA(WT). Furthermore, we have identified three key residues of CyPA involved in CD147(ECT) binding and found that mutations H70A, T107A, and R69A result in similar levels of reduction in CD147(ECT) binding ability and chemotaxis activity for CyPA, without affecting the PPIase activity. Our findings indicate that there exists a novel mechanism for CyPA to regulate cellular signaling processes, shedding new light on its applications in drug development and providing a new targeting site for drug design.

  2. Structural Insights into Substrate Binding by PvFKBP35, a Peptidylprolyl cis-trans Isomerase from the Human Malarial Parasite Plasmodium vivax

    PubMed Central

    Alag, Reema; Balakrishna, Asha Manikkoth; Rajan, Sreekanth; Qureshi, Insaf A.; Shin, Joon; Lescar, Julien; Grüber, Gerhard

    2013-01-01

    The immunosuppressive drug FK506 binding proteins (FKBPs), an immunophilin family with the immunosuppressive drug FK506 binding property, exhibit peptidylprolyl cis-trans isomerase (PPIase) activity. While the cyclophilin-catalyzed peptidylprolyl isomerization of X-Pro peptide bonds has been extensively studied, the mechanism of the FKBP-mediated peptidylprolyl isomerization remains uncharacterized. Thus, to investigate the binding of FKBP with its substrate and the underlying catalytic mechanism of the FKBP-mediated proline isomerization, here we employed the FK506 binding domain (FKBD) of the human malarial parasite Plasmodium vivax FK506 binding protein 35 (PvFKBP35) and examined the details of the molecular interaction between the isomerase and a peptide substrate. The crystallographic structures of apo PvFKBD35 and its complex with the tetrapeptide substrate succinyl-Ala-Leu-Pro-Phe-p-nitroanilide (sALPFp) determined at 1.4 Å and 1.65 Å resolutions, respectively, showed that the substrate binds to PvFKBD35 in a cis conformation. Nuclear magnetic resonance (NMR) studies demonstrated the chemical shift perturbations of D55, H67, V73, and I74 residues upon the substrate binding. In addition, the X-ray crystal structure, along with the mutational studies, shows that Y100 is a key residue for the catalytic activity. Taken together, our results provide insights into the catalytic mechanism of PvFKBP35-mediated cis-trans isomerization of substrate and ultimately might aid designing substrate mimetic inhibitors targeting the malarial parasite FKBPs. PMID:23435727

  3. 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

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

    PubMed

    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 Cu(2+), 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 Ca(2+)-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 Ca(2+) signaling and cyclophilin activity in Arabidopsis.

  5. Functional characterisation of parvulin-type peptidyl prolyl cis-trans isomerase, PinA in Dictyostelium discoideum.

    PubMed

    Haokip, Nemneineng; Naorem, Aruna

    2017-01-08

    Pin1-type parvulins are unique among PPIases that can catalyse an otherwise slow cis-trans isomerisation of phosphorylated peptide bond preceding proline in target proteins. This prolyl isomerisation process can regulate activity, stability and localisation of target proteins and thus control cellular processes like eukaryotic cell proliferation, cell cycle progression and gene regulation. Towards understanding the function of Pin1-type prolyl isomerisation in Dictyostelium discoideum, a slime mould with distinct growth and developmental phases, we identified PinA as a novel Pin1-type parvulin by its ability to complement the temperature sensitivity phenotype associated with a mutation in ESS1 in S. cerevisiae. In D. discoideum, pinA is temporally and spatially regulated during growth and development. PinA is both nuclear as well as cytoplasmic in the growing cells. We further show that loss of pinA (pinA(-)) leads to decreased growth rate, reduced spore formation and abnormal prespore-prestalk patterning. We conclude that PinA is required for normal growth as well as development in D. discoideum.

  6. Chicken FK506-binding protein, FKBP65, a member of the FKBP family of peptidylprolyl cis-trans isomerases, is only partially inhibited by FK506.

    PubMed Central

    Zeng, B; MacDonald, J R; Bann, J G; Beck, K; Gambee, J E; Boswell, B A; Bächinger, H P

    1998-01-01

    The chicken FK506-binding protein FKBP65, a peptidylprolyl cis-trans isomerase, is a rough endoplasmic reticulum protein that contains four domains homologous to FKBP13, another rough endoplasmic reticulum PPIase. Analytical ultracentrifugation suggests that in FKBP65 these four domains are arranged in a linear extended structure with a length of about 26 nm and a diameter of about 3 nm. All four domains are therefore expected to be accessible to substrates. The specificity of FKBP65 towards a number of peptide substrates was determined. The specific activity of FKBP65 is generally lower than that of FKBP12 when expressed as a per domain activity. The substrate specificity of FKBP65 also differs from that of FKBP12. Inhibition studies show that only one of the four domains can be inhibited by FK506, a powerful inhibitor of all other known FKBPs. Furthermore, the same domain seems to be susceptible to inhibition by cyclosporin A. No other FKBPs were shown to be inhibited by cyclosporin A. It is also shown that FKBP65 can catalyse the re-folding of type III collagen in vitro with a kcat/Km = 4.3 x 10(3) M-1.s-1. PMID:9461498

  7. Biochemical and Genetic Characterization of an FK506-Sensitive Peptidyl Prolyl cis-trans Isomerase from a Thermophilic Archaeon, Methanococcus thermolithotrophicus

    PubMed Central

    Furutani, Masahiro; Iida, Toshii; Yamano, Shigeyuki; Kamino, Kei; Maruyama, Tadashi

    1998-01-01

    A peptidyl prolyl cis-trans isomerase (PPIase) was purified from a thermophilic methanogen, Methanococcus thermolithotrophicus. The PPIase activity was inhibited by FK506 but not by cyclosporine. The molecular mass of the purified enzyme was estimated to be 16 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 42 kDa by gel filtration. The enzyme was thermostable, with the half-lives of its activity at 90 and 100°C being 90 and 30 min, respectively. The catalytic efficiencies (kcat/Km) measured at 15°C for the peptidyl substrates, N-succinyl-Ala-Leu-Pro-Phe-p-nitroanilide and N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, were 0.35 and 0.20 μM−1 s−1, respectively, in chymotrypsin-coupled assays. The purified enzyme was sensitive to FK506 and therefore was called MTFK (M. thermolithotrophicus FK506-binding protein). The MTFK gene (462 bp) was cloned from an M. thermolithotrophicus genomic library. The comparison of the amino acid sequence of MTFK with those of other FK506-binding PPIases revealed that MTFK has a 13-amino-acid insertion in the N-terminal region that is unique to thermophilic archaea. The relationship between the thermostable nature of MTFK and its structure is discussed. PMID:9440528

  8. Hepatitis C virus NS5A protein is a substrate for the peptidyl-prolyl cis/trans isomerase activity of cyclophilins A and B.

    PubMed

    Hanoulle, Xavier; Badillo, Aurélie; Wieruszeski, Jean-Michel; Verdegem, Dries; Landrieu, Isabelle; Bartenschlager, Ralf; Penin, François; Lippens, Guy

    2009-05-15

    We report here a biochemical and structural characterization of domain 2 of the nonstructural 5A protein (NS5A) from the JFH1 Hepatitis C virus strain and its interactions with cyclophilins A and B (CypA and CypB). Gel filtration chromatography, circular dichroism spectroscopy, and finally NMR spectroscopy all indicate the natively unfolded nature of this NS5A-D2 domain. Because mutations in this domain have been linked to cyclosporin A resistance, we used NMR spectroscopy to investigate potential interactions between NS5A-D2 and cellular CypA and CypB. We observed a direct molecular interaction between NS5A-D2 and both cyclophilins. The interaction surface on the cyclophilins corresponds to their active site, whereas on NS5A-D2, it proved to be distributed over the many proline residues of the domain. NMR heteronuclear exchange spectroscopy yielded direct evidence that many proline residues in NS5A-D2 form a valid substrate for the enzymatic peptidyl-prolyl cis/trans isomerase (PPIase) activity of CypA and CypB.

  9. Hepatitis C Virus NS5A Protein Is a Substrate for the Peptidyl-prolyl cis/trans Isomerase Activity of Cyclophilins A and B*S⃞

    PubMed Central

    Hanoulle, Xavier; Badillo, Aurélie; Wieruszeski, Jean-Michel; Verdegem, Dries; Landrieu, Isabelle; Bartenschlager, Ralf; Penin, François; Lippens, Guy

    2009-01-01

    We report here a biochemical and structural characterization of domain 2 of the nonstructural 5A protein (NS5A) from the JFH1 Hepatitis C virus strain and its interactions with cyclophilins A and B (CypA and CypB). Gel filtration chromatography, circular dichroism spectroscopy, and finally NMR spectroscopy all indicate the natively unfolded nature of this NS5A-D2 domain. Because mutations in this domain have been linked to cyclosporin A resistance, we used NMR spectroscopy to investigate potential interactions between NS5A-D2 and cellular CypA and CypB. We observed a direct molecular interaction between NS5A-D2 and both cyclophilins. The interaction surface on the cyclophilins corresponds to their active site, whereas on NS5A-D2, it proved to be distributed over the many proline residues of the domain. NMR heteronuclear exchange spectroscopy yielded direct evidence that many proline residues in NS5A-D2 form a valid substrate for the enzymatic peptidyl-prolyl cis/trans isomerase (PPIase) activity of CypA and CypB. PMID:19297321

  10. 1.88 A crystal structure of the C domain of hCyP33: A novel domain of peptidyl-prolyl cis-trans isomerase

    SciTech Connect

    Wang Tao; Yun Caihong; Gu Shenyan; Chang Wenrui; Liang Dongcai . E-mail: dcliang@sun5.ibp.ac.cn

    2005-08-05

    Cyclophilins (CyPs) are a widespreading protein family in living organisms and possess the activity of peptidyl-prolyl cis-trans isomerase (PPIase), which is inhibited by cyclosporin A (CsA). The human nuclear cyclophilin (hCyP33) is the first protein which was found to contain two RNA binding domains at the amino-terminus and a PPIase domain at the carboxyl-terminus. We isolated the hCyP33 gene from the human hematopoietic stem/progenitor cells and expressed it in Escherichia coli, and determined the crystal structure of the C domain of hCyP33 at 1.88 A resolution. The core structure is a {beta}-barrel covered by two {alpha}-helices. Superposition of the structure of the C domain of hCyP33 with the structure of CypA suggests that the C domain contains PPIase active site which binds to CsA. Furthermore, C domain seems to be able to bind with the Gag-encoded capsid (CA) of HIV-1 and may affect the viral replication of HIV-1. A key residue of the active site is changed from Ala-103-CypA to Ser-239-hCyP33, which may affect the PPIase domain/substrates interactions.

  11. The Wheat Peptidyl Prolyl cis-trans-Isomerase FKBP77 Is Heat Induced and Developmentally Regulated1

    PubMed Central

    Kurek, Isaac; Aviezer, Keren; Erel, Noa; Herman, Eliot; Breiman, Adina

    1999-01-01

    We isolated a cDNA encoding a 568-amino acid, heat-stress-induced peptidyl prolyl isomerase belonging to the FK506-binding-protein (FKBP) family. The open reading frame encodes for a peptidyl prolyl isomerase that possesses three FKBP-12-like domains, a putative tetratricopeptide motif, and a calmodulin-binding domain. Specific antibodies showed that the open reading frame encodes a heat-induced 77-kD protein, the wheat FKBP77 (wFKBP77), which exhibits 84% identity with the wFKBP73 and 42% identity with the human FKBP59. Because of the high similarity in sequence to wFKBP73, wFKBP77 was designated as the heat-induced isoform. The wFKBP77 mRNA steady-state level was 14-fold higher at 37°C than at 25°C. The wFKBP77 transcript abundance was the highest in mature embryos that had imbibed and 2-d-old green shoots exposed to 37°C, and decreased to 6% in 6-d-old green shoots. The transcript level returned to the level detected at 25°C after recovery of the embryos for 90 min at 25°C. We compared wFKBP73 and wFKBP77 with the heat-shock proteins having cognate and heat-stress-induced counterparts. PMID:9952466

  12. Peptidyl prolyl cis/trans-isomerases: comparative reactivities of cyclophilins, FK506-binding proteins, and parvulins with fluorinated oligopeptide and protein substrates.

    PubMed

    Golbik, Ralph; Yu, Chao; Weyher-Stingl, Elisabeth; Huber, Robert; Moroder, Luis; Budisa, Nediljko; Schiene-Fischer, Cordelia

    2005-12-13

    Peptidyl prolyl cis/trans-isomerases catalyze the cis-trans isomerization of prolyl bonds in oligopeptides and various folding states of proteins. The proline residue in PPIase substrates at the P1' subsite, which follows the isomerizing peptide bond, appears to be the common recognition element for all subfamilies of this enzyme class. The molecular principles that govern substrate specificity at the P1' subsite were analyzed using 4-fluoroproline-containing tetrapeptide 4-nitroanilides and barstar Cys40Ala/Cys82Ala/Pro27Ala/Pro48-->4-fluoroproline quadruple variants. Generally, PPIase catalysis demonstrated stereospecificity for monofluoro substitutions at the 4-position of the pyrrolidine ring. However, the replacement of hydrogens with fluoro atoms did not impair productive interactions for the majority of PPIase-substrate complexes. Comparison of specificity constants for oligopeptide and protein substrates revealed striking differences in the 4-fluoroproline substituent effects between members of the PPIase families. Introduction of 4(R)-fluoroproline resulted in an oligopeptide substrate completely resistant to catalytic effects of FKBP-like PPIases. By contrast, the 4(R)-fluoroproline barstar variant demonstrated only slightly reduced or even better catalytic susceptibility when compared to the parent barstar Cys40Ala/Cys82Ala/Pro27Ala/Pro48 substrate. On the other hand, Suc-Ala-Ser-4(S)-FPro-Phe-pNA exhibits a discriminating specificity toward the prototypic parvulin, the Escherichia coli Par10. The E. coli trigger factor, in the extreme, catalyzes Cys40Ala/Cys82Ala/Pro27Ala/4-F(2)Pro48 with a more than 20-fold higher efficiency when compared to the proline-containing congener. These findings support the combined subsite concept for PPIase catalysis in which the positioning of a substrate in the active cleft must activate a still unknown number of remote subsites in the transition state of the reaction. The number of critical subsites was shown to vary

  13. 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

  14. Complementary DNA encoding the human T-cell FK506-binding protein, a peptidylprolyl cis-trans isomerase distinct from cyclophilin

    SciTech Connect

    Maki, Noboru; Sekiguchi, Fumiko; Nishimaki, Junichi; Miwa, Keiko; Hayano, Toshiya; Takahashi, Nobuhiro; Suzuki, Masanori )

    1990-07-01

    The recently discovered macrolide FK506 has been demonstrated to have potent immunosuppressive activity at concentrations 100-fold lower than cyclosporin A, a cyclic undecapeptide that is used to prevent rejection after transplantation of bone marrow and organs, such as kidney, heart, and liver. After the recent discovery that the cylcosporin A-binding protein cyclophilin is identical to peptidylprolyl cis-trans isomerase, a cellular binding protein for FK506 was found to be distinct from cyclophilin but to have the same enzymatic activity. In this study, the authors isolated a cDNA coding for FK506-binding protein (FKBP) from human peripheral blood T cells by using mixed 20-mer oligonucleotide probes synthesized on the basis of the sequence, Glu-Asp-Gly-Lys-Lys-Phe-Asp, reported for bovine FKBP. The DNA isolated contained an open reading frame encoding 108 amino acid residues. The first 40 residues of the deduced amino acid sequence were identical to those of the reported amino-terminal sequence of bovine FKBP, indicating that the DNA sequence isolated represents the gene coding for FKBP. This result suggests that two catalytically similar proteins, cyclophilin and FKBP, evolved independently. In Northern blot analysis, mRNA species of {approx}1.8 kilobases that hybridized with human FKBP cDNA were detected in poly(A){sup +} RNAs from brain, lung, liver, and placental cells and leukocytes. Induction of Jurkat leukemic T cells with phorbol 12-myristate 13-acetate and ionomycin did not affect the level of FKBP mRNA.

  15. Parvulin (Par14), a peptidyl-prolyl cis-trans isomerase, is a novel rRNA processing factor that evolved in the metazoan lineage.

    PubMed

    Fujiyama-Nakamura, Sally; Yoshikawa, Harunori; Homma, Keiichi; Hayano, Toshiya; Tsujimura-Takahashi, Teruko; Izumikawa, Keiichi; Ishikawa, Hideaki; Miyazawa, Naoki; Yanagida, Mitsuaki; Miura, Yutaka; Shinkawa, Takashi; Yamauchi, Yoshio; Isobe, Toshiaki; Takahashi, Nobuhiro

    2009-07-01

    Although parvulin (Par14/eukaryotic parvulin homolog), a peptidyl-prolyl cis-trans isomerase, is found associated with the preribosomal ribonucleoprotein (pre-rRNP) complexes, its roles in ribosome biogenesis remain undetermined. In this study, we describe a comprehensive proteomics analysis of the Par14-associated pre-rRNP complexes using LC-MS/MS and a knockdown analysis of Par14. Together with our previous results, we finally identified 115 protein components of the complexes, including 39 ribosomal proteins and 54 potential trans-acting factors whose yeast homologs are found in the pre-rRNP complexes formed at various stages of ribosome biogenesis. We give evidence that, although Par14 exists in both the phosphorylated and unphosphorylated forms in the cell, only the latter form is associated with the pre-40 S and pre-60 S ribosomal complexes. We also show that Par14 co-localizes with the nucleolar protein B23 during the interphase and in the spindle apparatus during mitosis and that actinomycin D treatment results in the exclusion of Par14 from the nucleolus. Finally we demonstrate that knockdown of Par14 mRNA decelerates the processing of pre-rRNA to 18 and 28 S rRNAs. We propose that Par14 is a component of the pre-rRNA complexes and functions as an rRNA processing factor in ribosome biogenesis. As the amino acid sequence of Par14 including that in the amino-terminal pre-rRNP binding region is conserved only in metazoan homologs, we suggest that its roles in ribosome biogenesis have evolved in the metazoan lineage.

  16. Identification of homologs for thioredoxin, peptidyl prolyl cis-trans isomerase, and glycerophosphodiester phosphodiesterase in outer membrane fractions from Treponema pallidum, the syphilis spirochete.

    PubMed Central

    Shevchenko, D V; Akins, D R; Robinson, E J; Li, M; Shevchenko, O V; Radolf, J D

    1997-01-01

    In this study, we characterized candidate rare outer membrane (OM) proteins with apparent molecular masses of 19, 27, 38, and 38.5 kDa, which had been identified previously in OM fractions from Treponema pallidum (J. D. Radolf et al., Infect. Immun. 63:4244-4252, 1995). Using N-terminal and internal amino acid sequences, a probe for the 19-kDa candidate was PCR amplified and used to screen a T. pallidum genomic library in Lambda Zap II. The corresponding gene (tlp) encoded a homolog for periplasmic thioredoxin-like proteins (Tlp), which reduce c-type cytochromes. A degenerate oligonucleotide derived from the N terminus of the 27-kDa protein was used to PCR amplify a duplex probe from a T. pallidum genomic library in pBluescript II SK+. With this probe, the corresponding gene (ppiB) was identified and found to code for a presumptive periplasmic cyclophilin B-type peptidyl prolyl cis-trans isomerase (PpiB). We postulate that PpiB assists the folding of proteins within the T. pallidum periplasmic space. The N terminus of the 38-kDa candidate was blocked to Edman degradation. However, internal sequence data revealed that it was basic membrane protein (Bmp), a previously characterized, signal peptidase I-processed protein. Triton X-114 phase partitioning revealed that despite its name, Bmp is hydrophilic and therefore likely to be periplasmic. The final candidate was also blocked to Edman degradation; as before, a duplex probe was PCR amplified with degenerate primers derived from internal sequences. The corresponding gene (glpQ) coded for a presumptively lipid-modified homolog of glycerophosphodiester phosphodiesterase (GlpQ). Based upon findings with other treponemal lipoproteins, the hydrophilic GlpQ polypeptide is thought to be anchored by N-terminal lipids to the periplasmic leaflet(s) of the cytoplasmic membrane and/or OM. The discovery of T. pallidum periplasmic proteins with potentially defined functions provides fresh insights into a poorly understood aspect of

  17. An Intracellular Peptidyl-Prolyl cis/trans Isomerase Is Required for Folding and Activity of the Staphylococcus aureus Secreted Virulence Factor Nuclease.

    PubMed

    Wiemels, Richard E; Cech, Stephanie M; Meyer, Nikki M; Burke, Caleb A; Weiss, Andy; Parks, Anastacia R; Shaw, Lindsey N; Carroll, Ronan K

    2017-01-01

    Staphylococcus aureus is an important human pathogen that relies on a large repertoire of secreted and cell wall-associated proteins for pathogenesis. Consequently, the ability of the organism to cause disease is absolutely dependent on its ability to synthesize and successfully secrete these proteins. In this study, we investigate the role of peptidyl-prolyl cis/trans isomerases (PPIases) on the activity of the S. aureus secreted virulence factor nuclease (Nuc). We identify a staphylococcal cyclophilin-type PPIase (PpiB) that is required for optimal activity of Nuc. Disruption of ppiB results in decreased nuclease activity in culture supernatants; however, the levels of Nuc protein are not altered, suggesting that the decrease in activity results from misfolding of Nuc in the absence of PpiB. We go on to demonstrate that PpiB exhibits PPIase activity in vitro, is localized to the bacterial cytosol, and directly interacts with Nuc in vitro to accelerate the rate of Nuc refolding. Finally, we demonstrate an additional role for PpiB in S. aureus hemolysis and demonstrate that the S. aureus parvulin-type PPIase PrsA also plays a role in the activity of secreted virulence factors. The deletion of prsA leads to a decrease in secreted protease and phospholipase activity, similar to that observed in other Gram-positive pathogens. Together, these results demonstrate, for the first time to our knowledge, that PPIases play an important role in the secretion of virulence factors in S. aureus IMPORTANCE: Staphylococcus aureus is a highly dangerous bacterial pathogen capable of causing a variety of infections throughout the human body. The ability of S. aureus to cause disease is largely due to an extensive repertoire of secreted and cell wall-associated proteins, including adhesins, toxins, exoenzymes, and superantigens. These virulence factors, once produced, are typically transported across the cell membrane by the secretory (Sec) system in a denatured state. Consequently

  18. Inhibition of Ca2(+)-induced large-amplitude swelling of liver and heart mitochondria by cyclosporin is probably caused by the inhibitor binding to mitochondrial-matrix peptidyl-prolyl cis-trans isomerase and preventing it interacting with the adenine nucleotide translocase.

    PubMed Central

    Halestrap, A P; Davidson, A M

    1990-01-01

    1. Isolated rat liver and heart mitochondria incubated in 150 mM-KSCN or sucrose medium in the presence of respiratory-chain inhibitors showed a large increase in swelling when exposed to 250 microM-Ca2+. Swelling was inhibited by bongkrekic acid and cyclosporin A in both media and by ADP in KSCN medium; the effect of ADP was reversed by carboxyatractyloside. These results demonstrate that this is a suitable technique with which to study the opening of the Ca2(+)-induced non-specific pore of the mitochondrial inner membrane and implicate the adenine nucleotide carrier in this process. 2. Titration of the rate of swelling with increasing concentrations of cyclosporin showed the number of cyclosporin-binding sites (+/- S.E.M.) in liver and heart mitochondria to be respectively 113.7 +/- 5.0 (n = 9) and 124.3 +/- 11.2 (n = 10) pmol/mg of protein, with a Ki of about 5 nM. 3. Liver and heart mitochondrial-matrix fractions were prepared free of membrane and cytosolic contamination and shown to contain cyclosporin-sensitive peptidyl-prolyl cis-trans isomerase (cyclophilin) activity. Titration of isomerase activity with cyclosporin gave values (+/- S.E.M.) of 110.6 +/- 10.1 (n = 5) and 165.4 +/- 15.0 (n = 3) pmol of enzyme/mg of liver and heart mitochondrial protein respectively, with a Ki of 2.5 nM. The similarity of these results to those from the swelling experiments suggest that the isomerase may be involved in the Ca2(+)-induced swelling. 4. The rapid light-scattering change induced in energized heart mitochondria exposed to submicromolar Ca2+ [Halestrap (1987) Biochem. J. 244, 159-164] was inhibited by ADP and bongkrekic acid, the former effect being reversed by carboxyatractyloside. These results suggest an interaction of Ca2+ with the adenine nucleotide carrier when the 'c' conformation. 5. A model is proposed in which mitochondrial peptidyl-prolyl cis-trans isomerase interacts with the adenine nucleotide carrier in the presence of Ca2+ to cause non-specific pore

  19. Mutation in Cyclophilin B That Causes Hyperelastosis Cutis in American Quarter Horse Does Not Affect Peptidylprolyl cis-trans Isomerase Activity but Shows Altered Cyclophilin B-Protein Interactions and Affects Collagen Folding*

    PubMed Central

    Ishikawa, Yoshihiro; Vranka, Janice A.; Boudko, Sergei P.; Pokidysheva, Elena; Mizuno, Kazunori; Zientek, Keith; Keene, Douglas R.; Rashmir-Raven, Ann M.; Nagata, Kazuhiro; Winand, Nena J.; Bächinger, Hans Peter

    2012-01-01

    The rate-limiting step of folding of the collagen triple helix is catalyzed by cyclophilin B (CypB). The G6R mutation in cyclophilin B found in the American Quarter Horse leads to autosomal recessive hyperelastosis cutis, also known as hereditary equine regional dermal asthenia. The mutant protein shows small structural changes in the region of the mutation at the side opposite the catalytic domain of CypB. The peptidylprolyl cis-trans isomerase activity of the mutant CypB is normal when analyzed in vitro. However, the biosynthesis of type I collagen in affected horse fibroblasts shows a delay in folding and secretion and a decrease in hydroxylysine and glucosyl-galactosyl hydroxylysine. This leads to changes in the structure of collagen fibrils in tendon, similar to those observed in P3H1 null mice. In contrast to cyclophilin B null mice, where little 3-hydroxylation was found in type I collagen, 3-hydroxylation of type I collagen in affected horses is normal. The mutation disrupts the interaction of cyclophilin B with the P-domain of calreticulin, with lysyl hydroxylase 1, and probably other proteins, such as the formation of the P3H1·CypB·cartilage-associated protein complex, resulting in less effective catalysis of the rate-limiting step in collagen folding in the rough endoplasmic reticulum. PMID:22556420

  20. Prolyl cis-trans isomerization as a molecular timer.

    PubMed

    Lu, Kun Ping; Finn, Greg; Lee, Tae Ho; Nicholson, Linda K

    2007-10-01

    Proline is unique in the realm of amino acids in its ability to adopt completely distinct cis and trans conformations, which allows it to act as a backbone switch that is controlled by prolyl cis-trans isomerization. This intrinsically slow interconversion can be catalyzed by the evolutionarily conserved group of peptidyl prolyl cis-trans isomerase enzymes. These enzymes include cyclophilins and FK506-binding proteins, which are well known for their isomerization-independent role as cellular targets for immunosuppressive drugs. The significance of enzyme-catalyzed prolyl cis-trans isomerization as an important regulatory mechanism in human physiology and pathology was not recognized until the discovery of the phosphorylation-specific prolyl isomerase Pin1. Recent studies indicate that both phosphorylation-dependent and phosphorylation-independent prolyl cis-trans isomerization can act as a novel molecular timer to help control the amplitude and duration of a cellular process, and prolyl cis-trans isomerization might be a new target for therapeutic interventions.

  1. Aluminum(III) interferes with the structure and the activity of the peptidyl-prolyl cis-trans isomerase (Pin1): a new mechanism contributing to the pathogenesis of Alzheimer's disease and cancers?

    PubMed

    Wang, Jing-Zhang; Liu, Ji; Lin, Tao; Han, Yong-Guang; Luo, Yue; Xi, Lei; Du, Lin-Fang

    2013-09-01

    The enzyme peptidyl-prolyl cis-trans isomerase (Pin1) may play an important role in preventing the development of Alzheimer's disease (AD). The structural and functional stability of Pin1 is extremely important. Previously, we have determined the stability of Pin1 under stressed conditions, such as thermal treatment and acidic-pH. Considering that aluminum (Al(III)) is well known for its potential neurotoxicity in the pathogenesis of AD, we examined whether Al(III) affects the structure and function of Pin1, by means of a PPIase activity assay, intrinsic fluorescence, circular dichroism (CD) spectroscopy, FTIR, and differential scanning calorimetry (DSC). The intrinsic tryptophan fluorescence measurements mainly show that Al(III) may bind to the clusters nearby W11 and W34 in the WW domain of Pin1, quenching the intrinsic fluorescence of the two tryptophan residues, which possibly results in the decreased binding affinity of Pin1 to substrates. The secondary structural analysis as revealed by FTIR and CD measurements indicate that Al(III) induces the increase in β-sheet and the decrease in α-helix in Pin1. Furthermore, the changes of the thermodynamic parameters for Pin1 as monitored by DSC confirm that the thermal stability of Pin1 significantly increases in the presence of Al(III). The Al(III)-induced structural changes of Pin1 result in a sharp decrease of the PPIase activity of Pin1. To some extent, our research is suggestive that Al(III) may inhibit the isomerization activity of Pin1 in vivo, which may contribute to the pathogenesis of AD. Copyright © 2013. Published by Elsevier Inc.

  2. Analysis of peptidyl-propyl-cis/trans isomerase 1 (PIN1) gene -842(G > C) and -667(T > C) polymorphic variants in relation to breast cancer risk and clinico-pathological parameters.

    PubMed

    Naidu, Rakesh; Har, Yip C; Taib, Nur A M

    2011-10-01

    The purpose of this study was to investigate the association between the peptidyl-propyl-cis/trans isomerase 1 (PIN1) -842(G > C) and -667(T > C) polymorphic variants and breast cancer risk among Malaysian ethnic groups namely the Malays, Chinese and Indians, as well as clinico-pathological characteristics of the patients. The polymerase chain reaction-restriction fragment length polymorphism was used to genotype 387 breast cancer patients and 252 normal and healthy women who had no history of any malignancy. The distribution of -842(G > C) and -667(T > C) genotypes and alleles frequencies between breast cancer cases and normal individuals showed lack of statistical significance among the Malays (p > 0.05), Chinese (p > 0.05) and Indians (p > 0.05), respectively. Multivariate logistic regression analysis showed that the Malay, Chinese and Indian women who were -842CC homozygotes (p = 0.198, 0.089, 0.620), -842GC heterozygotes (p = 0.492, 0.176, 0.377) and -842C allele carriers (P = 0.226, 0.059, 0.669), respectively, were not associated with breast cancer risk. Furthermore Malay, Chinese and Indian women who were heterozygous (p = 0.777, 0.319, 0.710) and homozygous (p = 0.864, 0.986, 0.954) for -667C allele or carriers of -667C allele (p = 0.977, 0.915, 0.880), respectively, were not associated with an increased risk of breast cancer. None of the -842C and -667C allele genotypes were significantly associated with the clinico-pathological characteristics. Our findings suggest that the polymorphic variants of -842(G > C) and -667(T > C) genes may not appear to have an influence on breast cancer risk among Malaysian Malay, Chinese and Indian women.

  3. Homodimerization of the G Protein Srbeta in the Nucleotide-Free State Involves Proline cis/trans Isomerication in the Switch II Region

    SciTech Connect

    Schwartz,T.; Schmidt, D.; Brohawn, S.; Blobel, G.

    2006-01-01

    Protein translocation across and insertion into membranes is essential to all life forms. Signal peptide-bearing nascent polypeptide chains emerging from the ribosome are first sampled by the signal-recognition particle (SRP), then targeted to the membrane via the SRP receptor (SR), and, finally, transferred to the protein-conducting channel. In eukaryotes, this process is tightly controlled by the concerted action of three G proteins, the 54-kD subunit of SRP and the {alpha}- and {beta}-subunits of SR. We have determined the 2.2-Angstroms crystal structure of the nucleotide-free SR{beta} domain. Unexpectedly, the structure is a homodimer with a highly intertwined interface made up of residues from the switch regions of the G domain. The remodeling of the switch regions does not resemble any of the known G protein switch mechanisms. Biochemical analysis confirms homodimerization in vitro, which is incompatible with SR{alpha} binding. The switch mechanism involves cis/trans isomerization of a strictly conserved proline, potentially implying a new layer of regulation of cotranslational transport.

  4. Homodimerization of the G protein SRβ in the nucleotide-free state involves proline cis/trans isomerization in the switch II region

    PubMed Central

    Schwartz, Thomas U.; Schmidt, Daniel; Brohawn, Stephen G.; Blobel, Günter

    2006-01-01

    Protein translocation across and insertion into membranes is essential to all life forms. Signal peptide-bearing nascent polypeptide chains emerging from the ribosome are first sampled by the signal-recognition particle (SRP), then targeted to the membrane via the SRP receptor (SR), and, finally, transferred to the protein-conducting channel. In eukaryotes, this process is tightly controlled by the concerted action of three G proteins, the 54-kD subunit of SRP and the α- and β-subunits of SR. We have determined the 2.2-Å crystal structure of the nucleotide-free SRβ domain. Unexpectedly, the structure is a homodimer with a highly intertwined interface made up of residues from the switch regions of the G domain. The remodeling of the switch regions does not resemble any of the known G protein switch mechanisms. Biochemical analysis confirms homodimerization in vitro, which is incompatible with SRα binding. The switch mechanism involves cis/trans isomerization of a strictly conserved proline, potentially implying a new layer of regulation of cotranslational transport. PMID:16627619

  5. The intriguing Cyclophilin A-HIV-1 Vpr interaction: prolyl cis/trans isomerisation catalysis and specific binding

    PubMed Central

    2010-01-01

    Background Cyclophilin A (CypA) represents a potential target for antiretroviral therapy since inhibition of CypA suppresses human immunodeficiency virus type 1 (HIV-1) replication, although the mechanism through which CypA modulates HIV-1 infectivity still remains unclear. The interaction of HIV-1 viral protein R (Vpr) with the human peptidyl prolyl isomerase CypA is known to occur in vitro and in vivo. However, the nature of the interaction of CypA with Pro-35 of N-terminal Vpr has remained undefined. Results Characterization of the interactions of human CypA with N-terminal peptides of HIV-1 Vpr has been achieved using a combination of nuclear magnetic resonace (NMR) exchange spectroscopy and surface plasmon resonance spectroscopy (SPR). NMR data at atomic resolution indicate prolyl cis/trans isomerisation of the highly conserved proline residues Pro-5, -10, -14 and -35 of Vpr are catalyzed by human CypA and require only very low concentrations of the isomerase relative to that of the peptide substrates. Of the N-terminal peptides of Vpr only those containing Pro-35 bind to CypA in a biosensor assay. SPR studies of specific N-terminal peptides with decreasing numbers of residues revealed that a seven-residue motif centred at Pro-35 consisting of RHFPRIW, which under membrane-like solution conditions comprises the loop region connecting helix 1 and 2 of Vpr and the two terminal residues of helix 1, is sufficient to maintain strong specific binding. Conclusions Only N-terminal peptides of Vpr containing Pro-35, which appears to be vital for manifold functions of Vpr, bind to CypA in a biosensor assay. This indicates that Pro-35 is essential for a specific CypA-Vpr binding interaction, in contrast to the general prolyl cis/trans isomerisation observed for all proline residues of Vpr, which only involve transient enzyme-substrate interactions. Previously suggested models depicting CypA as a chaperone that plays a role in HIV-1 virulence are now supported by our data

  6. Catalysis of cis/trans isomerization in native HIV-1 capsid by human cyclophilin A

    PubMed Central

    Bosco, Daryl A.; Eisenmesser, Elan Z.; Pochapsky, Susan; Sundquist, Wesley I.; Kern, Dorothee

    2002-01-01

    Packaging of cyclophilin A (CypA) into HIV-1 virions is essential for efficient replication; however, the reason for this is unknown. Incorporation is mediated through binding to the Gly-89–Pro-90 peptide bond of the N-terminal domain of HIV-1 capsid (CAN). Despite the fact that CypA is a peptidyl-prolyl cis/trans isomerase, catalytic activity on CAN has not been observed previously. We show here, using NMR exchange spectroscopy, that CypA does not only bind to CAN but also catalyzes efficiently the cis/trans isomerization of the Gly-89–Pro-90 peptide bond. In addition, conformational changes in CAN distal to the CypA binding loop are observed on CypA binding and catalysis. The results provide experimental evidence for efficient CypA catalysis on a natively folded and biologically relevant protein substrate. PMID:11929983

  7. Refolding additive, dimethylbenzylammonium propane sulfonate (NDSB- 256), accelerates gly-pro cis-trans isomerization.

    PubMed

    Wang, Haimei; Hosoda, Kazuo; Terawaki, Shin-Ichi; Wakamatsu, Kaori

    2014-01-01

    Proline cis-trans isomerization plays a key role in the rate-determining steps of protein folding, and many different peptide-proline cis-trans isomerases (PPIases) catalyze this reaction. The acceleration of isomerization would be beneficial for in vitro refolding of protein preparations for industrial and research purposes. So we analyzed whether low-molecular-weight compounds that have been reported to enhance protein refolding have the activity to accelerate the isomerization. To evaluate the effects of chemicals on the isomerization rate, we set up a new NMR (EXSY) method that is invulnerable to their inhibitory activity, if any, and to their large NMR signals. With this method, we found that dimethylbenzylammonium propane sulfonate (NDSB-256) increase the isomerization rate in a concentration-dependent manner for the first time. Acceleration by imidazole (suggested but not experimentally confirmed) was also demonstrated. Arginine, a most popular refolding additive, did not show any significant effects on the isomerization reaction as expected.

  8. Flavylium based dual photochromism: addressing cis-trans isomerization and ring opening-closure by different light inputs.

    PubMed

    Gago, Sandra; Basílio, Nuno; Moro, Artur J; Pina, Fernando

    2015-04-30

    The multistate system of 4',7-dihydroxy-3-methoxyflavylium is constituted by a multiequilibrium involving trans-chalcone, cis-chalcone, hemiketal, flavylium cation and quinoidal base. This system possesses two independently addressable inter-connected photochromic systems based on the cis-trans isomerization and ring opening-closure of the hemiketal.

  9. Randomized Subspace Learning for Proline Cis-Trans Isomerization Prediction.

    PubMed

    Al-Jarrah, Omar Y; Yoo, Paul D; Taha, Kamal; Muhaidat, Sami; Shami, Abdallah; Zaki, Nazar

    2015-01-01

    Proline residues are common source of kinetic complications during folding. The X-Pro peptide bond is the only peptide bond for which the stability of the cis and trans conformations is comparable. The cis-trans isomerization (CTI) of X-Pro peptide bonds is a widely recognized rate-limiting factor, which can not only induces additional slow phases in protein folding but also modifies the millisecond and sub-millisecond dynamics of the protein. An accurate computational prediction of proline CTI is of great importance for the understanding of protein folding, splicing, cell signaling, and transmembrane active transport in both the human body and animals. In our earlier work, we successfully developed a biophysically motivated proline CTI predictor utilizing a novel tree-based consensus model with a powerful metalearning technique and achieved 86.58 percent Q2 accuracy and 0.74 Mcc, which is a better result than the results (70-73 percent Q2 accuracies) reported in the literature on the well-referenced benchmark dataset. In this paper, we describe experiments with novel randomized subspace learning and bootstrap seeding techniques as an extension to our earlier work, the consensus models as well as entropy-based learning methods, to obtain better accuracy through a precise and robust learning scheme for proline CTI prediction.

  10. 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

  11. Possible involvement of peptidylprolyl isomerase Pin1 in rheumatoid arthritis.

    PubMed

    Nagaoka, Akiko; Takizawa, Naohiro; Takeuchi, Ryohei; Inaba, Yutaka; Saito, Izumi; Nagashima, Yoji; Saito, Tomoyuki; Aoki, Ichiro

    2011-02-01

    The peptidylprolyl isomerase Pin1 is over-expressed in some human diseases including malignancies and chronic inflammatory diseases, this suggests that it contributes to the constitutive activation of certain intracellular signaling pathways that promote cell proliferation and cell invasion. Here, we investigate the possible role of Pin1 in rheumatoid arthritis (RA). Pin1 expression was immunohistochemically analyzed in synovial tissue (ST) obtained from patients with RA and osteoarthritis (OA). To investigate the correlation between Pin1 and motility and proliferation of synovial cells, Pin1 localization was immunohistochemically compared with matrix metalloproteinase (MMP)-1, MMP-3, and proliferating cell nuclear antigen (PCNA). Double immunofluorescent staining for Pin1 and p65 was performed to determine whether Pin1 is involved in nuclear factor κB (NF-κB) activation in RA-ST. Results showed Pin1 expression was significantly higher in RA-ST than in OA-ST. The expression of MMP-1, MMP-3, and PCNA was also significantly elevated in RA-ST. Double immunofluorescent staining revealed colocalization of Pin1 and p65 in the nuclei of RA-ST. These results suggest that Pin1 may be involved in the pathogenesis of RA binding with p65 to activate the proteins MMP-1, MMP-3, and PCNA. Therefore, Pin1 may play a pivotal role in the pathogenesis of RA. © 2010 The Authors. Pathology International © 2010 Japanese Society of Pathology and Blackwell Publishing Asia Pty Ltd.

  12. IRIS Toxicological Review of Cis-& Trans-1,2-Dichloroethylene (Final Report)

    EPA Science Inventory

    EPA has finalized the Toxicological Review of cis- & trans-1,2-Dichloroethylene: in support of the Integrated Risk Information System (IRIS). Now final, this assessment may be used by EPA’s program and regional offices to inform decisions to protect human health.

  13. IRIS Toxicological Review of Cis-& Trans-1,2-Dichloroethylene (Final Report)

    EPA Science Inventory

    EPA has finalized the Toxicological Review of cis- & trans-1,2-Dichloroethylene: in support of the Integrated Risk Information System (IRIS). Now final, this assessment may be used by EPA’s program and regional offices to inform decisions to protect human health.

  14. 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

  15. Characteristic structural features of indolicidin: effects of the cis-trans isomerism on its conformation.

    PubMed

    Leitgeb, Balázs

    2014-01-01

    Indolicidin is an antimicrobial peptide showing a broad spectrum of antibacterial and antifungal activities, and according to the cis-trans isomerism of three Xaa-Pro peptide bonds, eight different stereoisomers could be distinguished for this peptide. As the cis-trans isomerism about the Xaa-Pro peptide bonds was not considered in previous studies, the structural features of distinct stereoisomeric forms were not characterized in detail, so far. In this theoretical study, the influences of cis-trans isomerism on the conformation of indolicidin were investigated, as well as the typical structural properties of each stereoisomer were determined, focusing on the secondary structures and intramolecular interactions. Based on the results derived from the molecular dynamics simulations, it could be concluded that the eight different stereoisomeric forms of indolicidin adopted characteristic conformational features. Nevertheless, the appearance of various turn structures and intramolecular interactions proved to be dependent on the cis or trans nature of Xaa-Pro peptide bonds, indicating the relevant role of Pro amino acids in determining the three-dimensional structure of this peptide.

  16. Structural Elucidation of cis/trans Dicaffeoylquinic Acid Photoisomerization Using Ion Mobility Spectrometry-Mass Spectrometry.

    PubMed

    Zheng, Xueyun; Renslow, Ryan S; Makola, Mpho M; Webb, Ian K; Deng, Liulin; Thomas, Dennis G; Govind, Niranjan; Ibrahim, Yehia M; Kabanda, Mwadham M; Dubery, Ian A; Heyman, Heino M; Smith, Richard D; Madala, Ntakadzeni E; Baker, Erin S

    2017-04-06

    Due to the recently uncovered health benefits and anti-HIV activities of dicaffeoylquinic acids (diCQAs), understanding their structures and functions is of great interest for drug discovery efforts. DiCQAs are analytically challenging to identify and quantify since they commonly exist as a diverse mixture of positional and geometric (cis/trans) isomers. In this work, we utilized ion mobility spectrometry coupled with mass spectrometry to separate the various isomers before and after UV irradiation. The experimental collision cross sections were then compared with theoretical structures to differentiate and identify the diCQA isomers. Our analyses found that naturally the diCQAs existed predominantly as trans/trans isomers, but after 3 h of UV irradiation, cis/cis, cis/trans, trans/cis, and trans/trans isomers were all present in the mixture. This is the first report of successful differentiation of cis/trans diCQA isomers individually, which shows the great promise of IMS coupled with theoretical calculations for determining the structure and activity relationships of different isomers in drug discovery studies.

  17. Communication: An accurate calculation of the S1 C2H2 cis-trans isomerization barrier height.

    PubMed

    Baraban, Joshua H; Matthews, Devin A; Stanton, John F

    2016-03-21

    A high level ab initio calculation of the cis-trans isomerization barrier height in the first excited singlet electronic state of acetylene is found to agree very well with a recent experimental determination.

  18. Cis-trans isomerization of the (5-nitro-2-furyl)acrylamide, AF-2, initiated by ascorbate, glutathione, Fe(II) and OH-.

    PubMed

    Clarke, E D; Wardman, P; Wilson, I

    1984-01-01

    The cis-trans isomerization of the (5-nitro-2-furyl)acrylamide, AF-2, has been investigated using some important biological reducing agents to initiate reaction. Physiological concentrations of L-ascorbic acid, glutathione and iron(II) all accomplish isomerization in a catalytic manner over a period of minutes. Base-catalysed isomerization has also been observed. In all cases, the presence of oxygen severely inhibits isomerization. It is proposed that the mechanism involves a free-radical chain process; AF-2 or analogues are thus extremely sensitive probes for the generation of nitro radicals in biochemical reducing systems because of the high efficiency of isomerization.

  19. Involvement of prolyl isomerase PIN1 in the cell cycle progression and proliferation of hepatic oval cells.

    PubMed

    Risal, Prabodh; Shrestha, Nirajan; Chand, Lokendra; Sylvester, Karl G; Jeong, Yeon Jun

    2017-04-01

    Liver regenerates remarkably after toxic injury or surgical resection. In the case of failure of resident hepatocytes to restore loss, repopulation is carried out by induction, proliferation, and differentiation of the progenitor cell. Although, some signaling pathways have been verified to contribute oval cell-mediated liver regeneration, role of Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1(Pin1) in the oval cells proliferation is unknown. In the present study, we evaluate the role of Pin1 in oval cells proliferation. In our study, the expression of Pin1 in the mice liver increased after three weeks feeding of 3, 5-diethoxycarbonyl-1, 4-dihydrocollidine (DDC) diet along with the proliferation of oval cells. The expression of Pin1 was higher in oval cells compared to the hepatocytes.Pin1 inhibition by Juglone reduced oval cell proliferation, which was restored to normal when oval cells were treated with IGF-1. Consistent with increased cell growth, expression of Pin1, β-catenin and PCNA were increased in IGF-1 treated cells in a time dependent manner. In FACS analysis, siRNA-mediated knockdown of the Pin1 protein in the oval cells significantly increased the numbers of cells in G0/G1 phase. Furthermore, hepatocyte when treated with TGF-β showed marked reduction in cell proliferation and expression of Pin1 whereas this effect was not seen in the oval cells treated with TGF-β. In conclusion, Pin1 plays important role in the cell cycle progression and increase oval cells proliferation which may be crucial in chronic liver injury. Copyright © 2017 Elsevier GmbH. All rights reserved.

  20. cis-trans-Isomerization of unsaturated fatty acids during /γ-irradiation of barley grains

    NASA Astrophysics Data System (ADS)

    Geißler, Christian; Brede, Ortwin; Reinhardt, Jürgen

    2003-06-01

    Gamma-irradiating barley grains with doses of 10-100 kGy, a dose dependent isomerization of the naturally occurring cis-unsaturated fatty acids such as oleic, cis-vaccenic, linoleic and also of linolenic acid was found. Whereas the effect was negligible up to 10 kGy, at 50 kGy the trans-fatty acid level became comparable to that of other natural products like butter fat which means that there is no essential nutrition danger. The cis-trans-isomerization found in barley grains is explained mainly by a thiyl radical driven process rather than direct isomerization.

  1. Prolyl isomerases in gene transcription

    PubMed Central

    Hanes, Steven D.

    2014-01-01

    Background Peptidyl-prolyl isomerases (PPIases) are enzymes that assist in the folding of newly-synthesized proteins and regulate the stability, localization, and activity of mature proteins. They do so by catalyzing reversible (cis-trans) rotation about the peptide bond that precedes proline, inducing conformational changes in target proteins. Scope of Review This review will discuss how PPIases regulate gene transcription by controlling the activity of (1) DNA-binding transcription regulatory proteins, (2) RNA polymerase II, and (3) chromatin and histone modifying enzymes. Major Conclusions Members of each family of PPIase (cyclophilins, FKBPs, and parvulins) regulate gene transcription at multiple levels. In all but a few cases, the exact mechanisms remain elusive. Structure studies, development of specific inhibitors, and new methodologies for studying cis/trans isomerization in vivo represent some of the challenges in this new frontier that merges two important fields. General Significance Prolyl isomerases have been found to play key regulatory roles in all phases of the transcription process. Moreover, PPIases control upstream signaling pathways that regulate gene-specific transcription during development, hormone response and environmental stress. More broadly, although transcription is often rate-limiting in the production of enzymes and structural proteins, post-transcriptional modifications are also critical, and PPIases play key roles here as well (see other reviews in this issue). PMID:25450176

  2. Solvent isotope effects on retinal cis-trans isomerization in the dark adaptation of bacteriorhodopsin

    SciTech Connect

    Seltzer, S.

    1992-04-22

    The solvent isotope effect on the first-order rate constant for dark adaptation of bacteriorhodopsin, near neutral pH (pD), is inverse; k{sub D}/k{sub H} = 1.24. The fit of the variation of isotope effect with the atom fraction of deuterium in the solvent to the Gross-Butler equation leads to the conclusion that the proton(s) in motion is (are) less tightly bound in the reactant than in a reactive intermediate, formed prior to the rate-controlling step of cis-trans isomerization. The near-unity isotope effect on the equilibrium between the bound 13-cis- and all-trans-retinals in the dark-adapted state mixture indicates that the isotope effects on the forward and reverse rate constants for isomerization are approximately equal to each other and equal to the isotope effect on the observed rate constant for dark adaptation. The results support a previously proposed mechanism of rate-controlling Asp-212 nucleophilic catalysis of retinal cis-trans isomerication in the dark adaptation process. 27 refs., 2 figs., 1 tab.

  3. Classical description of the dynamics and time-resolved spectroscopy of nonadiabatic cis- trans photoisomerization

    NASA Astrophysics Data System (ADS)

    Uspenskiy, Igor; Strodel, Birgit; Stock, Gerhard

    2006-10-01

    The mapping formulation of nonadiabatic quantum dynamics is applied to obtain a classical description of the ultrafast dynamics and time-resolved spectroscopy of a photochemical reaction. Adopting a previously studied dissipative two-state two-mode model of nonadiabatic cis-trans photoisomerization, classical mapping simulations are compared to quantum-mechanical reduced density matrix calculations. Overall, the simple classical method is found to reproduce the quantum reference calculations quite well. In particular, it is studied if the classical approach yields the correct long-time cis/trans localization of the wave packet and therefore the correct quantum yield of the photoreaction. As the long-time behavior of the classical mapping formulation suffers from the well-known zero point energy problem of classical mechanics, a new practical method is proposed to determine a zero point energy correction. Employing a second-order Franck-Condon-type approximation, the capability of the classical method to simulate time- and frequency-resolved pump-probe spectra of the nonadiabatic photoreaction is studied. The potential of the classical approach as a practical method to describe condensed-phase photoreactions is discussed.

  4. Identification and functional analysis of a novel parvulin-type peptidyl-prolyl isomerase from Gossypium hirsutum.

    PubMed

    Wang, Ping; Li, Xin-Zheng; Cui, Hao-Ran; Feng, Yue-guang; Wang, Xiao-Yun

    2014-03-01

    Plants have developed a variety of adaptive mechanisms to cope with stresses. A novel salt-induced gene was isolated during the screening of a NaCl-induced cDNA library of cotton seedlings. The gene was registered as accession number AY972810 in GenBank. Phylogenetic analysis suggested that the protein encoded by the gene belongs to the parvulin family of peptidyl-prolyl cis/trans isomerases (PPIases, EC 5.2.1.8). Northern blot analysis indicated that the mRNA accumulation of GhPPI was induced by salt stress. Subcellular localization revealed that GhPPI (Gossypium hirsutum peptidyl-prolyl isomerase) was localized in the nucleus. The purified recombinant GhPPI could accelerate the initial velocity of the cis-trans conversion of peptidyl-prolyl bonds of a tetrapeptide in a GhPPI concentration-dependent manner. Recombinant GhPPI also suppressed protein aggregation under denaturing conditions using Gdn-HCl (guanidine hydrochloride), suggesting an additional chaperone activity. Several amino acid residues in GhPPI were speculated to be involved in substrate binding or catalysis based on molecular modeling and docking results. The activity of the peptidyl-prolyl isomerase was affected when the relevant amino acids were mutated. Among the 11 mutants, five amino acids mutations led to the enzyme activities decreased to 30% as that of wild type, and two reduced to approximately 60%. To the best of our knowledge, this is the first report of a plant parvulin PPIase involved in the salt stress response. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  5. Cis/trans Fluorescent Recognition by Naphthalimide Dyes ⊂ CB [7] Assembly.

    PubMed

    Li, Junyong; Gu, Xiaomin; Yuan, Xiaosheng; Qiu, Qiqi; Sun, Jie; Wang, Haibo

    2016-07-01

    A novel method to recognize cis/trans isomers was studied here. The naphthalimide dye as guest could bind with host cucurbit [7]uril (CB [7]) and 1:1 naphthalimide dye ⊂ CB [7] assembly was formed. Moreover, this assembly was used as a fluorescent probe to recognized Fumaric acid (FA) and maleic acid (MA) via fluorescence titration. Two carboxyls in MA are in the same side, they could form stable interaction with the assembly and the fluorescence intensity decreased obviously when naphthalimide dye ⊂ CB [7] was titrated by MA (nearly quenched in 1.5 equiv). But two carboxyls in FA are in opposite sides, the interaction between FA and the assembly was weak and not stable, and the fluorescence intensity changed inconspicuously when the assembly was titrated by FA.

  6. Holographic recording materials development. [development of cis-trans isomerization for holographic memory

    NASA Technical Reports Server (NTRS)

    1974-01-01

    Developments in the area of organic cis-trans isomerization systems for holographic memory applications are reported. The chemical research effort consisted of photochemical studies leading to the selection of a stilbene derivative and a polymer matrix system which have greatly improved refractive index differences between the cis and trans isomers as well as demonstrated efficiency of the photoisomerization process. In work on lithium niobate effects of sample stoichiometry and of read and write beam polarizations on recording efficiency were investigated. LiNbO3 was used for a study of angular sensitivity and of capability for simultaneous recording of extended objects without interference. The current status of LiNbO3 as a holographic recording material is summarized.

  7. Multiple gas-phase conformations of proline-containing peptides: Is it always cis/trans isomerization?

    PubMed Central

    Lietz, Christopher B.; Chen, Zhengwei; Son, Chang Yun; Pang, Xueqin; Cui, Qiang; Li, Lingjun

    2016-01-01

    Ion mobility-mass spectrometry (IM-MS) is often employed to look at the secondary, tertiary, and quaternary structure of naked peptides and proteins in the gas-phase. Recently, it has offered a unique glimpse into proline-containing peptides and their cis/trans Xxx-Pro isomers. An experimental “signature” has been identified wherein a proline-containing peptide has its Pro residues substituted with another amino acid and the presence or absence of conformations in the IM-MS spectra are observed. Despite high probability that one could attribute these conformations to cis/trans isomers, it is also possible that cis/trans isomers are not the cause of the additional conformations in proline-containing peptides. However, the experimental evidence of such a system has not been demonstrated or reported. Herein, we present the IM-MS analysis of Neuropeptide Y’s wild-type (WT) signal sequence and Leu7Pro (L7P) mutant. Although comparison of arrival times and collision cross sections of [M+4H]4+ ions yield the cis/trans “signature”, molecular dynamics indicates that a cis-Pro7 is not very stable and that trans-Pro7 conformations of the same cross section arise with equal frequency. We believe this work further underscores the importance of theoretical calculations in IM-MS structural assignments. PMID:27434776

  8. Multiple gas-phase conformations of proline-containing peptides: is it always cis/trans isomerization?

    PubMed

    Lietz, Christopher B; Chen, Zhengwei; Yun Son, Chang; Pang, Xueqin; Cui, Qiang; Li, Lingjun

    2016-08-02

    Ion mobility-mass spectrometry (IM-MS) is often employed to look at the secondary, tertiary, and quaternary structures of naked peptides and proteins in the gas-phase. Recently, it has offered a unique glimpse into proline-containing peptides and their cis/trans Xxx-Pro isomers. An experimental "signature" has been identified wherein a proline-containing peptide has its Pro residues substituted with another amino acid and the presence or absence of conformations in the IM-MS spectra is observed. Despite the high probability that one could attribute these conformations to cis/trans isomers, it is also possible that cis/trans isomers are not the cause of the additional conformations in proline-containing peptides. However, the experimental evidence of such a system has not been demonstrated or reported. Herein, we present the IM-MS analysis of Neuropeptide Y's wild-type (WT) signal sequence and Leu7Pro (L7P) mutant. Although comparison of arrival times and collision cross-sections of [M + 4H](4+) ions yields the cis/trans "signature", molecular dynamics indicates that a cis-Pro7 is not very stable and that trans-Pro7 conformations of the same cross-section arise with equal frequency. We believe that this work further underscores the importance of theoretical calculations in IM-MS structural assignments.

  9. Characterization of an isopentenyl diphosphate isomerase involved in the juvenile hormone pathway in Aedes aegypti.

    PubMed

    Diaz, Miguel E; Mayoral, Jaime G; Priestap, Horacio; Nouzova, Marcela; Rivera-Perez, Crisalejandra; Noriega, Fernando G

    2012-10-01

    Isopentenyl diphosphate isomerase (IPPI) is an enzyme involved in the synthesis of juvenile hormone (JH) in the corpora allata (CA) of insects. IPPI catalyzes the conversion of isopentenyl pyrophosphate (IPP) to dimethylallyl pyrophosphate (DMAPP); afterward IPP and DMAPP condense in a head-to-tail manner to produce geranyl diphosphate (GPP), this head-to-tail condensation can be repeated, by the further reaction of GPP with IPP, yielding the JH precursor farnesyl diphosphate. An IPPI expressed sequence tag (EST) was obtained from an Aedes aegypti corpora-allata + corpora cardiaca library. Its full-length cDNA encodes a 244-aa protein that shows a high degree of similarity with type I IPPIs from other organisms, particularly for those residues that have important roles in catalysis, metal coordination and interaction with the diphosphate moiety of the IPP. Heterologous expression produced a recombinant protein that metabolized IPP into DMAPP; treatment of DMAPP with phosphoric acid produced isoprene, a volatile compound that was measured with an assay based on a solid-phase micro extraction protocol and direct analysis by gas chromatography. A. aegypti IPPI (AaIPPI) required Mg(2+) or Mn(2+) but not Zn(2+) for full activity and it was entirely inhibited by iodoacetamide. Real time PCR experiments showed that AaIPPI is highly expressed in the CA. Changes in AaIPPI mRNA levels in the CA in the pupal and adult female mosquito corresponded well with changes in JH synthesis (Li et al., 2003). This is the first molecular and functional characterization of an isopentenyl diphosphate isomerase involved in the production of juvenile hormone in the CA of an insect.

  10. Structure of the conical intersections driving the cis-trans photoisomerization of conjugated molecules.

    PubMed

    Ruiz, Diego Sampedro; Cembran, Alessandro; Garavelli, Marco; Olivucci, Massimo; Fuss, Werner

    2002-12-01

    High-level ab initio calculations show that the singlet photochemical cis-trans isomerization of organic molecules under isolated conditions can occur according to two distinct mechanisms. These mechanisms are characterized by the different structures of the conical intersection funnels controlling photoproduct formation. In nonpolar (e.g. hydrocarbon) polyenes the lowest-lying funnel corresponds to a (CH)3 kink with both double and adjacent single bonds twisted, which may initiate hula-twist (HT) isomerization. On the other hand, in polar conjugated systems such as protonated Schiff bases (PSB) the funnel shows a structure with just one twisted double bond. The ground-state relaxation paths departing from the funnels indicate that the HT motion may take place in nonpolar conjugated systems but also that the single-bond twist may be turned back, whereas in free conjugated polar molecules such as PSB a one-bond flip mechanism dominates from the beginning. The available experimental evidence either supports these predictions or is at least consistent with them.

  11. Energy resolved tandem mass spectrometry experiments for resolution of isobaric compounds: a case of cis/trans isomerism.

    PubMed

    Menicatti, Marta; Guandalini, Luca; Dei, Silvia; Floriddia, Elisa; Teodori, Elisabetta; Traldi, Pietro; Bartolucci, Gianluca

    A series of N-alkanol-N-cyclohexanol amine aryl esters cis/trans isomers that showed high efficacy to reverse the acquired resistance of cancer cells during chemotherapeutic therapy (MDR mechanism) was studied. These compounds were two 1,4 cyclohexane cis/trans derivatives (named ELF26A and ELF26B, respectively), and their positional isomers (named ELF34A and ELF34B, respectively) where the aryl-moieties were exchanged. In order to evaluate the behaviour of these compounds during biological tests, a method based on liquid chromatography coupled with mass spectrometry (LC-MS), operating in tandem mass spectrometry (MS/MS) mode, was developed. A unique chromatographic method suitable to separate the two pairs of cis/trans isomers was not achieved and the MS/MS experiments of the different compounds was not always able to characterise the different isomers. Therefore, a system of linear equations of deconvolution analysis (LEDA) tool was proposed to determine the relative proportions of individual cis/trans isomers in the sample. Considering the pharmaceutical interest of the compounds under investigation, the analytical method developed was tested to be effective at the active concentration levels, corresponding to a concentration of ng mL(-1) of compound in a processed sample. Precision and accuracy of the LEDA algorithm at three levels of relative concentrations of analytes were checked, i.e. low-level (about 25% in the mixture), mid-level (about 50% in the mixture) and high-level (about 70% in the mixture). Evaluation of performances of the algorithm proved that the accuracy (between 88.3% and 99.9%) and precision (between 2.0% and 3.7%) for simultaneous analysis of the mixtures of the four isomers is feasible. It is worth highlighting that the choice of characteristic product ions and optimal abundance ratios plays an important role in the application of the LEDA approach. Therefore, performing an investigation on the energetics of fragmentation pathway allowed

  12. Peptidyl prolyl isomerase Pin1-inhibitory activity of D-glutamic and D-aspartic acid derivatives bearing a cyclic aliphatic amine moiety.

    PubMed

    Nakagawa, Hidehiko; Seike, Suguru; Sugimoto, Masatoshi; Ieda, Naoya; Kawaguchi, Mitsuyasu; Suzuki, Takayoshi; Miyata, Naoki

    2015-12-01

    Pin1 is a peptidyl prolyl isomerase that specifically catalyzes cis-trans isomerization of phosphorylated Thr/Ser-Pro peptide bonds in substrate proteins and peptides. Pin1 is involved in many important cellular processes, including cancer progression, so it is a potential target of cancer therapy. We designed and synthesized a novel series of Pin1 inhibitors based on a glutamic acid or aspartic acid scaffold bearing an aromatic moiety to provide a hydrophobic surface and a cyclic aliphatic amine moiety with affinity for the proline-binding site of Pin1. Glutamic acid derivatives bearing cycloalkylamino and phenylthiazole groups showed potent Pin1-inhibitory activity comparable with that of known inhibitor VER-1. The results indicate that steric interaction of the cyclic alkyl amine moiety with binding site residues plays a key role in enhancing Pin1-inhibitory activity.

  13. cis-trans photoisomerization of 1,3,5,7-octatetraene in n-hexane at 4.2 K

    PubMed Central

    Granville, Mark F.; Holtom, Gary R.; Kohler, Bryan E.

    1980-01-01

    Photoisomerization of the linear polyene 1,3,5,7-octatetraene has been observed in an n-hexane matrix maintained at the boiling point of helium. To a good approximation, only the trans,trans and cis,trans isomers participate in the photochemistry. These compounds have been unambiguously identified by comparing the observed high-resolution fluorescence spectra to those of chromatographically purified reference compounds. Although the quantum yield of this process is probably low, its microscopic rate seems to compete favorably with vibrational deactivation. PMID:16592751

  14. Cyclophilin 20 is involved in mitochondrial protein folding in cooperation with molecular chaperones Hsp70 and Hsp60.

    PubMed

    Rassow, J; Mohrs, K; Koidl, S; Barthelmess, I B; Pfanner, N; Tropschug, M

    1995-05-01

    We studied the role of mitochondrial cyclophilin 20 (CyP20), a peptidyl-prolyl cis-trans isomerase, in preprotein translocation across the mitochondrial membranes and protein folding inside the organelle. The inhibitory drug cyclosporin A did not impair membrane translocation of preproteins, but it delayed the folding of an imported protein in wild-type mitochondria. Similarly, Neurospora crassa mitochondria lacking CyP20 efficiently imported preproteins into the matrix, but folding of an imported protein was significantly delayed, indicating that CyP20 is involved in protein folding in the matrix. The slow folding in the mutant mitochondria was not inhibited by cyclosporin A. Folding intermediates of precursor molecules reversibly accumulated at the molecular chaperones Hsp70 and Hsp60 in the matrix. We conclude that CyP20 is a component of the mitochondrial protein folding machinery and that it cooperates with Hsp70 and Hsp60. It is speculated that peptidyl-prolyl cis-trans isomerases in other cellular compartments may similarly promote protein folding in cooperation with chaperone proteins.

  15. Mechanistic control of product selectivity. Reactions between cis-/trans cis-/trans-[OsVI(tpy)(Cl)2(N)]+ and triphenylphosphine sulfide.

    PubMed

    Huynh, M H; White, P S; Meyer, T J

    2000-06-26

    Reactions between the Os(VI)-nitrido complexes cis- and trans-[Os(VI)(tpy)(Cl)2(N)]+ (tpy is 2,2':6',2"-terpyridine) and triphenylphosphine sulfide, SPPh3, give the corresponding Os(IV)-phosphoraniminato, [Os(IV)(tpy)(Cl)2(NPPh3)]+, and Os(II)-thionitrosyl, [Os(II)(tpy)(Cl)2(NS)]+, complexes as products. The Os-N bond length and Os-N-P angle in cis-[Os(IV)(tpy)(Cl)2(NPPh3)](PF6) are 2.077(6) A and 138.4(4) degrees. The rate law for formation of cis- and trans-[Os(IV)(tpy)(Cl)2(NPPh3)]+ is first order in both [Os(VI)(tpy)(Cl)2(N)]+ and SPPh3 with ktrans(25 degrees C, CH3CN) = 24.6 +/- 0.6 M(-1) s(-1) and kcis(25 degrees C, CH3CN) = 0.84 +/- 0.09 M(-1) s(-1). As found earlier for [Os(II)(tpm)(Cl)2(NS)]+, both cis- and trans-[Os(II)(tpy)(Cl)2(NS)]+ react with PPh3 to give [Os(IV)(tpy)(Cl)2(NPPh3)]+ and SPPh3. For both complexes, the reaction is first order in each reagent with ktrans(25 degrees C, CH3CN) = (6.79 +/- 0.08) x 10(2) M(-1) s(-1) and kcis(25 degrees C, CH3CN) = (2.30 +/- 0.07) x 10(2) M(-1) s(-1). The fact that both reactions occur rules out mechanisms involving S atom transfer. These results can be explained by invoking a common intermediate, [Os(IV)(tpy)(Cl)2(NSPPh3)]+, which undergoes further reaction with PPh3 to give [Os(IV)(tpy)(Cl)2(NPPh3)]+ and SPPh3 or with [Os(VI)(tpy)(Cl)2(N)]+ to give [Os(IV)(tpy)(Cl)2(NPPh3)]+ and [Os(II)(tpy)(Cl)2(NS)]+.

  16. Structural and dynamic implications of an effector-induced backbone amide cis-trans isomerization in cytochrome P450cam

    PubMed Central

    Asciutto, Eliana K.; Madura, Jeffry D.; Pochapsky, Susan Sondej; OuYang, Bo; Pochapsky, Thomas C.

    2009-01-01

    Experimental evidence has been provided for a functionally relevant cis-trans isomerization of the Ile 88-Pro 89 peptide bond in cytochrome P450cam (CYP101). The isomerization is proposed to be a key element of the structural reorganization leading to the catalytically competent form of CYP101 upon binding of the effector protein putidaredoxin (Pdx). A detailed comparison of the results of molecular dynamics simulations on the cis and trans conformations of substrate- and carbonmonoxy-bound ferrous CYP101 with sequence-specific Pdx-induced structural perturbations identified by nuclear magnetic resonance is presented, providing insight into the structural and dynamic consequences of the isomerization. The mechanical coupling between the Pdx binding site on the proximal face of CYP101 and the site of isomerization is described. PMID:19327368

  17. Mechanism elucidation of the cis-trans isomerization of an azole ruthenium-nitrosyl complex and its osmium counterpart.

    PubMed

    Gavriluta, Anatolie; Büchel, Gabriel E; Freitag, Leon; Novitchi, Ghenadie; Tommasino, Jean Bernard; Jeanneau, Erwann; Kuhn, Paul-Steffen; González, Leticia; Arion, Vladimir B; Luneau, Dominique

    2013-06-03

    Synthesis and X-ray diffraction structures of cis and trans isomers of ruthenium and osmium metal complexes of general formulas (nBu4N)[cis-MCl4(NO)(Hind)], where M = Ru (1) and Os (3), and (nBu4N)[trans-MCl4(NO)(Hind)], where M = Ru (2) and Os (4) and Hind = 1H-indazole are reported. Interconversion between cis and trans isomers at high temperatures (80-130 °C) has been observed and studied by NMR spectroscopy. Kinetic data indicate that isomerizations correspond to reversible first order reactions. The rates of isomerization reactions even at 110 °C are very low with rate constants of 10(-5) s(-1) and 10(-6) s(-1) for ruthenium and osmium complexes, respectively, and the estimated rate constants of isomerization at room temperature are of ca. 10(-10) s(-1). The activation parameters, which have been obtained from fitting the reaction rates at different temperatures to the Eyring equation for ruthenium [ΔH(cis-trans)‡ = 122.8 ± 1.3; ΔH(trans-cis)‡ = 138.8 ± 1.0 kJ/mol; ΔS(cis-trans)‡ = -18.7 ± 3.6; ΔS(trans-cis)‡ = 31.8 ± 2.7 J/(mol·K)] and osmium [ΔH(cis-trans)‡ = 200.7 ± 0.7; ΔH(trans-cis)‡ = 168.2 ± 0.6 kJ/mol; ΔS(cis-trans)‡ = 142.7 ± 8.9; ΔS(trans-cis)‡ = 85.9 ± 3.9 J/(mol·K)] reflect the inertness of these systems. The entropy of activation for the osmium complexes is highly positive and suggests the dissociative mechanism of isomerization. In the case of ruthenium, the activation entropy for the cis to trans isomerization is negative [-18.6 J/(mol·K)], while being positive [31.0 J/(mol·K)] for the trans to cis conversion. The thermodynamic parameters for cis to trans isomerization of [RuCl4(NO)(Hind)]-, viz. ΔH° = 13.5 ± 1.5 kJ/mol and ΔS° = -5.2 ± 3.4 J/(mol·K) indicate the low difference between the energies of cis and trans isomers. The theoretical calculation has been carried out on isomerization of ruthenium complexes with DFT methods. The dissociative, associative, and intramolecular twist isomerization

  18. Pump-Probe Spectroscopy of Thermal Cis-Trans Isomerization Process of Methyl Red in 1-Octanol

    NASA Astrophysics Data System (ADS)

    Joukai, Tatsuya; Kita, Kenji; Yano, Ryuzi

    Thermal cis-trans isomerization process of Methyl Red in 1-Octanol at room temperature was studied by pump-probe spectroscopy. The isomerization rate increased as the dye concentration was increased. However, at concentrations above ~ 3 × 10-5 mol/L, the isomerization rate became almost constant regardless of the dye concentration. We interpreted that the concentration dependence of the thermal isomerization rate was caused by the creation of hydrogen-bonded dimer molecules. When a dimer is formed, one of the dye molecules which form the dimer easily changes its form from the cis isomer to the trans isomer. As the dye concentration is increased, the ratio of dyes which form dimer molecules will increase. When the dye concentration is high enough, most dye molecules form dimer molecules. Thus the isomerization rate averaged over the dye molecules is constant.

  19. A semiclassical study of cis-trans isomerization in HONO using an interpolating moving least-squares potential

    NASA Astrophysics Data System (ADS)

    Pham, Phong; Guo, Yin

    2013-04-01

    The interpolating moving least-squares (IMLS) approach for constructing potential energy surfaces has been developed and employed in standard classical trajectory simulations in the past few years. We extend the approach to the tunneling regime by combining the IMLS fitting method and the semiclassical scheme that incorporates tunneling into classical trajectory calculations. Dynamics of cis-trans isomerization in nitrous acid (HONO) is studied as a test case to investigate various aspects of the approach such as the strategy for growing the surface, the basis set employed, the scaling of the IMLS fits, and the accuracy of the surface required for obtaining converged rate coefficients. The validity of the approach is demonstrated through comparison with other semiclassical and quantum mechanical studies on HONO.

  20. The peptidyl-prolyl isomerase motif is lacking in PmpA, the PrsA-like protein involved in the secretion machinery of Lactococcus lactis.

    PubMed

    Drouault, Sophie; Anba, Jamila; Bonneau, Sophie; Bolotin, Alexander; Ehrlich, S Dusko; Renault, Pierre

    2002-08-01

    The prsA-like gene from Lactococcus lactis encoding its single homologue to PrsA, an essential protein triggering the folding of secreted proteins in Bacillus subtilis, was characterized. This gene, annotated pmpA, encodes a lipoprotein of 309 residues whose expression is increased 7- to 10-fold when the source of nitrogen is limited. A slight increase in the expression of the PrsA-like protein (PLP) in L. lactis removed the degradation products previously observed with the Staphylococcus hyicus lipase used as a model secreted protein. This shows that PmpA either triggers the folding of the secreted lipase or activates its degradation by the cell surface protease HtrA. Unlike the case for B. subtilis, the inactivation of the gene encoding PmpA reduced only slightly the growth rate of L. lactis in standard conditions. However, it almost stopped its growth when the lipase was overexpressed in the presence of salt in the medium. Like PrsA of B. subtilis and PrtM of L. lactis, the L. lactis PmpA protein could thus have a foldase activity that facilitates protein secretion. These proteins belong to the third family of peptidyl-prolyl cis/trans-isomerases (PPIases) for which parvulin is the prototype. Almost all PLP from gram-positive bacteria contain a domain with the PPIase signature. An exception to this situation was found only in Streptococcaceae, the family to which L. lactis belongs. PLP from Streptococcus pneumoniae and Enterococcus faecalis possess this signature, but those of L. lactis, Streptococcus pyogenes, and Streptococcus mutans do not. However, secondary structure predictions suggest that the folding of PLP is conserved over the entire length of the proteins, including the unconserved signature region. The activity associated with the expression of PmpA in L. lactis and these genomic data show that either the PPIase motif is not necessary for PPIase activity or, more likely, PmpA foldase activity does not necessarily require PPIase activity.

  1. 40 CFR 180.545 - Prallethrin (RS)-2-methyl-4-oxo-3-(2-propynyl)cyclopent-2-enyl (1RS)-cis, trans-chrysanthemate...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific Tolerances § 180.545 Prallethrin (RS)-2-methyl-4...-methyl-4-oxo-3-(2-propynyl)cyclopent-2-enyl (1RS)-cis, trans-chrysanthemate as follows: (2) In or on food commodities in food handling establishments where food and food products are held, processed, prepared and/or...

  2. Transient-Absorption Spectroscopy of Cis-Trans Isomerization of N,N-dimethyl-4,4'-Azodianiline with 3D-Printed Temperature-Controlled Sample Holder

    ERIC Educational Resources Information Center

    Kosenkov, Dmytro; Shaw, James; Zuczek, Jennifer; Kholod, Yana

    2016-01-01

    The laboratory unit demonstrates a project based approach to teaching physical chemistry laboratory where upper-division undergraduates carry out a transient-absorption experiment investigating the kinetics of cis-trans isomerization of N,N-dimethyl-4,4'-azodianiline. Students participate in modification of a standard flash-photolysis spectrometer…

  3. 1H-13C HSQC NMR spectroscopy for estimating procyanidin/prodelphinidin and cis/trans flavan-3-ol ratios of condensed tannin samples: correlation with thiolysis

    USDA-ARS?s Scientific Manuscript database

    Studies with a diverse array of 22 condensed tannin (CT) fractions from 9 plant species demonstrated that procyanidin/prodelphinidin (PC/PD) and cis/trans flavan-3-ol ratios can be appraised by 1H-13C HSQC NMR. The method was developed from fractions containing 44 to ~100% CT, PC/PD ratios ranging f...

  4. Transient-Absorption Spectroscopy of Cis-Trans Isomerization of N,N-dimethyl-4,4'-Azodianiline with 3D-Printed Temperature-Controlled Sample Holder

    ERIC Educational Resources Information Center

    Kosenkov, Dmytro; Shaw, James; Zuczek, Jennifer; Kholod, Yana

    2016-01-01

    The laboratory unit demonstrates a project based approach to teaching physical chemistry laboratory where upper-division undergraduates carry out a transient-absorption experiment investigating the kinetics of cis-trans isomerization of N,N-dimethyl-4,4'-azodianiline. Students participate in modification of a standard flash-photolysis spectrometer…

  5. Structures and functions of protein disulfide isomerase family members involved in proteostasis in the endoplasmic reticulum.

    PubMed

    Okumura, Masaki; Kadokura, Hiroshi; Inaba, Kenji

    2015-06-01

    The endoplasmic reticulum (ER) is an essential cellular compartment in which an enormous number of secretory and cell surface membrane proteins are synthesized and subjected to cotranslational or posttranslational modifications, such as glycosylation and disulfide bond formation. Proper maintenance of ER protein homeostasis (sometimes termed proteostasis) is essential to avoid cellular stresses and diseases caused by abnormal proteins. Accumulating knowledge of cysteine-based redox reactions catalyzed by members of the protein disulfide isomerase (PDI) family has revealed that these enzymes play pivotal roles in productive protein folding accompanied by disulfide formation, as well as efficient ER-associated degradation accompanied by disulfide reduction. Each of PDI family members forms a protein-protein interaction with a preferential partner to fulfill a distinct function. Multiple redox pathways that utilize PDIs appear to function synergistically to attain the highest quality and productivity of the ER, even under various stress conditions. This review describes the structures, physiological functions, and cooperative actions of several essential PDIs, and provides important insights into the elaborate proteostatic mechanisms that have evolved in the extremely active and stress-sensitive ER. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Involvement of cyclophilin D in mitochondrial permeability transition induction in intact cells.

    PubMed

    Tazawa, Hidejiro; Fujita, Chisako; Machida, Kiyotaka; Osada, Hiroyuki; Ohta, Yoshihiro

    2009-01-01

    The mitochondrial permeability transition (MPT) is involved in both Ca(2+) signaling and cell death. The present study aimed to clarify the involvement of cyclophilin D, a peptidyl prolyl cis-trans isomerase (PPIase), in MPT induction in intact cells. To achieve this, we used C6 cells overexpressing wild-type or PPIase-deficient cyclophilin D, and measured the inner mitochondrial membrane permeability to calcein, a 623-Da hydrophilic fluorescent molecule, to evaluate MPT induction. In vector control cells, the percentage of MPT induction by ionomycin increased as the Ca(2+) concentration in the extracellular medium increased. This result indicates that the present method is valid for numerical evaluation of MPT induction. In C6 cells expressing the PPIase-deficient mutant, the percentage of MPT induction was significantly decreased compared with wild-type CypD-overexpressing cells or vector control cells. These results suggest that cyclophilin D is involved in MPT induction by Ca(2+) in intact cells.

  7. Unexpectedly fast cis/trans isomerization of Xaa-Pro peptide bonds in disulfide-constrained cyclic peptides.

    PubMed

    Shi, Tiesheng; Spain, Stephen M; Rabenstein, Dallas L

    2004-01-28

    Acyclic dithiol and cyclic disulfide forms of the peptides Ac-Cys-Pro-Xaa-Cys-NH2 (Xaa = Phe, His, Tyr, Gly, and Thr) and Ac-Cys-Gly-Pro-Cys-NH2 and the peptide Ac-Ala-Gly-Pro-Ala-NH2 were synthesized and characterized by mass spectrometry and NMR spectroscopy. Rate constants kct and ktc for cis-to-trans and trans-to-cis isomerization, respectively, across the Cys-Pro or Gly-Pro peptide bonds were determined by magnetization transfer NMR techniques over a range of temperatures, and activation parameters were derived from the temperature dependence of the rate constants. It was found that constraints imposed by the disulfide bond confer an unexpected rate enhancement for cis/trans isomerization, ranging from a factor of 2 to 13. It is proposed that the rate enhancements are a result of an intramolecular catalysis mechanism in which the NH proton of the Pro-Xaa peptide bond hydrogen bonds to the proline nitrogen in the transition state. The peptides Ac-Cys-Pro-Xaa-Cys-NH2 and Ac-Cys-Gly-Pro-Cys-NH2 are model compounds for proline-containing active sites of the thioredoxin superfamily of oxidoreductase enzymes; the results suggest that the backbones of the active sites of the oxidized form of these enzymes may have unusual conformational flexibility.

  8. Does the cis/trans configuration of peptide bonds in bioactive tripeptides play a role in ACE-1 enzyme inhibition?

    PubMed Central

    Siltari, Aino; Viitanen, Riikka; Kukkurainen, Sampo; Vapaatalo, Heikki; Valjakka, Jarkko

    2014-01-01

    Background The milk casein-derived bioactive tripeptides isoleucine-proline-proline (IPP) and valine-proline-proline (VPP) have been shown to prevent development of hypertension in animal models and to lower blood pressure in moderately hypertensive subjects in most but not all clinical trials. Inhibition of angiotensin-converting enzyme 1 (ACE-1) has been suggested as the explanation for these antihypertensive and beneficial vascular effects. Previously, human umbilical vein endothelial cells (HUVEC) have not been used to test ACE-1 inhibiting properties of casein derived tripeptides in vasculature. Purpose We focused on the cis/trans configurations of the peptide bonds in proline-containing tripeptides in order to discover whether the different structural properties of these peptides influence their activity in ACE-1 inhibition. We hypothesized that the configuration of proline-containing peptides plays a significant role in enzyme inhibition. Methods AutoDock 4.2 docking software was used to predict suitable peptide bond configurations of the tripeptides. Besides modeling studies, we completed ACE-1 activity measurements in vitro using HUVEC cultures. Results In HUVEC cells, both IPP and VPP inhibited ACE-1. Based on molecular docking studies, we propose that in ACE-1 inhibition IPP and VPP share a similar cis configuration between the first aliphatic (isoleucine or valine) and the second (proline) amino acid residues and more different configurations between two proline residues. In vivo experiments are needed to validate the significance of the present findings. PMID:24596454

  9. Synthesis, DFT and antimicrobial activity assays in vitro for novel cis/trans-but-2-enedioic acid esters

    NASA Astrophysics Data System (ADS)

    Ma, Yan-Long; Zhou, Ru-Jin; Zeng, Xing-Ye; An, Ya-Xiong; Qiu, Song-Shan; Nie, Li-Jun

    2014-04-01

    Six novel cis/trans-but-2-enedioic acid esters had been synthesized to discover the new bioactive molecules that could kill food-related bacteria and fungi. Their structures were analyzed by melting point, LC-MS, 1H NMR and 13C NMR. 4-(Methoxycarbonyl) phenyl ethyl fumarate (6b) was also characterized by single-crystal X-ray diffraction. Their antimicrobial activities were evaluated in vitro by measuring the minimal inhibitory concentration (MIC). Compared with the single monomethyl fumarate and methyl 4-hydroxybenzoate, these compounds had stronger antimicrobial activity against all the eight microorganisms. Among the antibacterial and antifungal compounds, 4-(methoxycarbonyl) phenyl methyl fumarate (6a) showed the best antimicrobial activity. The electronic properties of these compounds were calculated by the density functional theory (DFT) method with 6-31G (d, p) basis set. DFT studies indicated that molecular electrostatic potential (MEP) map, ELUMO, energy gap, electronegativity and electrophilicity index could be helpful to understand the various antimicrobial activities among these compounds. The antimicrobial activity of compound 6a was evaluated in vitro against Salmonellacholeraesuis subsp. choleraesuis, Lactococcus lactis subsp. lactis and Saccharomyces cerevisiae by time-kill, and it was found that compound 6a exhibited significant microbiocidal activity against the three microorganisms.

  10. Quantum coherence effects in natural light-induced processes: cis-trans photoisomerization of model retinal under incoherent excitation.

    PubMed

    Tscherbul, Timur V; Brumer, Paul

    2015-12-14

    We present a theoretical study of quantum coherence effects in the primary cis-trans photoisomerization of retinal in rhodopsin induced by incoherent solar light. Using the partial secular Bloch-Redfield quantum master equation approach based on a two-state two-mode linear vibronic coupling model of the retinal chromophore [S. Hahn and G. Stock, J. Phys. Chem. B, 2000, 104, 1146-1149], we show that a sudden turn-on of incoherent pumping can generate substantial Fano coherences among the excited states of retinal. These coherences are the most pronounced in the regime where the matrix elements of the transition dipole moment between the ground and excited eigenstates are parallel to one another. We show that even when the transition dipole moments are perpendicular (implying the absence of light-induced Fano coherence) a small amount of excited-state coherence is still generated due to the coupling to intramolecular vibrational modes and the protein environment, causing depopulation of the excited eigenstates. The overall effect of the coherences on the steady-state population and on the photoproduct quantum yield is shown to be small; however we observe a significant transient effect on the formation of the trans photoproduct, enhancing the photoreaction quantum yield by ∼11% at 200 fs. These calculations suggest that coupling to intramolecular vibrational modes and the protein environment play an important role in photoreaction dynamics, suppressing oscillations in the quantum yield associated with Fano interference.

  11. On the Structure and Function of the Phytoene Desaturase CRTI from Pantoea ananatis, a Membrane-Peripheral and FAD-Dependent Oxidase/Isomerase

    PubMed Central

    Gemmecker, Sandra; Poussin-Courmontagne, Pierre; Mailliot, Justine; McEwen, Alastair G.; Ghisla, Sandro; Al-Babili, Salim; Cavarelli, Jean; Beyer, Peter

    2012-01-01

    CRTI-type phytoene desaturases prevailing in bacteria and fungi can form lycopene directly from phytoene while plants employ two distinct desaturases and two cis-tans isomerases for the same purpose. This property renders CRTI a valuable gene to engineer provitamin A-formation to help combat vitamin A malnutrition, such as with Golden Rice. To understand the biochemical processes involved, recombinant CRTI was produced and obtained in homogeneous form that shows high enzymatic activity with the lipophilic substrate phytoene contained in phosphatidyl-choline (PC) liposome membranes. The first crystal structure of apo-CRTI reveals that CRTI belongs to the flavoprotein superfamily comprising protoporphyrinogen IX oxidoreductase and monoamine oxidase. CRTI is a membrane-peripheral oxidoreductase which utilizes FAD as the sole redox-active cofactor. Oxygen, replaceable by quinones in its absence, is needed as the terminal electron acceptor. FAD, besides its catalytic role also displays a structural function by enabling the formation of enzymatically active CRTI membrane associates. Under anaerobic conditions the enzyme can act as a carotene cis-trans isomerase. In silico-docking experiments yielded information on substrate binding sites, potential catalytic residues and is in favor of single half-site recognition of the symmetrical C40 hydrocarbon substrate. PMID:22745782

  12. Rhein exhibits antitumorigenic effects by interfering with the interaction between prolyl isomerase Pin1 and c-Jun.

    PubMed

    Cho, Jin Hyoung; Chae, Jung-Il; Shim, Jung-Hyun

    2017-03-01

    The Pin1 protein (or peptidyl-prolyl cis/trans isomerase) specifically catalyzes the cis/trans isomerization of phosphorylated serine/threonine-proline (Ser/Thr-Pro) bonds and plays an important role in many cellular events through the effects of conformational change in the function of c-Jun, its biological substrate. Pin1 expression is involved in essential cellular pathways that mediate cell proliferation, cell cycle progression, tumorigenesis and apoptosis by altering their stability and function, and it is overexpressed in various types of tumors. Pin1 phosphorylation has been regarded as a marker of Pin1 isomerase activity, and the phosphorylation of Ser/Thr-Pro on protein substrates is prerequisite for its binding activity with Pin1 and subsequent isomerization. Since phosphorylation of proteins on Ser/Thr-Pro is a key regulatory mechanism in the control of cell proliferation and transformation, Pin1 has become an attractive molecule in cancer research. Many inhibitors of Pin1 have been discovered, including several classes of both designed inhibitors and natural products. Anthraquinone compounds possess antitumor properties and have therefore been applied in human and veterinary therapeutics as active substances in medicinal products. Among the anthraquinones, rhein (4,5-dihydroxy-9,10-dioxoanthracene-2-carboxylic acid) is a monomeric anthraquinone derivative found mainly in plants in the Polygonaceae family, such as rhubarb and Polygonum cuspidatum. Recent studies have shown that rhein has numerous pharmacological activities, including antitumor effects. Here, we demonstrated the antitumorigenic effect of rhein using cell proliferation assay, anchorage-independent cell transformation, pull-down assay, luciferase promoter activity, fluorescence-activated cell sorting and western blot analysis. The rhein/Pin1 association was found to play a regulatory role in cell proliferation and neoplastic cell transformation and the binding of phosphorylated c-Jun (Ser

  13. NMR spectroscopic studies of a TAT-derived model peptide in imidazolium-based ILs: influence on chemical shifts and the cis/trans equilibrium state.

    PubMed

    Wiedemann, Christoph; Ohlenschläger, Oliver; Mrestani-Klaus, Carmen; Bordusa, Frank

    2017-09-13

    NMR spectroscopy was used to study systematically the impact of imidazolium-based ionic liquid (IL) solutions on a TAT-derived model peptide containing Xaa-Pro peptide bonds. The selected IL anions cover a wide range of the Hofmeister series of ions. Based on highly resolved one- and two-dimensional NMR spectra individual (1)H and (13)C peptide chemical shift differences were analysed and a classification of IL anions according to the Hofmeister series was derived. The observed chemical shift changes indicate significant interactions between the peptide and the ILs. In addition, we examined the impact of different ILs towards the cis/trans equilibrium state of the Xaa-Pro peptide bonds. In this context, the IL cations appear to be of exceptional importance for inducing an alteration of the native cis/trans equilibrium state of Xaa-Pro bonds in favour of the trans-isomers.

  14. Au nanoparticle scaffolds modulating intermolecular interactions among the conjugated azobenzenes chemisorbed on curved surfaces: tuning the kinetics of cis-trans isomerisation

    NASA Astrophysics Data System (ADS)

    Raimondo, Corinna; Kenens, Bart; Reinders, Federica; Mayor, Marcel; Uji-I, Hiroshi; Samorì, Paolo

    2015-08-01

    π-π Intermolecular interactions among adjacent conjugated azobenzenes chemisorbed on (non-)flat Au surfaces can be tuned by varying the curvature of the Au nanoparticles. Here we show that such interactions rule the thermal cis-trans isomerization kinetics, towards a better control on the azobenzene bistability for its optimal integration as a responsive material.π-π Intermolecular interactions among adjacent conjugated azobenzenes chemisorbed on (non-)flat Au surfaces can be tuned by varying the curvature of the Au nanoparticles. Here we show that such interactions rule the thermal cis-trans isomerization kinetics, towards a better control on the azobenzene bistability for its optimal integration as a responsive material. Electronic supplementary information (ESI) available: Physico-chemical characterization of the different sizes of nanoparticles, UV-Vis, Surface Enhanced Raman Spectroscopy (SERS), materials and methods. See DOI: 10.1039/c5nr03688g

  15. Asymmetric epoxidation of cis/trans-β-methylstyrene catalysed by immobilised Mn(salen) with different linkages: heterogenisation of homogeneous asymmetric catalysis.

    PubMed

    Zhang, Haidong; Zou, Yu; Wang, Yi-Meng; Shen, Yu; Zheng, Xuxu

    2014-06-16

    Immobilised Mn(salen) catalysts with two different linkages were studied in the asymmetric epoxidation of cis/trans-β-methylstyrene using NaClO as oxidant. The immobilised Mn(salen) complexes inside nanopores can lead to different catalytic behaviour compared with that of homogeneous Jacobsen catalyst. The rigidity of the linkage was found to be a key factor affecting the catalytic performance of immobilised catalysts. The immobilised catalyst with a rigid linkage exhibited comparable chemical selectivity, enantioselectivity and cis/trans ratio of product formation to that obtained with homogeneous Jacobsen catalysts. In contrast, the immobilised catalyst with a flexible linkage gave remarkably lower chemical selectivity, enantioselectivity and inverted cis/trans ratio compared with the results obtained with the homogeneous Jacobsen catalyst and the immobilised catalyst with rigid linkage. Thus, for immobilised Mn(salen) catalysts, a rigid linkage connecting active centres to the support is essential to obtain activity and enantioselectivity as high as those obtained in homogeneous systems.

  16. Cis-trans isomerase gene in psychrophilic Pseudomonas syringae is constitutively expressed during growth and under conditions of temperature and solvent stress.

    PubMed

    Kiran, Madanahally D; Annapoorni, Sampath; Suzuki, Iwane; Murata, Norio; Shivaji, Sisinthy

    2005-04-01

    In a recent study, we established that psychrophilic Pseudomonas syringae (Lz4W) requires trans-monounsaturated fatty acid for growth at higher temperatures (Kiran et al. in Extremophiles, 2004). It was also demonstrated that the cti gene was highly conserved and exhibited high sequence identity with cti of other Pseudomonas spp. (Kiran et al. in Extremophiles, 2004). Therefore it would be interesting to understand the expression of the cti gene so as to unravel the molecular basis of adaptation of microorganisms to high temperature. In the present study, the expression of cti was monitored by RT-PCR analysis during different growth stages and under conditions of high temperature and solvent stress in P. syringae. Results indicated that the cti gene is constitutively expressed during different stages of growth and the transcript level is unaltered even under conditions of temperature and solvent stress implying that the observed increase in trans-monounsaturated fatty acids (Kiran et al. in Extremophiles, 2004) is not under transcriptional control. A putative promoter present in the intergenic region of the metH and cti gene has also been characterized. The translation start site ATG, the Shine-Dalgarno sequence AGGA and the transcription start site "C" were also identified. These results provide evidence for the first time that the cti gene is constitutively expressed under normal conditions of growth and under conditions of temperature and solvent stress thus implying that the Cti enzyme is post-transcriptionally regulated.

  17. "There's no chasing involved": cis/trans relationships, "tranny chasers," and the future of a sex-positive trans politics.

    PubMed

    Tompkins, Avery Brooks

    2014-01-01

    This article adds to a small, but growing, body of work on trans sexualities and partnerships, and provides a much-needed inquiry into the complex and contested politics of desire when we take trans identities, bodies, and sexualities into account. Using digital ethnographic data from YouTube videos along with in-person observational data from LGBTQ and trans conferences in the U.S., Tompkins argues that a sex-positive trans politics cannot emerge in trans and trans-allied communities if the rhetoric of the "tranny chaser" continues to inform discourses of desire and attraction to trans people.

  18. Conformations of heterochiral and homochiral proline-pseudoproline segments in peptides: context dependent cis-trans peptide bond isomerization.

    PubMed

    Raghothama, Srinivasarao; Raghavender, Upadhyayula Surya; Aravinda, Subrayashastry; Shamala, Narayanaswamy; Balaram, Padmanabhan

    2009-01-01

    The pseudoproline residue (Psi Pro, L-2,2-dimethyl-1,3-thiazolidine-4-carboxylic acid) has been introduced into heterochiral diproline segments that have been previously shown to facilitate the formation of beta-hairpins, containing central two and three residue turns. NMR studies of the octapeptide Boc-Leu-Phe-Val-(D)Pro-Psi Pro-Leu-Phe-Val-OMe (1), Boc-Leu-Val-Val-(D)Pro-Psi Pro-Leu-Val-Val-OMe (2), and the nonapeptide sequence Boc-Leu-Phe-Val-(D)Pro-Psi Pro-(D)Ala-Leu-Phe-Val-OMe (3) established well-registered beta-hairpin structures in chloroform solution, with the almost exclusive population of the trans conformation for the peptide bond preceding the Psi Pro residue. The beta-hairpin conformation of 1 is confirmed by single crystal X-ray diffraction. Truncation of the strand length in Boc-Val-(D)Pro-Psi Pro-Leu-OMe (4) results in an increase in the population of the cis conformer, with a cis/trans ratio of 3.65. Replacement of Psi Pro in 4 by (L)Pro in 5, results in almost exclusive population of the trans form, resulting in an incipient beta-hairpin conformation, stabilized by two intramolecular hydrogen bonds. Further truncation of the sequence gives an appreciable rise in the population of cis conformers in the tripeptide Piv-(D)Pro-Psi Pro-Leu-OMe (6). In the homochiral segment Piv-Pro-Psi Pro-Leu-OMe (7) only the cis form is observed with the NMR evidence strongly supporting a type VIa beta-turn conformation, stabilized by a 4-->1 hydrogen bond between the Piv (CO) and Leu (3) NH groups. The crystal structure of the analog peptide 7a (Piv-Pro-Psi(H,CH3)Pro-Leu-NHMe) confirms the cis peptide bond geometry for the Pro-Psi(H,CH3)Pro peptide bond, resulting in a type VIa beta-turn conformation.

  19. Cis-trans isomerization in the S1 state of acetylene: identification of cis-well vibrational levels.

    PubMed

    Merer, Anthony J; Steeves, Adam H; Baraban, Joshua H; Bechtel, Hans A; Field, Robert W

    2011-06-28

    acetylene that was previously thought to be unobservable, as well as the first high resolution spectroscopic results describing cis-trans isomerization.

  20. The ULTRACURVATA2 Gene of Arabidopsis Encodes an FK506-Binding Protein Involved in Auxin and Brassinosteroid Signaling1

    PubMed Central

    Pérez-Pérez, José Manuel; Ponce, María Rosa; Micol, José Luis

    2004-01-01

    The dwarf ucu (ultracurvata) mutants of Arabidopsis display vegetative leaves that are spirally rolled downwards and show reduced expansion along the longitudinal axis. We have previously determined that the UCU1 gene encodes a SHAGGY/GSK3-like kinase that participates in the signaling pathways of auxins and brassinosteroids. Here, we describe four recessive alleles of the UCU2 gene, whose homozygotes display helical rotation of several organs in addition to other phenotypic traits shared with ucu1 mutants. Following a map-based strategy, we identified the UCU2 gene, which was found to encode a peptidyl-prolyl cis/trans-isomerase of the FK506-binding protein family, whose homologs in metazoans are involved in cell signaling and protein trafficking. Physiological and double mutant analyses suggest that UCU2 is required for growth and development and participates in auxin and brassinosteroid signaling. PMID:14730066

  1. Theoretical study on the torsional direction of simple ethylenoids after electronic relaxation at the conical intersection in the cis-trans photoisomerization

    NASA Astrophysics Data System (ADS)

    Amatatsu, Yoshiaki

    2015-07-01

    The conical intersections (CIXs) for the cis-trans photoisomerization of simple ethylenoids, such as ethylene, styrene and stilbene, have been calculated by complete active space self-consistent-field method. This is because we check if a simple relationship which is found in the case of fluorene-based ethylenoids is also true for simple ethylenoids. Thereby, the four CIXs, which are distinguished by the directions of wagging and rocking motions of the anionic part against the ethylenic bond, are found to be related with the torsional direction of the ethylenic bond after electronic relaxation at each CIX.

  2. MNDO barrier heights for catalyzed bicycle-pedal, hula-twist, and ordinary cis-trans isomerizations of protonated retinal Schiff base

    SciTech Connect

    Seltzer, S.

    1987-03-18

    Energy barriers to dark cis-trans isomerization in a protonated retinal Schiff base model in the presence and absence of electrostatic and nucleophilic catalysts have been calculated by the MNDO method. Three general processes - ordinary double bond isomerization, concerted isomerization about two double bonds by bicycle-pedal motion, and one-step double bond and adjacent single bond isomerization by hula-twist motion - are considered. Point negative charges or negatively charged nucleophiles near the protonated nitrogen substantially increase the barrier to cis-trans isomerization over what they would be in the absence of these agents. Negative charge or a nucleophile near C13 lowers the barrier to bicycle-pedal isomerization. Dark isomerization by a hula-twist motion required greater energy and is not substantially aided by the placement of a negative charge or nucleophile near any of the skeletal atoms in the isomerizing system. The importance of this to the mechanism of dark-light adaptation of bacteriorhodopsin is discussed.

  3. Assembly of heterodimeric luciferase after de novo synthesis of subunits in rabbit reticulocyte lysate involves hsc70 and hsp40 at a post-translational stage.

    PubMed

    Tyedmers, J; Kruse, M; Lerner, M; Demand, J; Höhfeld, J; Solsbacher, J; Volkmer, J; Zimmermann, R

    2000-06-01

    Heterodimeric luciferase from Vibrio harveyi had been established as a unique model enzyme for direct measurements of the effects of molecular chaperones and folding catalysts on protein folding and subunit assembly after de novo synthesis of subunits in rabbit reticulocyte lysate. It was observed that luciferase assembly can be separated in time from synthesis of the two subunits and that under these post-translational conditions assembly was inhibited by either ATP depletion or inhibition of peptidylprolyl cis/trans isomerases, that is, by addition of cyclosporin A or FK506. Furthermore, it was observed that the inhibitory effect of FK506 on luciferase assembly can be suppressed by addition of purified cyclophilin, thereby providing the first direct evidence for the involvement of peptidylprolyl cis/trans isomerases in protein biogenesis in the eukaryotic cytosol. Here the ATP requirement in luciferase assembly has been characterized. Depletion of either Hsp90 or CCT from reticulocyte lysate did not interfere with luciferase assembly. However, addition of purified Hsc70 stimulated luciferase assembly. While addition of purified Hsp40 did not have any effect on luciferase assembly, the stimulatory effect of Hsc70 was further increased by Hsp40. Thus, after synthesis of the two subunits in reticulocyte lysate assembly of heterodimeric luciferase involves Hsc70 and its co-chaperone Hsp40. Therefore, Hsc70 aids protein biogenesis in the eukaryotic cytosol not only at the levels of nascent polypeptide chains and precursor proteins that have to be kept competent for transport into cell organelles, but also at the level of subunits that have to be kept competent for assembly.

  4. 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.

  5. An additional function of the rough endoplasmic reticulum protein complex prolyl 3-hydroxylase 1·cartilage-associated protein·cyclophilin B: the CXXXC motif reveals disulfide isomerase activity in vitro.

    PubMed

    Ishikawa, Yoshihiro; Bächinger, Hans Peter

    2013-11-01

    Collagen biosynthesis occurs in the rough endoplasmic reticulum, and many molecular chaperones and folding enzymes are involved in this process. The folding mechanism of type I procollagen has been well characterized, and protein disulfide isomerase (PDI) has been suggested as a key player in the formation of the correct disulfide bonds in the noncollagenous carboxyl-terminal and amino-terminal propeptides. Prolyl 3-hydroxylase 1 (P3H1) forms a hetero-trimeric complex with cartilage-associated protein and cyclophilin B (CypB). This complex is a multifunctional complex acting as a prolyl 3-hydroxylase, a peptidyl prolyl cis-trans isomerase, and a molecular chaperone. Two major domains are predicted from the primary sequence of P3H1: an amino-terminal domain and a carboxyl-terminal domain corresponding to the 2-oxoglutarate- and iron-dependent dioxygenase domains similar to the α-subunit of prolyl 4-hydroxylase and lysyl hydroxylases. The amino-terminal domain contains four CXXXC sequence repeats. The primary sequence of cartilage-associated protein is homologous to the amino-terminal domain of P3H1 and also contains four CXXXC sequence repeats. However, the function of the CXXXC sequence repeats is not known. Several publications have reported that short peptides containing a CXC or a CXXC sequence show oxido-reductase activity similar to PDI in vitro. We hypothesize that CXXXC motifs have oxido-reductase activity similar to the CXXC motif in PDI. We have tested the enzyme activities on model substrates in vitro using a GCRALCG peptide and the P3H1 complex. Our results suggest that this complex could function as a disulfide isomerase in the rough endoplasmic reticulum.

  6. A cis/trans Test of the Effect of the First Enzyme for Histidine Biosynthesis on Regulation of the Histidine Operon

    PubMed Central

    Kovach, John S.; Ballesteros, Antonio O.; Meyers, Marilyn; Soria, Marco; Goldberger, Robert F.

    1973-01-01

    Previous studies showed that when triazolalanine was added to a derepressed culture of a histidine auxotroph, repression of the histidine operon occurred as though histidine had been added (6). However, when triazolalanine was added to a derepressed culture of a strain with a mutation in the first gene of the histidine operon which rendered the first enzyme for histidine biosynthesis resistant to inhibition by histidine, repression did not occur. The studies reported here represent a cis/trans test of this effect of mutations to feedback resistance. Using specially constructed merodiploid strains, we were able to show that the wild-type allele is dominant to the mutant (feedback resistant) allele and that the effect operates in trans. We conclude that the enzyme encoded by the first gene of the histidine operon exerts its regulatory effect on the operon not by acting locally at its site of synthesis, but by acting as a freely diffusible protein. PMID:4572718

  7. Interactions of Arabidopsis RS domain containing cyclophilins with SR proteins and U1 and U11 small nuclear ribonucleoprotein-specific proteins suggest their involvement in pre-mRNA Splicing.

    PubMed

    Lorkovic, Zdravko J; Lopato, Sergiy; Pexa, Monika; Lehner, Reinhard; Barta, Andrea

    2004-08-06

    Ser/Arg (SR)-rich proteins are important splicing factors in both general and alternative splicing. By binding to specific sequences on pre-mRNA and interacting with other splicing factors via their RS domain they mediate different intraspliceosomal contacts, thereby helping in splice site selection and spliceosome assembly. While characterizing new members of this protein family in Arabidopsis, we have identified two proteins, termed CypRS64 and CypRS92, consisting of an N-terminal peptidyl-prolyl cis/trans isomerase domain and a C-terminal domain with many SR/SP dipeptides. Cyclophilins possess a peptidyl-prolyl cis/trans isomerase activity and are implicated in protein folding, assembly, and transport. CypRS64 interacts in vivo and in vitro with a subset of Arabidopsis SR proteins, including SRp30 and SRp34/SR1, two homologs of mammalian SF2/ASF, known to be important for 5' splice site recognition. In addition, both cyclophilins interact with U1-70K and U11-35K, which in turn are binding partners of SRp34/SR1. CypRS64 is a nucleoplasmic protein, but in most cells expressing CypRS64-GFP fusion it was also found in one to six round nuclear bodies. However, co-expression of CypRS64 with its binding partners resulted in re-localization of CypRS64 from the nuclear bodies to nuclear speckles, indicating functional interactions. These findings together with the observation that binding of SRp34/SR1 to CypRS64 is phosphorylation-dependent indicate an involvement of CypRS64 in nuclear pre-mRNA splicing, possibly by regulating phosphorylation/dephosphorylation of SR proteins and other spliceosomal components. Alternatively, binding of CypRS64 to proteins important for 5' splice site recognition suggests its involvement in the dynamics of spliceosome assembly.

  8. Reversible equilibrium unfolding of triosephosphate isomerase from Trypanosoma cruzi in guanidinium hydrochloride involves stable dimeric and monomeric intermediates.

    PubMed

    Chánez-Cárdenas, María Elena; Pérez-Hernández, Gerardo; Sánchez-Rebollar, Brenda Guadalupe; Costas, Miguel; Vázquez-Contreras, Edgar

    2005-08-16

    The reversible guanidinium hydrochloride-induced unfolding of Trypanosoma cruzi triosephosphate isomerase (TcTIM) was characterized under equilibrium conditions. The catalytic activity was followed as a native homodimeric functional probe. Circular dichroism, intrinsic fluorescence, and size-exclusion chromatography were used as secondary, tertiary, and quaternary structural probes, respectively. The change in ANS fluorescence intensity with increasing denaturant concentrations was also determined. The results show that two stable intermediates exist in the transition from the homodimeric native enzyme to the unfolded monomers: one (N(2*)) is a slightly more expanded, non-native, and active dimer, and the other is a partially expanded monomer (M) that binds ANS. Spectroscopic and activity data were used to reach a thermodynamic characterization. The results indicate that the Gibbs free energies for the partial reactions are 4.5 (N(2) <==> N(2*)), 65.8 (N(2*) <==> 2M), and 17.8 kJ/mol (M <==> U). It appears that TcTIM monomers are more stable than those found for other TIM species (except yeast TIM), where monomer stability is only marginal. These results are compared with those for the guanidinium hydrochloride-induced denaturation of TIM from different species, where despite the functional and three-dimensional similarities, a remarkable heterogeneity exists in the unfolding pathways.

  9. Active site mutants of human cyclophilin A separate peptidyl-prolyl isomerase activity from cyclosporin A binding and calcineurin inhibition.

    PubMed Central

    Zydowsky, L. D.; Etzkorn, F. A.; Chang, H. Y.; Ferguson, S. B.; Stolz, L. A.; Ho, S. I.; Walsh, C. T.

    1992-01-01

    Based on recent X-ray structural information, six site-directed mutants of human cyclophilin A (hCyPA) involving residues in the putative active site--H54, R55, F60, Q111, F113, and H126--have been constructed, overexpressed, and purified from Escherichia coli to homogeneity. The proteins W121A (Liu, J., Chen, C.-M., & Walsh, C.T., 1991a, Biochemistry 30, 2306-2310), H54Q, R55A, F60A, Q111A, F113A, and H126Q were assayed for cis-trans peptidyl-prolyl isomerase (PPIase) activity, their ability to bind the immunosuppressive drug cyclosporin A (CsA), and protein phosphatase 2B (calcineurin) inhibition in the presence of CsA. Results indicate that H54Q, Q111A, F113A, and W121A retain 3-15% of the catalytic efficiency (kcat/Km) of wild-type recombinant hCyPA. The remaining three mutants (R55A, F60A, and H126Q) each retain less than 1% of the wild-type catalytic efficiency, indicating participation by these residues in PPIase catalysis. Each of the mutants bound to a CsA affinity matrix. The mutants R55A, F60A, F113A, and H126Q inhibited calcineurin in the presence of CsA, whereas W121A did not. Although CsA is a competitive inhibitor of PPIase activity, it can complex with enzymatically inactive cyclophilins and inhibit the phosphatase activity of calcineurin. PMID:1338979

  10. Fatty acids attached to all-trans-astaxanthin alter its cis-trans equilibrium, and consequently its stability, upon light-accelerated autoxidation.

    PubMed

    de Bruijn, Wouter J C; Weesepoel, Yannick; Vincken, Jean-Paul; Gruppen, Harry

    2016-03-01

    Fatty acid esterification, common in naturally occurring astaxanthin, has been suggested to influence both colour stability and degradation of all-trans-astaxanthin. Therefore, astaxanthin stability was studied as influenced by monoesterification and diesterification with palmitate. Increased esterification decelerated degradation of all-trans-astaxanthin (RP-UHPLC-PDA), whereas, it had no influence on colour loss over time (spectrophotometry). This difference might be explained by the observation that palmitate esterification influenced the cis-trans equilibrium. Free astaxanthin produced larger amounts of 9-cis isomer whereas monopalmitate esterification resulted in increased 13-cis isomerization. The molar ratios of 9-cis:13-cis after 60min were 1:1.7 (free), 1:4.8 (monopalmitate) and 1:2.6 (dipalmitate). The formation of 9-cis astaxanthin, with its higher molar extinction coefficient than that of all-trans-astaxanthin, might compensate for colour loss induced by conjugated double bond cleavage. As such, it was concluded that spectrophotometry is not an accurate measure of the degradation of the all-trans-astaxanthin molecule. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Effect of the medium on intramolecular H-atom tunneling: cis-trans conversion of formic acid in solid matrixes of noble gases.

    PubMed

    Trakhtenberg, Leonid I; Fokeyev, Anatoly A; Zyubin, Alexander S; Mebel, Alexander M; Lin, S H

    2010-12-30

    Intramolecular tunneling of a hydrogen atom in formic acid at low temperatures has been studied theoretically on the basis of quantum-chemical modeling of HCOOH@Nb(12) clusters. Three noble matrixes (Ar, Kr, and Xe) are considered. Energetic and geometric parameters as well as vibrational frequencies for the formic acid in cis and trans configurations surrounded by 12 Nb atoms are calculated within the frame of the MP2 approach with extended basis sets. The rate constant of HCOOH cis-trans conversion is analyzed by taking into account matrix reorganization and the change of HCOOH position in the cluster. The matrix reorganization is considered within the Debye model of lattice vibrations, whereas the external motion of HCOOH in the cluster is treated using the Einstein model of solids. It has been shown that the literature experimental data on the cis to trans tunneling reaction in the formic acid can be accounted for within the proposed mechanism, which describes the matrix reorganization and the change of the HCOOH position in the noble gas matrix, with fitting parameters of the suggested theoretical model attaining reasonable values.

  12. Effect of processing conditions on the content of cis/trans carotene isomers as provitamin A carotenoids in Korean sweet potato varieties.

    PubMed

    Kim, Heon Woong; Kim, Jung Bong; Poovan, Shanmugavelan; Chung, Mi Nam; Cho, Soo Muk; Lee, Young Min; Cho, Young Sook; Kim, Jae Hyun; Kim, Haeng Ran

    2014-11-01

    The present investigation intends to evaluate the changes in the content of cis/trans carotene isomers as provitamin A carotenoids by steaming and roasting processes in the roots of four Korean sweet potato varieties viz. Shinzami, Younwhangmi, Chuwhangmi and Jinhongmi using a liquid chromatography with diode array detection and the negative ion atmospheric pressure chemical ionization mass spectrometric (LC-DAD-APCI/MS) method and UV spectral pattern library created from several reference data. Except Shinzami, the content of all trans β-carotenes was found to slightly decreased or remained constant when steamed or roasted. The content of cis α-/β-carotenes was potentially increased about 2-fold or greater when raw or steamed and the content was slightly decreased while roasted. In Chuwhangmi, the content of 13-cis α-carotene and all trans α-carotenes were rapidly increased when steamed and slightly decreased when roasted. Chuwhangmi exhibited 27.2 mg/100 g DW content of all trans β-carotenes when roasted and thus, it was considered as a relatively superior cultivar.

  13. Prolyl isomerase Pin1 promotes proplatelet formation of megakaryocytes via tau.

    PubMed

    Shimizu, Taiki; Uchida, Chiyoko; Shimizu, Ritsuko; Motohashi, Hozumi; Uchida, Takafumi

    2017-11-18

    Here we show that Pin1, a peptidyl-prolyl cis/trans isomerase which catalyzes the isomerization of phosphorylated Ser/Thr-Pro, is a regulatory molecule of thrombopoiesis. We found that mice lacking the Pin1 gene (Pin1(-⁄-) mice) formed more megakaryocytes (MKs) than wild type mice (WT mice), and that the proplatelet formation of MKs was poorer in Pin1(-⁄-) mice than WT mice. Treatment of Meg-01 cells, a megakaryoblastic floating cell line, with shRNA against Pin1 suppressed the proplatelet formation. Expression of tau, a microtubule associated protein was induced in MKs during proplatelet formation. Pin1 bound tau and promoted microtubule polymerization. Our results show that Pin1 serves as a positive regulatory molecule of proplatelet formation of MKs by enhancing the function of phosphorylated tau. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Trypanosomatid pin1-type peptidyl-prolyl isomerase is cytosolic and not essential for cell proliferation.

    PubMed

    Erben, Esteban D; Nardelli, Sheila C; de Jesus, Teresa C L; Schenkman, Sergio; Tellez-Iñon, Maria T

    2013-01-01

    Pin1-type peptidyl-prolyl cis/trans isomerases (PPIases) isomerise the peptide bond of specific phosphorylated (Ser/Thr)-Pro residues, regulating various cellular events. Previously, we reported a Pin1-type PPIase in Trypanosoma cruzi, but little is known about its function and subcellular localization. Immunofluorescence analysis revealed that in contrast with Pin1-like proteins from diverse organisms, TcPin1 mainly localized in the cytoplasm and was excluded from the nuclei. In addition, RNAi-mediated downregulation of TbPin1 in Trypanosoma brucei did not abolish cell proliferation. Using yeast two-hybrid assay, we identified a MORN domain-containing protein as putative Pin1-binding partners. These data suggest that Pin1-mediated signaling mechanism plays a different role in protozoan parasites. © 2012 The Author(s) Journal of Eukaryotic Microbiology © 2012 International Society of Protistologists.

  15. Chiral Cyclobutane β-Amino Acid-Based Amphiphiles: Influence of Cis/Trans Stereochemistry on Condensed Phase and Monolayer Structure.

    PubMed

    Sorrenti, Alessandro; Illa, Ona; Ortuño, Rosa M; Pons, Ramon

    2016-07-12

    New diastereomeric nonionic amphiphiles, cis- and trans-1, based on an optically pure cyclobutane β-amino ester moiety have been investigated to gain insight into the influence exerted by cis/trans stereochemistry and stereochemical constraints on the physicochemical behavior, molecular organization, and morphology of their Langmuir monolayers and dry solid states. All these features are relevant to the rational design of functional materials. trans-1 showed a higher thermal stability than cis-1. For the latter, a higher fluidity of its monolayers was observed when compared with the films formed by trans-1 whose BAM images revealed the formation of condensed phase domains with a dendritic shape, which are chiral, and all of them feature the same chiral sign. Although the formation of LC phase domains was not observed by BAM for cis-1, compact dendritic crystals floating on a fluid subphase were observed beyond the collapse, which are attributable to multilayered 3D structures. These differences can be explained by the formation of hydrogen bonds between the amide groups of consecutive molecules allowing the formation of extended chains for trans-1 giving ordered arrangements. However, for cis-1, this alignment coexists with another one that allows the simultaneous formation of two hydrogen bonds between the amide and the ester groups of adjacent molecules. In addition, the propensity to form intramolecular hydrogen bonds must be considered to justify the formation of different patterns of hydrogen bonding and, consequently, the formation of less ordered phases. Those characteristics are congruent also with the results obtained from SAXS-WAXS experiments which suggest a more bent configuration for cis-1 than for trans-1.

  16. Chiral Cyclobutane β-Amino Acid-Based Amphiphiles: Influence of Cis/Trans Stereochemistry on Solution Self-Aggregation and Recognition.

    PubMed

    Sorrenti, Alessandro; Illa, Ona; Pons, Ramon; Ortuño, Rosa M

    2015-09-08

    Novel diastereomeric anionic amphiphiles based on the rigid cyclobutane β-amino acid scaffold have been synthesized and deeply investigated with the aim of generating new functional supramolecular architectures on the basis of the rational design of original amphiphilic molecules and the control of their self-assembly. The main interest has been focused on the effect that cis/trans stereochemistry exerts on their molecular organization and recognition. In diluted solutions, the relative stereochemistry mainly influences the headgroup solvation and anionic-charge stabilization, i.e., better stabilized in the cis diastereoisomer due to intramolecular hydrogen-bonding and/or charge-dipole interactions. This provokes differences in their physicochemical behavior (pKa, cmc, conductivity) as well as in the structural parameters of the spherical micelles formed. Although both diastereoisomers form fibers that evolve with time from the spherical micelles, they display markedly different morphology and kinetics of formation. In the lyotropic liquid crystal domain, the greatest differences are observed at the highest concentrations and can be ascribed to different hydrogen-bonding and molecular packing imposed by the stereochemical constraints. Remarkably, the spherical micelles of the two anionic surfactants show dramatically diverse enantioselection ability for bilirubin enantiomers. In addition, both the surfactants form heteroaggregates with bilirubin at submicellar concentrations but with a different expression of supramolecular chirality. This points out that the unlike relative configuration of the two surfactants influences their chiral recognition ability as well as the fashion in which chirality is expressed at the supramolecular level by controlling the molecular organization in both micellar aggregates and surfactant/bilirubin heteroaggregates. All these differential features can be appropriate and useful for the design and development of new soft materials with

  17. A Crystallographic Study of Bright Far-Red Fluorescent Protein mKate Reveals pH-induced cis-trans Isomerization of the Chromophore

    SciTech Connect

    Pletnev, Sergei; Shcherbo, Dmitry; Chudakov, Dmitry M.; Pletneva, Nadezhda; Merzlyak, Ekaterina M.; Wlodawer, Alexander; Dauter, Zbigniew; Pletnev, Vladimir

    2008-11-03

    The far-red fluorescent protein mKate {lambda}{sup ex}, 588 nm; {lambda}{sub em}, 635 nm; chromophore-forming triad Met{sup 63}-Tyr{sup 64}-Gly{sup 65}, originating from wild-type red fluorescent progenitor eqFP578 (sea anemone Entacmaea quadricolor), is monomeric and characterized by the pronounced pH dependence of fluorescence, relatively high brightness, and high photostability. The protein has been crystallized at a pH ranging from 2 to 9 in three space groups, and four structures have been determined by x-ray crystallography at the resolution of 1.75--2.6 {angstrom}. The pH-dependent fluorescence of mKate has been shown to be due to reversible cis-trans isomerization of the chromophore phenolic ring. In the non-fluorescent state at pH 2.0, the chromophore of mKate is in the trans-isomeric form. The weakly fluorescent state of the protein at pH 4.2 is characterized by a mixture of trans and cis isomers. The chromophore in a highly fluorescent state at pH 7.0/9.0 adopts the cis form. Three key residues, Ser{sup 143}, Leu{sup 174}, and Arg{sup 197} residing in the vicinity of the chromophore, have been identified as being primarily responsible for the far-red shift in the spectra. A group of residues consisting of Val{sup 93}, Arg{sup 122}, Glu{sup 155}, Arg{sup 157}, Asp{sup 159}, His{sup 169}, Ile{sup 171}, Asn{sup 173}, Val{sup 192}, Tyr{sup 194}, and Val{sup 216}, are most likely responsible for the observed monomeric state of the protein in solution.

  18. Synthesis of 7-azabicyclo[2.2.1]heptane and 2-oxa-4-azabicyclo[3.3.1]non-3-ene derivatives by base-promoted heterocyclization of alkyl N-(cis(trans)-3,trans(cis)-4-dibromocyclohex-1-yl)carbamates and N-(cis(trans)-3,trans(cis)-4-dibromocyclohex-1-yl)-2,2,2-trifluoroacetamides.

    PubMed

    Gómez-Sanchez, Elena; Soriano, Elena; Marco-Contelles, José

    2007-11-09

    We have studied the base-promoted heterocyclization of alkyl N-(cis(trans)-3,trans(cis)-4-dibromocyclohex-1-yl)carbamates and N-(cis(trans)-3,trans(cis)-4-dibromocyclohex-1-yl)-2,2,2-trifluoroacetamides, investigating the effect of the nitrogen protecting group and the relative configuration of the leaving group at C3 and C4 on the outcome of this reaction. We have observed that the sodium hydride-promoted heterocyclization of alkyl N-(cis-3,trans-4-dibromocyclohex-1-yl)carbamates (10, 12, 14, 16, 18) is a convenient method for the synthesis of 7-azabicyclo[2.2.1]heptane derivatives. For instance, the reaction of tert-butyl N-(cis-3,trans-4-dibromocyclohex-1-yl)carbamate (10) with sodium hydride in DMF at room temperature provides 2-bromo-7-[(tert-butoxy)carbonyl]-7-azabicyclo[2.2.1]heptane (2) (52% yield), whose t-BuOK-promoted hydrogen bromide elimination affords 7-[(tert-butoxy)carbonyl]-7-azabicyclo[2.2.1]hept-2-ene (31) in 78% yield, an intermediate in the total synthesis of epibatidine (1). However, the NaH/DMF-mediated heterocyclization of alkyl N-(trans-3,cis-4-dibromocyclohex-1-yl)carbamates (11, 13) is a more structure dependent reaction, where the nucleophilic attack of the oxygen atom of the protecting group controls the outcome of the reaction, giving rise to benzooxazolone and 2-oxa-4-azabicyclo[3.3.1]non-3-ene derivatives, respectively, from low to moderate yields, in complex reaction mixtures. Conversely, the NaH/DMF heterocyclizations of N-(cis-3,trans-4-dibromocyclohex-1-yl)-2,2,2-trifluoroacetamide (40) or N-(trans-3,cis-4-dibromocyclohex-1-yl)-2,2,2-trifluoroacetamide (42) are very clean reactions giving 7-azabicyclo[2.2.1]heptane or 2-oxa-4-azabicyclo[3.3.1]non-3-ene derivatives, respectively, in good yields. Finally, a mechanistic investigation, based on DFT calculations, has been carried out to rationalize the formation of the different adducts.

  19. 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

  20. An on-line SPE-HPLC method for effective sample preconcentration and determination of fenoxycarb and cis, trans-permethrin in surface waters.

    PubMed

    Šatínský, Dalibor; Naibrtová, Linda; Fernández-Ramos, Carolina; Solich, Petr

    2015-09-01

    A new on-line SPE-HPLC method using fused-core columns for on-line solid phase extraction and large volume sample injection for increasing the sensitivity of detection was developed for the determination of insecticides fenoxycarb and cis-, trans-permethrin in surface waters. The separation was carried out on fused-core column Phenyl-Hexyl (100×4.6 mm), particle size 2.7 µm with mobile phase acetonitrile:water in gradient mode at flow rate 1.0 mL min(-1), column temperature 45°C. Large volume sample injection (1500 µL) to the extraction dimension using short precolumn Ascentis Express RP C-18 (5×4.6 mm); fused-core particle size 2.7 µm allowed effective sample preconcentration and efficient ballast sample matrix removal. The washing mobile phase consisting of a mixture of acetonitrile:water; 30:70, (v/v) was pumped at flow rate of 0.5 mL min(-1) through the extraction precolumn to the waste. Time of the valve switch for transferring the preconcentrated sample zone from the extraction to the separation column was set at 3rd min. Elution of preconcentrated insecticides from the extraction precolumn and separation on the analytical column was performed in gradient mode. Linear gradient elution started from 40% of acetonitrile at time of valve switch from SPE column (3rd min) to 95% of acetonitrile at 7th min. Synthetic dye sudan I was chosen as an internal standard. UV detection at wavelength 225 nm was used and the method reached the limits of detection (LOD) at ng mL(-1) levels for both insecticides. The method showing on-line sample pretreatment and preconcentration with highly sensitive determination of insecticides was applied for monitoring of fenoxycarb and both permethrin isomers in different surface water samples in Czech Republic. The time of whole analysis including on-line extraction, interferences removal, chromatography separation and system equilibration was less than 8 min. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. The Prolyl Isomerase Pin1 Promotes the Herpesvirus-Induced Phosphorylation-Dependent Disassembly of the Nuclear Lamina Required for Nucleocytoplasmic Egress

    PubMed Central

    Milbradt, Jens; Hutterer, Corina; Bahsi, Hanife; Wagner, Sabrina; Sonntag, Eric; Kaufer, Benedikt B.; Mori, Yasuko; Sticht, Heinrich; Fossen, Torgils; Marschall, Manfred

    2016-01-01

    The nuclear lamina lines the inner nuclear membrane providing a structural framework for the nucleus. Cellular processes, such as nuclear envelope breakdown during mitosis or nuclear export of large ribonucleoprotein complexes, are functionally linked to the disassembly of the nuclear lamina. In general, lamina disassembly is mediated by phosphorylation, but the precise molecular mechanism is still not completely understood. Recently, we suggested a novel mechanism for lamina disassembly during the nuclear egress of herpesviral capsids which involves the cellular isomerase Pin1. In this study, we focused on mechanistic details of herpesviral nuclear replication to demonstrate the general importance of Pin1 for lamina disassembly. In particular, Ser22-specific lamin phosphorylation consistently generates a Pin1-binding motif in cells infected with human and animal alpha-, beta-, and gammaherpesviruses. Using nuclear magnetic resonance spectroscopy, we showed that binding of Pin1 to a synthetic lamin peptide induces its cis/trans isomerization in vitro. A detailed bioinformatic evaluation strongly suggests that this structural conversion induces large-scale secondary structural changes in the lamin N-terminus. Thus, we concluded that a Pin1-induced conformational change of lamins may represent the molecular trigger responsible for lamina disassembly. Consistent with this concept, pharmacological inhibition of Pin1 activity blocked lamina disassembly in herpesvirus-infected fibroblasts and consequently impaired virus replication. In addition, a phospho-mimetic Ser22Glu lamin mutant was still able to form a regular lamina structure and overexpression of a Ser22-phosphorylating kinase did not induce lamina disassembly in Pin1 knockout cells. Intriguingly, this was observed in absence of herpesvirus infection proposing a broader importance of Pin1 for lamina constitution. Thus, our results suggest a functional model of similar events leading to disassembly of the nuclear

  2. Synthesis and characterization of fac-[M(CO)3(P)(OO)] and cis-trans-[M(CO)2(P)2(OO)] complexes (M = Re, (99m)Tc) with acetylacetone and curcumin as OO donor bidentate ligands.

    PubMed

    Triantis, Charalampos; Tsotakos, Theodoros; Tsoukalas, Charalampos; Sagnou, Marina; Raptopoulou, Catherine; Terzis, Aris; Psycharis, Vassilis; Pelecanou, Maria; Pirmettis, Ioannis; Papadopoulos, Minas

    2013-11-18

    The synthesis and characterization of neutral mixed ligand complexes fac-[M(CO)3(P)(OO)] and cis-trans-[M(CO)2(P)2(OO)] (M = Re, (99m)Tc), with deprotonated acetylacetone or curcumin as the OO donor bidentate ligands and a phosphine (triphenylphosphine or methyldiphenylphosphine) as the monodentate P ligand, is described. The complexes were synthesized through the corresponding fac-[M(CO)3(H2O)(OO)] (M = Re, (99m)Tc) intermediate aqua complex. In the presence of phosphine, replacement of the H2O molecule of the intermediate complex at room temperature generates the neutral tricarbonyl monophosphine fac-[Re(CO)3(P)(OO)] complex, while under reflux conditions further replacement of the trans to the phosphine carbonyl generates the new stable dicarbonyl bisphosphine complex cis-trans-[Re(CO)2(P)2(OO)]. The Re complexes were fully characterized by elemental analysis, spectroscopic methods, and X-ray crystallography showing a distorted octahedral geometry around Re. Both the monophosphine and the bisphosphine complexes of curcumin show selective binding to β-amyloid plaques of Alzheimer's disease. At the (99m)Tc tracer level, the same type of complexes, fac-[(99m)Tc(CO)3(P)(OO)] and cis-trans-[(99m)Tc(CO)2(P)2(OO)], are formed introducing new donor combinations for (99m)Tc(I). Overall, β-diketonate and phosphine constitute a versatile ligand combination for Re(I) and (99m)Tc(I), and the successful employment of the multipotent curcumin as β-diketone provides a solid example of the pharmacological potential of this system.

  3. Evidence for the Involvement of Acid/Base Chemistry in the Reaction Catalyzed by the Type II Isopentenyl Diphosphate/Dimethylallyl Diphosphate Isomerase from Staphylococcus aureus†

    PubMed Central

    Thibodeaux, Christopher J.; Mansoorabadi, Steven O.; Kittleman, William; Chang, Wei-chen; Liu, Hung-wen

    2011-01-01

    The type II isopentenyl diphosphate/dimethylallyl diphosphate isomerase (IDI-2) is a flavin mononucleotide (FMN)-dependent enzyme that catalyzes the reversible isomerization of isopentenyl pyrophosphate (IPP) to dimethylallyl pyrophosphate (DMAPP), a reaction with no net change in redox state of the coenzyme or substrate. Here, UV-vis spectral analysis of the IDI-2 reaction revealed the accumulation of a reduced neutral dihydroflavin intermediate when the reduced enzyme was incubated with IPP or DMAPP. When IDI-2 was reconstituted with 1-deazaFMN and 5-deazaFMN, similar reduced neutral forms of the deazaflavin analogues were observed in the presence of IPP. Single turnover stopped-flow absorbance experiments indicated that this flavin intermediate formed and decayed at kinetically competent rates in the pre-steady-state and, thus, most likely represents a true intermediate in the catalytic cycle. UV-vis spectra of the reaction mixtures reveal trace amounts of a neutral semiquinone, but evidence for the presence of IPP-based radicals could not be obtained by EPR spectroscopy. Rapid-mix chemical quench experiments show no burst in DMAPP formation, suggesting that the rate determining step in the forward direction (IPP to DMAPP) occurs prior to DMAPP formation. A solvent deuterium kinetic isotope effect (D2OVmax = 1.5) was measured on vo in steady-state kinetic experiments at saturating substrate concentrations. A substrate deuterium kinetic isotope effect was also measured on the initital velocity (DVmax = 1.8) and on the decay rate of the flavin intermediate (Dks = 2.3) in single-turnover stopped-flow experiments using (R)-[2-2H]-IPP. Taken together, these data suggest that the C2–H bond of IPP is cleaved in the rate determining step and that general acid/base catalysis may be involved during turnover. Possible mechanisms for the IDI-2 catalyzed reaction are presented and discussed in terms of the available X-ray crystal structures. PMID:18229948

  4. Autophagy regulated by prolyl isomerase Pin1 and phospho-Ser-GSK3αβ involved in protection of oral squamous cell carcinoma against cadmium toxicity

    SciTech Connect

    So, Keum-Young; Ahn, Sang-Gun; Oh, Seon-Hee

    2015-10-23

    Prolyl isomerase Pin1 plays an important role in cell proliferation and is overexpressed in many human tumors. However, its role in autophagy induction remains undefined. Here we show that Pin1 regulates cell survival via autophagy in cadmium (Cd)-exposed oral squamous cell carcinoma (OSCC). OSCC exposure to Cd induced autophagy, as demonstrated by the formation of green fluorescent punctae in transfected cells expressing GFP-conjugated microtubule-associated protein light chain 3 (LC3) and by LC3 flux in the presence of autophagy inhibitors. Suppression of Atg5 enhanced Cd-induced apoptosis, indicating that autophagy is involved in cell protection. In dose–response experiments, cleavage of procaspase-3, PARP-1, and LC3-II was induced by Cd with an IC{sub 50} of 45 μM. Expression of Pin1 was decreased at or above the Cd IC{sub 50} value and was inversely correlated with the level of phospho(p)-Ser-GSK3αβ. Genetic or pharmacologic inhibition of Pin1 suppressed Cd-induced autophagy, but increased p-Akt-mediated p-Ser-GSK3αβ; this was reversed by overexpression of Pin1. However, suppression of GSK3αβ inhibited Cd-induced autophagy and induced apoptosis, which could be reversed by overexpression of GSK3β. The PI3K inhibitor Ly294002 blocked p-Akt-mediated increases in p-Ser-GSK3αβ and autophagy and induced apoptosis. Therefore, p-Ser-GSK3αβ can directly regulate Cd-induced autophagy, although its function is suppressed by Pin1. Collectively, the present results indicate that targeting Pin1 and GSK3αβ at the same time could be an effective therapeutic tool for Cd-induced carcinogenesis. - Highlights: • Pin1 regulated autophagy to protect cells from cadmium toxicity. • Pin1 suppression inhibited cadmium-induced autophagy and induced apoptosis. • Pin1 inhibited the function of p-Ser-GSK3αβ in autophagy regulation. • p-Ser-GSK3αβ regulated autophagy independently of Pin1.

  5. Development of a mariner-based transposon and identification of Listeria monocytogenes determinants, including the peptidyl-prolyl isomerase PrsA2, that contribute to its hemolytic phenotype.

    PubMed

    Zemansky, Jason; Kline, Benjamin C; Woodward, Joshua J; Leber, Jess H; Marquis, Hélène; Portnoy, Daniel A

    2009-06-01

    Listeriolysin O (LLO) is a pore-forming toxin that mediates phagosomal escape and cell-to-cell spread of the intracellular pathogen Listeria monocytogenes. In order to identify factors that control the production, activity, or secretion of this essential virulence factor, we constructed a Himar1 mariner transposon delivery system and screened 50,000 mutants for a hypohemolytic phenotype on blood agar plates. Approximately 200 hypohemolytic mutants were identified, and the 51 most prominent mutants were screened ex vivo for intracellular growth defects. Eight mutants with a phenotype were identified, and they contained insertions in the following genes: lmo0964 (similar to yjbH), lmo1268 (clpX), lmo1401 (similar to ymdB), lmo1575 (similar to ytqI), lmo1695 (mprF), lmo1821 (similar to prpC), lmo2219 (prsA2), and lmo2460 (similar to cggR). Some of these genes are involved in previously unexplored areas of research with L. monocytogenes: the genes yjbH and clpX regulate the disulfide stress response in Bacillus subtilis, and the prpC phosphatase has been implicated in virulence in other gram-positive pathogens. Here we demonstrate that prsA2, an extracytoplasmic peptidyl-prolyl cis/trans isomerase, is critical for virulence and contributes to the folding of LLO and to the activity of another virulence factor, the broad-range phospholipase C (PC-PLC). Furthermore, although it has been shown that prsA2 expression is linked to PrfA, the master virulence transcription factor in L. monocytogenes pathogenesis, we demonstrate that prsA2 is not directly controlled by PrfA. Finally, we show that PrsA2 is involved in flagellum-based motility, indicating that this factor likely serves a broad physiological role.

  6. Development of a mariner-Based Transposon and Identification of Listeria monocytogenes Determinants, Including the Peptidyl-Prolyl Isomerase PrsA2, That Contribute to Its Hemolytic Phenotype▿

    PubMed Central

    Zemansky, Jason; Kline, Benjamin C.; Woodward, Joshua J.; Leber, Jess H.; Marquis, Hélène; Portnoy, Daniel A.

    2009-01-01

    Listeriolysin O (LLO) is a pore-forming toxin that mediates phagosomal escape and cell-to-cell spread of the intracellular pathogen Listeria monocytogenes. In order to identify factors that control the production, activity, or secretion of this essential virulence factor, we constructed a Himar1 mariner transposon delivery system and screened 50,000 mutants for a hypohemolytic phenotype on blood agar plates. Approximately 200 hypohemolytic mutants were identified, and the 51 most prominent mutants were screened ex vivo for intracellular growth defects. Eight mutants with a phenotype were identified, and they contained insertions in the following genes: lmo0964 (similar to yjbH), lmo1268 (clpX), lmo1401 (similar to ymdB), lmo1575 (similar to ytqI), lmo1695 (mprF), lmo1821 (similar to prpC), lmo2219 (prsA2), and lmo2460 (similar to cggR). Some of these genes are involved in previously unexplored areas of research with L. monocytogenes: the genes yjbH and clpX regulate the disulfide stress response in Bacillus subtilis, and the prpC phosphatase has been implicated in virulence in other gram-positive pathogens. Here we demonstrate that prsA2, an extracytoplasmic peptidyl-prolyl cis/trans isomerase, is critical for virulence and contributes to the folding of LLO and to the activity of another virulence factor, the broad-range phospholipase C (PC-PLC). Furthermore, although it has been shown that prsA2 expression is linked to PrfA, the master virulence transcription factor in L. monocytogenes pathogenesis, we demonstrate that prsA2 is not directly controlled by PrfA. Finally, we show that PrsA2 is involved in flagellum-based motility, indicating that this factor likely serves a broad physiological role. PMID:19376879

  7. Epistasis in tomato color mutations involves regulation of phytoene synthase 1 expression by cis-carotenoids.

    PubMed

    Kachanovsky, David E; Filler, Shdema; Isaacson, Tal; Hirschberg, Joseph

    2012-11-13

    Tomato (Solanum lycopersicum) fruit accumulate the red carotenoid pigment lycopene. The recessive mutation yellow-flesh (locus r) in tomato eliminates fruit carotenoids by disrupting the activity of the fruit-specific phytoene synthase (PSY1), the first committed step in the carotenoid biosynthesis pathway. Fruits of the recessive mutation tangerine (t) appear orange due to accumulation of 7,9,7',9'-tetra-cis-lycopene (prolycopene) as a result of a mutation in the carotenoid cis-trans isomerase. It was established 60 y ago that tangerine is epistatic to yellow-flesh. This uncharacteristic epistasis interaction defies a paradigm in biochemical genetics arguing that mutations that disrupt enzymes acting early in a biosynthetic pathway are epistatic to other mutations that block downstream steps in the same pathway. To explain this conundrum, we have investigated the interaction between tangerine and yellow-flesh at the molecular level. Results presented here indicate that allele r(2997) of yellow-flesh eliminates transcription of PSY1 in fruits. In a genetic background of tangerine, transcription of PSY1 is partially restored to a level sufficient for producing phytoene and downstream carotenoids. Our results revealed the molecular mechanism underlying the epistasis of t over r and suggest the involvement of cis-carotenoid metabolites in a feedback regulation of PSY1 gene expression.

  8. Involvement of lipoprotein PpiA of Streptococcus gordonii in evasion of phagocytosis by macrophages.

    PubMed

    Cho, K; Arimoto, T; Igarashi, T; Yamamoto, M

    2013-10-01

    Streptococcus gordonii is a commensal gram-positive bacterium that resides in the human oral cavity, and is one of the most common causes of infective endocarditis (IE). Bacterial surface molecules play an important role in establishing IE, and several S. gordonii proteins have been implicated in binding to host cells during the establishment of IE. In this study, we identified a putative lipoprotein, peptidyl-prolyl cis/trans isomerase (PpiA), and clarified its role in evasion of phagocytosis by macrophages. Attenuation of the gene encoding prolipoprotein diacylglyceryl transferase (Lgt) altered the localization of PpiA from the cell surface to the culture supernatant, indicating that PpiA is lipid-anchored in the cell membrane by Lgt. Both human and murine macrophages showed higher phagocytic activity towards ppiA and lgt mutants than the wild-type, indicating that the presence of PpiA suppresses phagocytosis of S. gordonii. Human macrophages treated with dextran sulfate had significantly impaired phagocytosis of S. gordonii, suggesting that class A scavenger receptors in human macrophages are involved in the phagocytosis of S. gordonii. These results provide evidence that S. gordonii lipoprotein PpiA plays an important role in inhibiting phagocytic engulfment and in evasion of the host immune response.

  9. Determination of the cis-trans isomerization barriers of L-alanyl-L-proline in aqueous solutions and at water/hydrophobic interfaces by on-line temperature-jump relaxation HPLC and dynamic on-column reaction HPLC.

    PubMed

    Shibukawa, Masami; Miyake, Ayaka; Eda, Sayaka; Saito, Shingo

    2015-09-15

    Proline cis-trans isomerization is known to play a key role in the rate-determining steps of protein folding. It is thus very important to understand the influence of environments, not only bulk solutions but also microenvironments such as interfaces, on the isomerization reaction of proline peptides. Here we present two HPLC methods for measurements of kinetic and equilibrium parameters for the isomerization reactions in bulk solutions and at liquid/solid interfaces. On-line temperature-jump relaxation HPLC (T-jump HPLC) allows the determination of forward and reverse rate constants of the isomerization in a bulk solution by monitoring the whole time course of conversion of pure isomers from both sides of the reaction, in contrast to other HPLC and capillary zone electrophoresis as well as spectrometric and calorimetric methods, which use a mixture of the isomers. We can then determine cis-trans isomerization barriers of the peptide at liquid/solid interfaces from the kinetic data obtained by dynamic on-column reaction HPLC and T-jump HPLC. We observed that the interconversion around the peptide bond for l-alanyl-l-proline (Ala-Pro) in water is accelerated at the surfaces of an alkyl-bonded silica and a poly(styrene-divinylbenzene) copolymer resin, and this is caused by a remarkable decrease in the enthalpy of activation. The molecular structures of the cis and trans forms of Ala-Pro estimated by quantum mechanics calculation reveal that an equilibrium shift toward the cis form as well as the rapid isomerization of Ala-Pro at the water/hydrophobic interfaces can be attributed to the lower polarity of the interfacial water at the surfaces of the hydrophobic materials compared to that of bulk water.

  10. 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.

  11. A Burkholderia pseudomallei Macrophage Infectivity Potentiator-Like Protein Has Rapamycin-Inhibitable Peptidylprolyl Isomerase Activity and Pleiotropic Effects on Virulence ▿

    PubMed Central

    Norville, Isobel H.; Harmer, Nicholas J.; Harding, Sarah V.; Fischer, Gunter; Keith, Karen E.; Brown, Katherine A.; Sarkar-Tyson, Mitali; Titball, Richard W.

    2011-01-01

    Macrophage infectivity potentiators (Mips) are a group of virulence factors encoded by pathogenic bacteria such as Legionella, Chlamydia, and Neisseria species. Mips are part of the FK506-binding protein (FKBP) family, whose members typically exhibit peptidylprolyl cis-trans isomerase (PPIase) activity which is inhibitable by the immunosuppressants FK506 and rapamycin. Here we describe the identification and characterization of BPSS1823, a Mip-like protein in the intracellular pathogen Burkholderia pseudomallei. Recombinant BPSS1823 protein has rapamycin-inhibitable PPIase activity, indicating that it is a functional FKBP. A mutant strain generated by deletion of BPSS1823 in B. pseudomallei exhibited a reduced ability to survive within cells and significant attenuation in vivo, suggesting that BPSS1823 is important for B. pseudomallei virulence. In addition, pleiotropic effects were observed with a reduction in virulence mechanisms, including resistance to host killing mechanisms, swarming motility, and protease production. PMID:21859853

  12. The Phosphinomethylmalate Isomerase Gene pmi, Encoding an Aconitase-Like Enzyme, Is Involved in the Synthesis of Phosphinothricin Tripeptide in Streptomyces viridochromogenes

    PubMed Central

    Heinzelmann, E.; Kienzlen, G.; Kaspar, S.; Recktenwald, J.; Wohlleben, W.; Schwartz, D.

    2001-01-01

    Streptomyces viridochromogenes Tü494 produces the antibiotic phosphinothricin tripeptide (PTT). In the postulated biosynthetic pathway, one reaction, the isomerization of phosphinomethylmalate, resembles the aconitase reaction of the tricarboxylic acid (TCA) cycle. It was speculated that this reaction is carried out by the corresponding enzyme of the primary metabolism (C. J. Thompson and H. Seto, p. 197–222, in L. C. Vining and C. Stuttard, ed., Genetics and Biochemistry of Antibiotic Production, 1995). However, in addition to the TCA cycle aconitase gene, a gene encoding an aconitase-like protein (the phosphinomethylmalate isomerase gene, pmi) was identified in the PTT biosynthetic gene cluster by Southern hybridization experiments, using oligonucleotides which were derived from conserved amino acid sequences of aconitases. The deduced protein revealed high similarity to aconitases from plants, bacteria, and fungi and to iron regulatory proteins from eucaryotes. Pmi and the S. viridochromogenes TCA cycle aconitase, AcnA, have 52% identity. By gene insertion mutagenesis, a pmi mutant (Mapra1) was generated. The mutant failed to produce PTT, indicating the inability of AcnA to carry out the secondary-metabolism reaction. A His-tagged protein (Hispmi*) was heterologously produced in Streptomyces lividans. The purified protein showed no standard aconitase activity with citrate as a substrate, and the corresponding gene was not able to complement an acnA mutant. This indicates that Pmi and AcnA are highly specific for their respective enzymatic reactions. PMID:11472937

  13. Ziploc-ing the structure 2.0: Endoplasmic reticulum-resident peptidyl prolyl isomerases show different activities toward hydroxyproline.

    PubMed

    Ishikawa, Yoshihiro; Mizuno, Kazunori; Bächinger, Hans Peter

    2017-06-02

    Extracellular matrix proteins are biosynthesized in the rough endoplasmic reticulum (rER), and the triple-helical protein collagen is the most abundant extracellular matrix component in the human body. Many enzymes, molecular chaperones, and post-translational modifiers facilitate collagen biosynthesis. Collagen contains a large number of proline residues, so the cis/trans isomerization of proline peptide bonds is the rate-limiting step during triple-helix formation. Accordingly, the rER-resident peptidyl prolyl cis/trans isomerases (PPIases) play an important role in the zipper-like triple-helix formation in collagen. We previously described this process as "Ziploc-ing the structure" and now provide additional information on the activity of individual rER PPIases. We investigated the substrate preferences of these PPIases in vitro using type III collagen, the unhydroxylated quarter fragment of type III collagen, and synthetic peptides as substrates. We observed changes in activity of six rER-resident PPIases, cyclophilin B (encoded by the PPIB gene), FKBP13 (FKBP2), FKBP19 (FKBP11), FKBP22 (FKBP14), FKBP23 (FKBP7), and FKBP65 (FKBP10), due to posttranslational modifications of proline residues in the substrate. Cyclophilin B and FKBP13 exhibited much lower activity toward post-translationally modified substrates. In contrast, FKBP19, FKBP22, and FKBP65 showed increased activity toward hydroxyproline-containing peptide substrates. Moreover, FKBP22 showed a hydroxyproline-dependent effect by increasing the amount of refolded type III collagen in vitro and FKBP19 seems to interact with triple helical type I collagen. Therefore, we propose that hydroxyproline modulates the rate of Ziploc-ing of the triple helix of collagen in the rER. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Synergistic Interaction of Light Alcohol Administration in the Presence of Mild Iron Overload in a Mouse Model of Liver Injury: Involvement of Triosephosphate Isomerase Nitration and Inactivation.

    PubMed

    Gao, Wanxia; Zhao, Jie; Gao, Zhonghong; Li, Hailing

    2017-01-01

    It is well known that iron overload promotes alcoholic liver injury, but the doses of iron or alcohol used in studies are usually able to induce liver injury independently. Little attention has been paid to the coexistence of low alcohol consumption and mild iron overload when either of them is insufficient to cause obvious liver damage, although this situation is very common among some people. We studied the interactive effects and the underlining mechanism of mild doses of iron and alcohol on liver injury in a mouse model. Forty eight male Kunming mice were randomly divided into four groups: control, iron (300 mg/kg iron dextran, i.p.), alcohol (2 g/kg/day ethanol for four weeks i.g.), and iron plus alcohol group. After 4 weeks of treatment, mice were sacrificed and blood and livers were collected for biochemical analysis. Protein nitration level in liver tissue was determined by immunoprecipitation and Western blot analysis. Although neither iron overload nor alcohol consumption at our tested doses can cause severe liver injury, it was found that co-administration of the same doses of alcohol and iron resulted in liver injury and hepatic dysfunction, accompanied with elevated ratio of NADH/NAD+, reduced antioxidant ability, increased oxidative stress, and subsequent elevated protein nitration level. Further study revealed that triosephosphate isomerase, an important glycolytic enzyme, was one of the targets to be oxidized and nitrated, which was responsible for its inactivation. These data indicate that even under low alcohol intake, a certain amount of iron overload can cause significant liver oxidative damage, and the modification of triosephosphate isomerasemight be the important underlining mechanism of hepatic dysfunction.

  15. Synergistic Interaction of Light Alcohol Administration in the Presence of Mild Iron Overload in a Mouse Model of Liver Injury: Involvement of Triosephosphate Isomerase Nitration and Inactivation

    PubMed Central

    Gao, Wanxia; Zhao, Jie; Gao, Zhonghong

    2017-01-01

    It is well known that iron overload promotes alcoholic liver injury, but the doses of iron or alcohol used in studies are usually able to induce liver injury independently. Little attention has been paid to the coexistence of low alcohol consumption and mild iron overload when either of them is insufficient to cause obvious liver damage, although this situation is very common among some people. We studied the interactive effects and the underlining mechanism of mild doses of iron and alcohol on liver injury in a mouse model. Forty eight male Kunming mice were randomly divided into four groups: control, iron (300 mg/kg iron dextran, i.p.), alcohol (2 g/kg/day ethanol for four weeks i.g.), and iron plus alcohol group. After 4 weeks of treatment, mice were sacrificed and blood and livers were collected for biochemical analysis. Protein nitration level in liver tissue was determined by immunoprecipitation and Western blot analysis. Although neither iron overload nor alcohol consumption at our tested doses can cause severe liver injury, it was found that co-administration of the same doses of alcohol and iron resulted in liver injury and hepatic dysfunction, accompanied with elevated ratio of NADH/NAD+, reduced antioxidant ability, increased oxidative stress, and subsequent elevated protein nitration level. Further study revealed that triosephosphate isomerase, an important glycolytic enzyme, was one of the targets to be oxidized and nitrated, which was responsible for its inactivation. These data indicate that even under low alcohol intake, a certain amount of iron overload can cause significant liver oxidative damage, and the modification of triosephosphate isomerasemight be the important underlining mechanism of hepatic dysfunction. PMID:28103293

  16. The Ess1 prolyl isomerase: Traffic cop of the RNA polymerase II transcription

    PubMed Central

    Hanes, Steven D.

    2014-01-01

    Ess1 is a prolyl isomerase that regulates the structure and function of eukaryotic RNA polymerase II. Ess1 works by catalyzing the cis/trans conversion of pSer5–Pro6 bonds, and to a lesser extent pSer2–Pro3 bonds, within the carboxy-terminal domain (CTD) of Rpb1, the largest subunit of RNA pol II. Ess1 is conserved in organisms ranging from yeast to humans. In budding yeast, Ess1 is essential for growth and is required for efficient transcription initiation and termination, RNA processing, and suppression of cryptic transcription. In mammals, Ess1 (called Pin1) functions in a variety of pathways, including transcription, but it is not essential. Recent work has shown that Ess1 coordinates the binding and release of CTD-binding proteins that function as co-factors in the RNA pol II complex. In this way, Ess1 plays an integral role in writing (and reading) the so-called CTD code to promote production of mature RNA pol II transcripts including non-coding RNAs and mRNAs. PMID:24530645

  17. Probing cis-trans isomerization in the S{sub 1} state of C{sub 2}H{sub 2} via H-atom action and hot band-pumped IR-UV double resonance spectroscopies

    SciTech Connect

    Changala, P. Bryan; Baraban, Joshua H.; Field, Robert W.; Merer, Anthony J.

    2015-08-28

    We report novel experimental strategies that should prove instrumental in extending the vibrational and rotational assignments of the S{sub 1} state of acetylene, C{sub 2}H{sub 2}, in the region of the cis-trans isomerization barrier. At present, the assignments are essentially complete up to ∼500 cm{sup −1} below the barrier. Two difficulties arise when the assignments are continued to higher energies. One is that predissociation into C{sub 2}H + H sets in roughly 1100 cm{sup −1} below the barrier; the resulting quenching of laser-induced fluorescence (LIF) reduces its value for recording spectra in this region. The other difficulty is that tunneling through the barrier causes a staggering in the K-rotational structure of isomerizing vibrational levels. The assignment of these levels requires data for K values up to at least 3. Given the rotational selection rule K′ − ℓ{sup ′′} = ± 1, such data must be obtained via excited vibrational levels of the ground state with ℓ{sup ′′} > 0. In this paper, high resolution H-atom resonance-enhanced multiphoton ionization spectra are demonstrated to contain predissociated bands which are almost invisible in LIF spectra, while preliminary data using a hyperthermal pulsed nozzle show that ℓ{sup ′′} = 2 states can be selectively populated in a jet, giving access to K′ = 3 states in IR-UV double resonance.

  18. Zero-point Energy is Needed in Molecular Dynamics Calculations to Access the Saddle Point for H+HCN→H2CN* and cis/trans-HCNH* on a New Potential Energy Surface.

    PubMed

    Wang, Xiaohong; Bowman, Joel M

    2013-02-12

    We calculate the probabilities for the association reactions H+HCN→H2CN* and cis/trans-HCNH*, using quasiclassical trajectory (QCT) and classical trajectory (CT) calculations, on a new global ab initio potential energy surface (PES) for H2CN including the reaction channels. The surface is a linear least-squares fit of roughly 60 000 CCSD(T)-F12b/aug-cc-pVDZ electronic energies, using a permutationally invariant basis with Morse-type variables. The reaction probabilities are obtained at a variety of collision energies and impact parameters. Large differences in the threshold energies in the two types of dynamics calculations are traced to the absence of zero-point energy in the CT calculations. We argue that the QCT threshold energy is the realistic one. In addition, trajectories find a direct pathway to trans-HCNH, even though there is no obvious transition state (TS) for this pathway. Instead the saddle point (SP) for the addition to cis-HCNH is evidently also the TS for direct formation of trans-HCNH.

  19. 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.

  20. Evidence of Sulfur Mustard Exposure in Human Plasma by LC-ESI-MS-MS Detection of the Albumin-Derived Alkylated HETE-CP Dipeptide and Chromatographic Investigation of Its Cis/Trans Isomerism.

    PubMed

    Gandor, Felix; Gawlik, Michael; Thiermann, Horst; John, Harald

    2015-05-01

    Sulfur mustard (SM) is a chemical warfare agent that causes painful blisters and chemically modifies endogenous biomacromolecules by alkylation to hydroxyethylthioethyl (HETE) adducts representing valuable long-term markers for post-exposure analysis. The albumin adduct formed in human plasma in vitro (HETE bound to the side chain of cysteine 34) was isolated and cleaved by current lots of pronase primarily generating the internal modified dipeptide (HETE-cysteine-proline, HETE-CP) instead of the formerly reported HETE-CPF tripeptide. The analyte was detected by liquid chromatography-electrospray ionization tandem-mass spectrometry (LC-ESI-MS-MS). In principle, HETE-CP undergoes a dynamic on-column equilibrium of cis-trans isomerism thus requiring separation at 50°C to obtain one narrow peak. Accordingly, we developed both a novel longer lasting but more sensitive microbore (1 mm i.d., flow 30 µL/min, cycle time 60 min, LOD 50 nM) and a faster, less sensitive narrowbore (2.1 mm i.d., 200 µL/min, cycle time 16 min, LOD 100 nM, both on Atlantis T3 material at 50°C) LC-ESI-MS-MS method suitable for verification analysis. The corresponding tri- and tetrapeptide, Q(HETE)-CPF were monitored simultaneously. HETE-CP peak areas were directly proportional to SM concentrations added to plasma in vitro (0.05-100 µM). Albumin adducts formed by deuterated SM (d8-SM) served as internal standard.

  1. Unusual temperature dependence in the cis/trans-oxetane formation discloses competitive syn versus anti attack for the Paternò-Büchi reaction of triplet-excited ketones with cis- and trans-cylooctenes. Conformational control of diastereoselectivity in the cyclization and cleavage of preoxetane diradicals.

    PubMed

    Adam, Waldemar; Stegmann, Veit R

    2002-04-10

    Toluene-d(8) solutions of cis- and trans-cyclooctene (cis- and trans-1a) as well as (Z)- and (E)-1-methylcyclooctene (cis- and trans-1b) have been irradiated at temperatures between -95 and +110 degrees C in the presence of benzophenone (BP) to afford mixtures of the cis- and trans-configured oxetanes 2a,b and the regioisomeric 2b'. Correspondingly, benzoquinone (BQ) gave with cis- and trans-1a the cycloadducts cis- and trans-3a. The cis/trans diastereomeric ratios of the [2 + 2]-cycloadducts 2 and 3 display a strong temperature dependence; with cis- and trans-1a or cis-1b as starting materials, the diastereoselectivity of the oxetane formation is high at low temperature, under preservation of the initial cyclooctene configuration. With increasing temperature, the cis diastereoselectivity decreases continuously for the cis-cyclooctenes; in the case of the cis-1a, the diastereoselectivity is even switched to trans (cis/trans ca. 20:80) at very high temperatures. For the strained trans-1a, the trans-oxetanes are strongly preferred over the entire temperature range, with only minor leakage (up to 10%) to the cis-oxetanes at very high temperatures. Oxetane formation is accompanied by nonthermal trans-to-cis isomerization of the cyclooctene. The methyl-substituted trans-1b constitutes an exceptional substrate; it displays cis diastereoselectivity in the [2 + 2] photocycloaddition at low temperatures for both regioisomers 2b and 2b', and the trans selectivity increases at moderate temperature (cis/trans = 4:96), to decrease again at high temperature, especially for the minor regioisomer 2b'. This complex temperature behavior of the cis/trans diastereoselectivity may be rationalized in terms of the triplet-diradical mechanism of the Paternò-Büchi reaction. We propose that the cyclooctene may be competitively attacked by the triplet-excited ketone from the higher (syn) or the less (anti) substituted side; such syn and anti trajectories have hitherto not been considered

  2. 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 ...

  3. Allergens involved in the cross-reactivity of Aedes aegypti with other arthropods.

    PubMed

    Cantillo, Jose Fernando; Puerta, Leonardo; Lafosse-Marin, Sylvie; Subiza, Jose Luis; Caraballo, Luis; Fernandez-Caldas, Enrique

    2017-06-01

    Cross-reactivity between Aedes aegypti and mites, cockroaches, and shrimp has been previously suggested, but the involved molecular components have not been fully described. To evaluate the cross-reactivity between A aegypti and other arthropods. Thirty-four serum samples from patients with asthma and/or allergic rhinitis were selected, and specific IgE to A aegypti, Dermatophagoides pteronyssinus, Dermatophagoides farinae, Blomia tropicalis, Periplaneta americana. and Litopenaeus vannamei was measured by enzyme-linked immunosorbent assay. Cross-reactivity was investigated using pooled serum samples from allergic patients, allergenic extracts, and the recombinant tropomyosins (Aed a 10.0201, Der p 10, Blo t 10, Lit v 1, and Per a 7). Four IgE reactive bands were further characterized by matrix-assisted laser desorption/ionization tandem time of flight. Frequency of positive IgE reactivity was 82.35% to at least one mite species, 64.7% to A aegypti, 29.4% to P americana, and 23.5% to L vannamei. The highest IgE cross-reactivity was seen between A aegypti and D pteronyssinus (96.6%) followed by L vannamei (95.4%), B tropicalis (84.4%), and P americana (75.4%). Recombinant tropomyosins from mites, cockroach, or shrimp inhibited the IgE reactivity to the mosquito at a lower extent than the extracts from these arthropods. Several bands of A aegypti cross-reacted with arthropod extracts, and 4 of them were identified as odorant binding protein, mitochondrial cytochrome C, peptidyl-prolyl cis-trans isomerase, and protein with hypothetical magnesium ion binding function. We identified 4 novel cross-reactive allergens in A aegypti allergenic extract. These molecules could influence the manifestation of allergy to environmental allergens in the tropics. Copyright © 2017 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  4. Identification of phosphorylated proteins involved in the oncogenesis of prostate cancer via Pin1-proteomic analysis.

    PubMed

    Endoh, Kanji; Nishi, Mayuko; Ishiguro, Hitoshi; Uemura, Hiroji; Miyagi, Yohei; Aoki, Ichiro; Hirano, Hisashi; Kubota, Yoshinobu; Ryo, Akihide

    2012-05-01

    The peptidyl-prolyl isomerase Pin1 regulates a subset of phosphorylated proteins by catalyzing the cis-trans isomerization of their specific phosphorylated Ser/Thr-Pro motifs. Although Pin1 has been shown to be involved in cell transformation and the maintenance of the malignant phenotype in prostate cancer, its specific substrates during these processes have not yet been determined. Cancer-specific phosphorylated proteins were isolated from two human prostate cancer cell lines (PC-3, LNCaP) and the Dunning rat prostate cancer cell lines by GST-pull down analysis with recombinant GST-Pin1 protein. These proteins were then identified by the LC-MS/MS analysis using a Q-Tof micro mass spectrometer and processed for further functional analysis. We newly identified five prostate cancer-specific Pin1 binding proteins (PINBPs) in this screen. Among these, TRK-fused gene (TFG) was found to be preferentially up-regulated in prostate cancer cell lines and tissues. The targeted inhibition of TFG by specific siRNA resulted in the reduced cell proliferation and the induction of premature senescence in PC3 prostate cancer cells. We further found that TFG can facilitate the cell signaling mediated by NF-kappaB and androgen receptor (AR). Tissue micro-dissection based quantitative RT-PCR analysis of prostate cancer tissues following radical prostatectomy further revealed that TFG expression is closely associated with both a higher probability and shorter period of tumor recurrence following surgery. Pin1-based proteomics analysis is a useful tool for the identification of prostate cancer-specific phosphorylated proteins. TFG could be a potential diagnostic and/or prognostic marker and therapeutic target in prostate cancer. Copyright © 2011 Wiley Periodicals, Inc.

  5. 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

  6. Brown Algae Polyphenol, a Prolyl Isomerase Pin1 Inhibitor, Prevents Obesity by Inhibiting the Differentiation of Stem Cells into Adipocytes.

    PubMed

    Suzuki, Atsuko; Saeki, Toshiyuki; Ikuji, Hiroko; Uchida, Chiyoko; Uchida, Takafumi

    2016-01-01

    While screening for an inhibitor of the peptidyl prolyl cis/trans isomerase, Pin1, we came across a brown algae polyphenol that blocks the differentiation of fibroblasts into adipocytes. However, its effectiveness on the accumulation of fat in the body has never been studied. Oral administration of brown algae polyphenol to mice fed with a high fat diet, suppressed the increase in fat volume to a level observed in mice fed with a normal diet. We speculate that Pin1 might be required for the differentiation of stem cell to adipocytes. We established wild type (WT) and Pin1-/- (Pin1-KO) adipose-derived mesenchymal stem cell (ASC) lines and found that WT ASCs differentiate to adipocytes but Pin1-KO ASCs do not. Oral administration of brown algae polyphenol, a Pin1 inhibitor, reduced fat buildup in mice. We showed that Pin1 is required for the differentiation of stem cells into adipocytes. We propose that oral intake of brown algae polyphenol is useful for the treatment of obesity.

  7. Brown Algae Polyphenol, a Prolyl Isomerase Pin1 Inhibitor, Prevents Obesity by Inhibiting the Differentiation of Stem Cells into Adipocytes

    PubMed Central

    Suzuki, Atsuko; Saeki, Toshiyuki; Ikuji, Hiroko; Uchida, Chiyoko; Uchida, Takafumi

    2016-01-01

    Background While screening for an inhibitor of the peptidyl prolyl cis/trans isomerase, Pin1, we came across a brown algae polyphenol that blocks the differentiation of fibroblasts into adipocytes. However, its effectiveness on the accumulation of fat in the body has never been studied. Methodology/Principal Findings Oral administration of brown algae polyphenol to mice fed with a high fat diet, suppressed the increase in fat volume to a level observed in mice fed with a normal diet. We speculate that Pin1 might be required for the differentiation of stem cell to adipocytes. We established wild type (WT) and Pin1-/- (Pin1-KO) adipose-derived mesenchymal stem cell (ASC) lines and found that WT ASCs differentiate to adipocytes but Pin1-KO ASCs do not. Conclusion and Significance Oral administration of brown algae polyphenol, a Pin1 inhibitor, reduced fat buildup in mice. We showed that Pin1 is required for the differentiation of stem cells into adipocytes. We propose that oral intake of brown algae polyphenol is useful for the treatment of obesity. PMID:28036348

  8. 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

  9. Specific cross-linking of the proline isomerase cyclophilin to a non-proline-containing peptide.

    PubMed Central

    McNew, J A; Sykes, K; Goodman, J M

    1993-01-01

    A peptide corresponding to an efficient peroxisomal targeting sequence, the carboxy terminal 12 amino acids of PMP20 from Candida boidinii, was employed as an affinity ligand to search for a peroxisomal targeting receptor. Two proteins from yeast extracts with apparent molecular masses of 20 and 80 kDa were detected by chemical cross-linking to radioiodinated peptide. Both proteins were present in cytosolic supernatants. The 20-kDa species did not cross-link to a control peptide with reversed sequence, whereas the 80-kDa protein cross-linked to both peptides. The cross-linking assay was used to purify the 20-kDa protein from Saccharomyces cerevisiae. Partial protein sequencing identified this protein as cyclophilin, the product of the CYP1 gene. This protein, a peptidyl-prolyl cis-trans isomerase, is the yeast homologue of the protein that mediates the immunosuppressant effects of the drug cyclosporin A (CsA). Cross-linking of peptide to cyclophilin was inhibited by CsA. The cross-linking of cyclophilin to the PMP20-derived peptide was unanticipated because the peptide contains no prolines. The CYP1-encoded protein was not required to target proteins to peroxisomes because this organelle appeared to be assembled normally in a CYP1-disrupted strain. Furthermore, the final three amino acids of the peptide, which are critical for peroxisomal sorting, were not required for cross-linking to cyclophilin. We conclude that either cyclophilin is playing a nonessential facilitating role in peroxisomal targeting or that the interaction of the targeting peptide to cyclophilin is mimicking an interaction with an unidentified substrate or effector of cyclophilin. Images PMID:8443418

  10. Specific cross-linking of the proline isomerase cyclophilin to a non-proline-containing peptide.

    PubMed

    McNew, J A; Sykes, K; Goodman, J M

    1993-02-01

    A peptide corresponding to an efficient peroxisomal targeting sequence, the carboxy terminal 12 amino acids of PMP20 from Candida boidinii, was employed as an affinity ligand to search for a peroxisomal targeting receptor. Two proteins from yeast extracts with apparent molecular masses of 20 and 80 kDa were detected by chemical cross-linking to radioiodinated peptide. Both proteins were present in cytosolic supernatants. The 20-kDa species did not cross-link to a control peptide with reversed sequence, whereas the 80-kDa protein cross-linked to both peptides. The cross-linking assay was used to purify the 20-kDa protein from Saccharomyces cerevisiae. Partial protein sequencing identified this protein as cyclophilin, the product of the CYP1 gene. This protein, a peptidyl-prolyl cis-trans isomerase, is the yeast homologue of the protein that mediates the immunosuppressant effects of the drug cyclosporin A (CsA). Cross-linking of peptide to cyclophilin was inhibited by CsA. The cross-linking of cyclophilin to the PMP20-derived peptide was unanticipated because the peptide contains no prolines. The CYP1-encoded protein was not required to target proteins to peroxisomes because this organelle appeared to be assembled normally in a CYP1-disrupted strain. Furthermore, the final three amino acids of the peptide, which are critical for peroxisomal sorting, were not required for cross-linking to cyclophilin. We conclude that either cyclophilin is playing a nonessential facilitating role in peroxisomal targeting or that the interaction of the targeting peptide to cyclophilin is mimicking an interaction with an unidentified substrate or effector of cyclophilin.

  11. The prolyl isomerase Pin1 increases β-cell proliferation and enhances insulin secretion.

    PubMed

    Nakatsu, Yusuke; Mori, Keiichi; Matsunaga, Yasuka; Yamamotoya, Takeshi; Ueda, Koji; Inoue, Yuki; Mitsuzaki-Miyoshi, Keiko; Sakoda, Hideyuki; Fujishiro, Midori; Yamaguchi, Suguru; Kushiyama, Akifumi; Ono, Hiraku; Ishihara, Hisamitsu; Asano, Tomoichiro

    2017-07-14

    The prolyl isomerase Pin1 binds to the phosphorylated Ser/Thr-Pro motif of target proteins and enhances their cis-trans conversion. This report is the first to show that Pin1 expression in pancreatic β cells is markedly elevated by high-fat diet feeding and in ob/ob mice. To elucidate the role of Pin1 in pancreatic β cells, we generated β-cell-specific Pin1 KO (βPin1 KO) mice. These mutant mice showed exacerbation of glucose intolerance but had normal insulin sensitivity. We identified two independent factors underlying impaired insulin secretion in the βPin1 KO mice. Pin1 enhanced pancreatic β-cell proliferation, as indicated by a reduced β-cell mass in βPin1 KO mice compared with control mice. Moreover, a diet high in fat and sucrose failed to increase pancreatic β-cell growth in the βPin1 KO mice, an observation to which up-regulation of the cell cycle protein cyclin D appeared to contribute. The other role of Pin1 was to activate the insulin-secretory step: Pin1 KO β cells showed impairments in glucose- and KCl-induced elevation of the intracellular Ca(2+) concentration and insulin secretion. We also identified salt-inducible kinase 2 (SIK2) as a Pin1-binding protein that affected the regulation of Ca(2+) influx and found Pin1 to enhance SIK2 kinase activity, resulting in a decrease in p35 protein, a negative regulator of Ca(2+) influx. Taken together, our observations demonstrate critical roles of Pin1 in pancreatic β cells and that Pin1 both promotes β-cell proliferation and activates insulin secretion. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. 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 enzyme...

  13. 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 enzyme...

  14. 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 enzyme...

  15. 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 enzyme...

  16. 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 enzyme...

  17. Fine-tuning the extent and dynamics of binding cleft opening as a potential general regulatory mechanism in parvulin-type peptidyl prolyl isomerases

    NASA Astrophysics Data System (ADS)

    Czajlik, András; Kovács, Bertalan; Permi, Perttu; Gáspári, Zoltán

    2017-03-01

    Parvulins or rotamases form a distinct group within peptidyl prolyl cis-trans isomerases. Their exact mode of action as well as the role of conserved residues in the family are still not unambiguously resolved. Using backbone S2 order parameters and NOEs as restraints, we have generated dynamic structural ensembles of three distinct parvulins, SaPrsA, TbPin1 and CsPinA. The resulting ensembles are in good agreement with the experimental data but reveal important differences between the three enzymes. The largest difference can be attributed to the extent of the opening of the substrate binding cleft, along which motional mode the three molecules occupy distinct regions. Comparison with a wide range of other available parvulin structures highlights structural divergence along the bottom of the binding cleft acting as a hinge during the opening-closing motion. In the prototype WW-domain containing parvulin, Pin1, this region is also important in forming contacts with the WW domain known to modulate enzymatic activity of the catalytic domain. We hypothesize that modulation of the extent and dynamics of the identified ‘breathing motion’ might be one of the factors responsible for functional differences in the distinct parvulin subfamilies.

  18. Structure and Activity of the Peptidyl-Prolyl Isomerase Domain from the Histone Chaperone Fpr4 toward Histone H3 Proline Isomerization*

    PubMed Central

    Monneau, Yoan R.; Soufari, Heddy; Nelson, Christopher J.; Mackereth, Cameron D.

    2013-01-01

    The FK506-binding protein (FKBP) family of peptidyl-prolyl isomerases (PPIases) is characterized by a common catalytic domain that binds to the inhibitors FK506 and rapamycin. As one of four FKBPs within the yeast Saccharomyces cerevisiae, Fpr4 has been described as a histone chaperone, and is in addition implicated in epigenetic function in part due to its mediation of cis-trans conversion of proline residues within histone tails. To better understand the molecular details of this activity, we have determined the solution structure of the Fpr4 C-terminal PPIase domain by using NMR spectroscopy. This canonical FKBP domain actively increases the rate of isomerization of three decapeptides derived from the N terminus of yeast histone H3, whereas maintaining intrinsic cis and trans populations. Observation of the uncatalyzed and Fpr4-catalyzed isomerization rates at equilibrium demonstrate Pro16 and Pro30 of histone H3 as the major proline targets of Fpr4, with little activity shown against Pro38. This alternate ranking of the three target prolines, as compared with affinity determination or the classical chymotrypsin-based fluorescent assay, reveals the mechanistic importance of substrate residues C-terminal to the peptidyl-prolyl bond. PMID:23888048

  19. Structure and activity of the peptidyl-prolyl isomerase domain from the histone chaperone Fpr4 toward histone H3 proline isomerization.

    PubMed

    Monneau, Yoan R; Soufari, Heddy; Nelson, Christopher J; Mackereth, Cameron D

    2013-09-06

    The FK506-binding protein (FKBP) family of peptidyl-prolyl isomerases (PPIases) is characterized by a common catalytic domain that binds to the inhibitors FK506 and rapamycin. As one of four FKBPs within the yeast Saccharomyces cerevisiae, Fpr4 has been described as a histone chaperone, and is in addition implicated in epigenetic function in part due to its mediation of cis-trans conversion of proline residues within histone tails. To better understand the molecular details of this activity, we have determined the solution structure of the Fpr4 C-terminal PPIase domain by using NMR spectroscopy. This canonical FKBP domain actively increases the rate of isomerization of three decapeptides derived from the N terminus of yeast histone H3, whereas maintaining intrinsic cis and trans populations. Observation of the uncatalyzed and Fpr4-catalyzed isomerization rates at equilibrium demonstrate Pro(16) and Pro(30) of histone H3 as the major proline targets of Fpr4, with little activity shown against Pro(38). This alternate ranking of the three target prolines, as compared with affinity determination or the classical chymotrypsin-based fluorescent assay, reveals the mechanistic importance of substrate residues C-terminal to the peptidyl-prolyl bond.

  20. Fine-tuning the extent and dynamics of binding cleft opening as a potential general regulatory mechanism in parvulin-type peptidyl prolyl isomerases

    PubMed Central

    Czajlik, András; Kovács, Bertalan; Permi, Perttu; Gáspári, Zoltán

    2017-01-01

    Parvulins or rotamases form a distinct group within peptidyl prolyl cis-trans isomerases. Their exact mode of action as well as the role of conserved residues in the family are still not unambiguously resolved. Using backbone S2 order parameters and NOEs as restraints, we have generated dynamic structural ensembles of three distinct parvulins, SaPrsA, TbPin1 and CsPinA. The resulting ensembles are in good agreement with the experimental data but reveal important differences between the three enzymes. The largest difference can be attributed to the extent of the opening of the substrate binding cleft, along which motional mode the three molecules occupy distinct regions. Comparison with a wide range of other available parvulin structures highlights structural divergence along the bottom of the binding cleft acting as a hinge during the opening-closing motion. In the prototype WW-domain containing parvulin, Pin1, this region is also important in forming contacts with the WW domain known to modulate enzymatic activity of the catalytic domain. We hypothesize that modulation of the extent and dynamics of the identified ‘breathing motion’ might be one of the factors responsible for functional differences in the distinct parvulin subfamilies. PMID:28300139

  1. Subtractive hybridization analysis of gastric diseases-associated Helicobacter pylori identifies peptidyl-prolyl isomerase as a potential marker for gastric cancer.

    PubMed

    Gong, Yue-Hua; Chen, Moye; Xu, Ying; Dong, Nannan; Sang, Zhikun; Liu, Jun; Yuan, Yuan

    2011-07-01

    Helicobacter pylori, a microaerophilic Gram-negative bacterium, is known to cause chronic gastritis, peptic ulcer and gastric cancer. Genes that are present in certain isolates may determine strain-specific traits such as disease association and drug resistance. In order to understand the pathogenic mechanisms of gastric diseases, identify molecular markers of the diseases associated with H. pylori strains and provide clues for target treatment of H. pylori-related diseases, a subtracted DNA library was constructed from a gastric cancer-associated H. pylori strain and a superficial gastritis-associated H. pylori strain by suppression subtractive hybridization. The presence of gastric cancer-specific genes was identified by dot blot hybridization, DNA sequencing and PCR-based screening. Twelve gastric cancer-specific high-copy genes and nine low-copy genes were found in gastric cancer compared with the superficial gastritis strain. These genes were confirmed by PCR analysis of H. pylori isolates. Notably, peptidyl-prolyl cis-trans isomerase (PPIase) was detected positively in 11 out of 22 (50%) gastric cancer-associated H. pylori strains. In contrast, <24% of the H. pylori strains from superficial gastritis showed positive results. Given the potential role of PPIases in cell growth, apoptosis and oncogenic transformation, our results suggest that PPIase may represent a novel marker and potential therapeutic target for gastric cancer. © 2011 The First Affiliated Hospital, China Medical University. FEMS Microbiology Letters © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd.

  2. The prolyl isomerase Pin1 acts as a novel molecular switch for TNF-α–induced priming of the NADPH oxidase in human neutrophils

    PubMed Central

    Boussetta, Tarek; Gougerot-Pocidalo, Marie-Anne; Hayem, Gilles; Ciappelloni, Silvia; Raad, Houssam; Arabi Derkawi, Riad; Bournier, Odile; Kroviarski, Yolande; Zhou, Xiao Zhen; Malter, James S.; Lu, Ping K.; Bartegi, Aghleb; Dang, Pham My-Chan

    2010-01-01

    Neutrophils play a key role in host defense by releasing reactive oxygen species (ROS). However, excessive ROS production by neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase can damage bystander tissues, thereby contributing to inflammatory diseases. Tumor necrosis factor-α (TNF-α), a major mediator of inflammation, does not activate NADPH oxidase but induces a state of hyperresponsiveness to subsequent stimuli, an action known as priming. The molecular mechanisms by which TNF-α primes the NADPH oxidase are unknown. Here we show that Pin1, a unique cis-trans prolyl isomerase, is a previously unrecognized regulator of TNF-α–induced NADPH oxidase hyperactivation. We first showed that Pin1 is expressed in neutrophil cytosol and that its activity is markedly enhanced by TNF-α. Inhibition of Pin1 activity with juglone or with a specific peptide inhibitor abrogated TNF-α–induced priming of neutrophil ROS production induced by N-formyl-methionyl-leucyl-phenylalanine peptide (fMLF). TNF-α enhanced fMLF-induced Pin1 and p47phox translocation to the membranes and juglone inhibited this process. Pin1 binds to p47phox via phosphorylated Ser345, thereby inducing conformational changes that facilitate p47phox phosphorylation on other sites by protein kinase C. These findings indicate that Pin1 is critical for TNF-α–induced priming of NADPH oxidase and for excessive ROS production. Pin1 inhibition could potentially represent a novel anti-inflammatory strategy. PMID:20956805

  3. Induction of chalcone isomerase in elicitor-treated bean cells. Comparison of rates of synthesis and appearance of immunodetectable enzyme.

    PubMed

    Robbins, M P; Dixon, R A

    1984-11-15

    Chalcone isomerase, an enzyme involved in the formation of flavonoid-derived compounds in plants, has been purified nearly 600-fold from cell suspension cultures of dwarf French bean (Phaseolus vulgaris L.). Chromatofocussing yielded a single form of the enzyme of apparent pI 5.0. This preparation was used to raise rabbit anti-(chalcone isomerase) serum. Changes in the rate of synthesis of chalcone isomerase have been investigated by indirect immunoprecipitation of enzyme labelled in vivo with [35S]methionine in elicitor-treated cultures of P. vulgaris. Elicitor, heat-released from cell walls of the phytopathogenic fungus Colletotrichum lindemuthianum, the causal agent of anthracnose disease of bean, causes increased synthesis of the isomerase, with maximum synthetic rate occurring 11-12 h after exposure to elicitor. Immune blotting studies indicate that the elicitor-mediated increase in extractable activity of the isomerase is associated with increased appearance of immunodetactable isomerase protein of Mr 27 000. However, the maximum level of immunodetectable isomerase was attained approximately 6 h earlier than maximum extractable activity. Furthermore, a 2.8-fold increase in enzyme activity above basal levels at 12 h after elicitor-treatment was associated with a corresponding 5.8-fold increase in immunodetectable enzyme. It is concluded that elicitor induces the synthesis of both active and inactive chalcone isomerase of Mr 27 000, and that some activation of inactive enzyme occurs during the elicitor-mediated increase in isomerase activity. The presence of a pool of inactive chalcone isomerase in bean cell cultures has recently been suggested on the basis of density labelling experiments utilising 2H from 2H2O [Dixon et al. (1983) Planta (Berl.) 159, 561-569].

  4. 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

  5. Cyclophilin J is a novel peptidyl-prolyl isomerase and target for repressing the growth of hepatocellular carcinoma.

    PubMed

    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.

  6. The cloning, characterization, and functional analysis of a gene encoding an isopentenyl diphosphate isomerase involved in triterpene biosynthesis in the Lingzhi or reishi medicinal mushroom Ganoderma lucidum (higher Basidiomycetes).

    PubMed

    Wu, Feng-Li; Shi, Liang; Yao, Jian; Ren, Ang; Zhou, Chao; Mu, Da-Shuai; Zhao, Ming-Wen

    2013-01-01

    An isopentenyl diphosphate isomerase (IDI) gene, GlIDI, was isolated from Ganoderma lucidum, which produces triterpenes through the mevalonate pathway. The open reading frame of GlIDI encodes a 252 amino acid polypeptide with a theoretical molecular mass of 28.71 kDa and a theoretical isoelectric point of 5.36. GlIDI is highly homologous to other fungal IDIs and contains conserved active residues and nudix motifs shared by the IDI protein family. The color complementation assay indicated that GlIDI can accelerate the accumulation of β-carotene and confirmed that the cloned complementary DNA encoded a functional GlIDI protein. Gene expression analysis showed that the GlIDI transcription level was relatively low in the mycelia and reached a relatively high level in the mushroom primordia. In addition, its expression level could be up-regulated by 254 µM methyl jasmonate. Our results suggest that this enzyme may play an important role in triterpene biosynthesis.

  7. A Cluster of Genes Encodes the Two Types of Chalcone Isomerase Involved in the Biosynthesis of General Flavonoids and Legume-Specific 5-Deoxy(iso)flavonoids in Lotus japonicus1

    PubMed Central

    Shimada, Norimoto; Aoki, Toshio; Sato, Shusei; Nakamura, Yasukazu; Tabata, Satoshi; Ayabe, Shin-ichi

    2003-01-01

    Leguminous plants produce 5-deoxyflavonoids and 5-deoxyisoflavonoids that play essential roles in legume-microbe interactions. Together with chalcone polyketide reductase and cytochrome P450 2-hydroxyisoflavanone synthase, the chalcone isomerase (CHI) of leguminous plants is fundamental in the construction of these ecophysiologically active flavonoids. Although CHIs of nonleguminous plants isomerize only 6′-hydroxychalcone to 5-hydroxyflavanone (CHIs with this function are referred to as type I), leguminous CHIs convert both 6′-deoxychalcone and 6′-hydroxychalcone to 5-deoxyflavanone and 5-hydroxyflavanone, respectively (referred to as type II). In this study, we isolated multiple CHI cDNAs (cCHI1–cCHI3) from a model legume, Lotus japonicus. In contrast to previous observations, the amino acid sequence of CHI2 was highly homologous to nonleguminous CHIs, whereas CHI1 and CHI3 were the conventional leguminous type. Furthermore, genome sequence analysis revealed that four CHI genes (CHI1–3 and a putative gene, CHI4) form a tandem cluster within 15 kb. Biochemical analysis with recombinant CHIs expressed in Escherichia coli confirmed that CHI1 and CHI3 are type II CHIs and that CHI2 is a type I CHI. The occurrence of both types of CHIs is probably common in leguminous plants, and it was suggested that type II CHIs evolved from an ancestral CHI by gene duplication and began to produce 5-deoxy(iso)flavonoids along with the establishment of the Fabaceae. PMID:12644647

  8. 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.

  9. 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...

  10. 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... 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 of...

  11. 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 of...

  12. 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... 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 of...

  13. The chaperonin cycle cannot substitute for prolyl isomerase activity, but GroEL alone promotes productive folding of a cyclophilin-sensitive substrate to a cyclophilin-resistant form.

    PubMed

    von Ahsen, O; Tropschug, M; Pfanner, N; Rassow, J

    1997-08-01

    The chaperonin GroEL and the peptidyl-prolyl cis-trans isomerase cyclophilin are major representatives of two distinct cellular systems that help proteins to adopt their native three-dimensional structure: molecular chaperones and folding catalysts. Little is known about whether and how these proteins cooperate in protein folding. In this study, we have examined the action of GroEL and cyclophilin on a substrate protein in two distinct prolyl isomerization states. Our results indicate that: (i) GroEL binds the same substrate in different prolyl isomerization states. (ii) GroEL-ES does not promote prolyl isomerizations, but even retards isomerizations. (iii) Cyclophilin cannot promote the correct isomerization of prolyl bonds of a GroEL-bound substrate, but acts sequentially after release of the substrate from GroEL. (iv) A denatured substrate with all-native prolyl bonds is delayed in folding by cyclophilin due to isomerization to non-native prolyl bonds; a substrate that has proceeded in folding beyond a stage where it can be bound by GroEL is still sensitive to cyclophilin. (v) If a denatured cyclophilin-sensitive substrate is first bound to GroEL, however, productive folding to a cyclophilin-resistant form can be promoted, even without GroES. We conclude that GroEL and cyclophilin act sequentially and exert complementary functions in protein folding.

  14. TAL Effectors Target the C-Terminal Domain of RNA Polymerase II (CTD) by Inhibiting the Prolyl-Isomerase Activity of a CTD-Associated Cyclophilin

    PubMed Central

    de Oliveira, Maria Luiza Peixoto; de Mello, Uli Quirino; Benedetti, Celso Eduardo

    2012-01-01

    Transcriptional activator-like (TAL) effectors of plant pathogenic bacteria function as transcription factors in plant cells. However, how TAL effectors control transcription in the host is presently unknown. Previously, we showed that TAL effectors of the citrus canker pathogen Xanthomonas citri, named PthAs, targeted the citrus protein complex comprising the thioredoxin CsTdx, ubiquitin-conjugating enzymes CsUev/Ubc13 and cyclophilin CsCyp. Here we show that CsCyp complements the function of Cpr1 and Ess1, two yeast cyclophilins that regulate transcription by the isomerization of proline residues of the regulatory C-terminal domain (CTD) of RNA polymerase II. We also demonstrate that CsCyp, CsTdx, CsUev and four PthA variants interact with the citrus CTD and that CsCyp co-immunoprecipitate with the CTD in citrus cell extracts and with PthA2 transiently expressed in sweet orange epicotyls. The interactions of CsCyp with the CTD and PthA2 were inhibited by cyclosporin A (CsA), a cyclophilin inhibitor. Moreover, we present evidence that PthA2 inhibits the peptidyl-prolyl cis-trans isomerase (PPIase) activity of CsCyp in a similar fashion as CsA, and that silencing of CsCyp, as well as treatments with CsA, enhance canker lesions in X. citri-infected leaves. Given that CsCyp appears to function as a negative regulator of cell growth and that Ess1 negatively regulates transcription elongation in yeast, we propose that PthAs activate host transcription by inhibiting the PPIase activity of CsCyp on the CTD. PMID:22911812

  15. Metabolic engineering of a xylose-isomerase-expressing Saccharomyces cerevisiae strain for rapid anaerobic xylose fermentation.

    PubMed

    Kuyper, Marko; Hartog, Miranda M P; Toirkens, Maurice J; Almering, Marinka J H; Winkler, Aaron A; van Dijken, Johannes P; Pronk, Jack T

    2005-02-01

    After an extensive selection procedure, Saccharomyces cerevisiae strains that express the xylose isomerase gene from the fungus Piromyces sp. E2 can grow anaerobically on xylose with a mu(max) of 0.03 h(-1). In order to investigate whether reactions downstream of the isomerase control the rate of xylose consumption, we overexpressed structural genes for all enzymes involved in the conversion of xylulose to glycolytic intermediates, in a xylose-isomerase-expressing S. cerevisiae strain. The overexpressed enzymes were xylulokinase (EC 2.7.1.17), ribulose 5-phosphate isomerase (EC 5.3.1.6), ribulose 5-phosphate epimerase (EC 5.3.1.1), transketolase (EC 2.2.1.1) and transaldolase (EC 2.2.1.2). In addition, the GRE3 gene encoding aldose reductase was deleted to further minimise xylitol production. Surprisingly the resulting strain grew anaerobically on xylose in synthetic media with a mu(max) as high as 0.09 h(-1) without any non-defined mutagenesis or selection. During growth on xylose, xylulose formation was absent and xylitol production was negligible. The specific xylose consumption rate in anaerobic xylose cultures was 1.1 g xylose (g biomass)(-1) h(-1). Mixtures of glucose and xylose were sequentially but completely consumed by anaerobic batch cultures, with glucose as the preferred substrate.

  16. 21 CFR 862.1570 - Phosphohexose isomerase test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    .... Measurements of phosphohexose isomerase are used in the diagnosis and treatment of muscle diseases such as muscular dystrophy, liver diseases such as hepatitis or cirrhosis, and metastatic carcinoma....

  17. 21 CFR 862.1570 - Phosphohexose isomerase test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    .... Measurements of phosphohexose isomerase are used in the diagnosis and treatment of muscle diseases such as muscular dystrophy, liver diseases such as hepatitis or cirrhosis, and metastatic carcinoma....

  18. 21 CFR 862.1570 - Phosphohexose isomerase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    .... Measurements of phosphohexose isomerase are used in the diagnosis and treatment of muscle diseases such as muscular dystrophy, liver diseases such as hepatitis or cirrhosis, and metastatic carcinoma....

  19. 21 CFR 862.1570 - Phosphohexose isomerase test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    .... Measurements of phosphohexose isomerase are used in the diagnosis and treatment of muscle diseases such as muscular dystrophy, liver diseases such as hepatitis or cirrhosis, and metastatic carcinoma....

  20. The Periplasmic Chaperone Network of Campylobacter jejuni: Evidence that SalC (Cj1289) and PpiD (Cj0694) Are Involved in Maintaining Outer Membrane Integrity

    PubMed Central

    Taylor, Aidan J.; Zakai, Shadi A. I.; Kelly, David J.

    2017-01-01

    The outer membrane (OM) of Gram-negative pathogenic bacteria is a key structure in host–pathogen interactions that contains a plethora of proteins, performing a range of functions including adhesion, nutrient uptake, export of effectors and interaction with innate and adaptive components of the immune system. In addition, the OM can exclude drugs and thus contribute to antimicrobial resistance. The OM of the food-borne pathogen Campylobacter jejuni contains porins, adhesins and other virulence factors that must be specifically localized to this membrane, but the protein sorting mechanisms involved are only partially understood. In particular, chaperones are required to ferry OM proteins across the periplasm after they emerge from the Sec translocation system. The SurA-related chaperone PEB4 (Cj0596) is the only protein with a proven role in OM biogenesis and integrity in C. jejuni. In this work, we have constructed a set of isogenic deletion mutants in genes encoding both known and predicted chaperones (cj0596, cj0694, cj1069, cj1228c, and cj1289) using NCTC 11168H as the parental strain. These mutants were characterized using a range of assays to determine effects on growth, agglutination, biofilm formation, membrane permeability and hydrophobicity. We focused on Cj1289 and Cj0694, which our previous work suggested possessed both chaperone and peptidyl-proyl cis/trans isomerase (PPIase) domains. Mutants in either cj1289 or cj0694 showed growth defects, increased motility, agglutination and biofilm formation and severe OM permeability defects as measured by a lysozyme accessibility assay, that were comparable to those exhibited by the isogenic peb4 mutant. 2D-gel comparisons showed a general decrease in OM proteins in these mutants. We heterologously overproduced and purified Cj0694 and obtained evidence that this protein was an active PPIase, as judged by its acceleration of the refolding rate of reduced and alkylated ribonuclease T1 and that it also possessed

  1. 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.

  2. Segmental movement: definition of the structural requirements for loop closure in catalysis by triosephosphate isomerase.

    PubMed

    Sampson, N S; Knowles, J R

    1992-09-15

    To determine what drives the closure of the active-site loop in the reaction catalyzed by triosephosphate isomerase, several residues involved in hydrogen bonding between the loop and the bulk of the protein have been altered. It was known from earlier work that the loop serves two functions: to stabilize the reaction intermediate (and the two transition states that flank it) and to prevent the loss of this unstable species into free solution. To discover what elements of the protein are necessary for proper closure of the loop, selective destabilization of the "open" and the "closed" forms of the enzyme with respect to one another has been attempted. The mutant Y164F isomerase has been prepared to evaluate the importance of the structure of the "open" form, and the mutant E129Q, Y208F, and S211A enzymes have allowed investigation of the "closed" form. The integrity of the loop itself has been destabilized by making the T172A isomerase. We have found that only those mutations that destabilize the "closed" form of the enzyme significantly perturb the catalytic properties of the isomerase. The second-order rate constants (kcat/Km) of the S211A and E129Q enzymes are reduced 30-fold, and that of the mutant Y208F enzyme is reduced 2000-fold, from the level of the wild-type enzyme. The dramatic drop in activity of the Y208F enzyme is accompanied by a 200-fold increase in the dissociation constant of the intermediate analogue phosphoglycolohydroxamate. The most important property of the mobile loop of triosephosphate isomerase lies, therefore, in the stability of the system when the active site contains ligand and the loop is closed.

  3. 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... defined in § 170.3(o)(9) of this chapter, to convert glucose to fructose. (2) The ingredient is used in...

  4. Involvement of FKBP6 in hepatitis C virus replication

    PubMed Central

    Kasai, Hirotake; Kawakami, Kunihiro; Yokoe, Hiromasa; Yoshimura, Kentaro; Matsuda, Masanori; Yasumoto, Jun; Maekawa, Shinya; Yamashita, Atsuya; Tanaka, Tomohisa; Ikeda, Masanori; Kato, Nobuyuki; Okamoto, Toru; Matsuura, Yoshiharu; Sakamoto, Naoya; Enomoto, Nobuyuki; Takeda, Sen; Fujii, Hideki; Tsubuki, Masayoshi; Kusunoki, Masami; Moriishi, Kohji

    2015-01-01

    The chaperone system is known to be exploited by viruses for their replication. In the present study, we identified the cochaperone FKBP6 as a host factor required for hepatitis C virus (HCV) replication. FKBP6 is a peptidyl prolyl cis-trans isomerase with three domains of the tetratricopeptide repeat (TPR), but lacks FK-506 binding ability. FKBP6 interacted with HCV nonstructural protein 5A (NS5A) and also formed a complex with FKBP6 itself or FKBP8, which is known to be critical for HCV replication. The Val121 of NS5A and TPR domains of FKBP6 were responsible for the interaction between NS5A and FKBP6. FKBP6 was colocalized with NS5A, FKBP8, and double-stranded RNA in HCV-infected cells. HCV replication was completely suppressed in FKBP6-knockout hepatoma cell lines, while the expression of FKBP6 restored HCV replication in FKBP6-knockout cells. A treatment with the FKBP8 inhibitor N-(N′, N′-dimethylcarboxamidomethyl)cycloheximide impaired the formation of a homo- or hetero-complex consisting of FKBP6 and/or FKBP8, and suppressed HCV replication. HCV infection promoted the expression of FKBP6, but not that of FKBP8, in cultured cells and human liver tissue. These results indicate that FKBP6 is an HCV-induced host factor that supports viral replication in cooperation with NS5A. PMID:26567527

  5. 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.

  6. Current studies on sucrose isomerase and biological isomaltulose production using sucrose isomerase.

    PubMed

    Mu, Wanmeng; Li, Wenjing; Wang, Xiao; Zhang, Tao; Jiang, Bo

    2014-08-01

    Isomaltulose is a natural isomer of sucrose. It is widely used as a functional sweetener with promising properties, including slower digestion, lower glycemic index, prolonged energy release, lower insulin reaction, and less cariogenicity. It has been approved as a safe sucrose substitute by the Food and Drug Administration of the US; Ministry of Health, Labor and Welfare of Japan; and the Commission of the European Communities. This article presents a review of recent studies on the properties, physiological effects, and food application of isomaltulose. In addition, the biochemical properties of sucrose isomerases producing isomaltulose are compared; the heterologous expression, fermentation optimization, structural determination, and catalysis mechanism of sucrose isomerase are reviewed; and the biotechnological production of isomaltulose from sucrose is summarized.

  7. Automated Yeast Transformation Protocol to Engineer S. cerevisiae Strains for Cellulosic Ethanol Production with Open Reading Frames that Express Proteins Binding to Xylose Isomerase Identified using Robotic Two-hybrid Screen

    USDA-ARS?s Scientific Manuscript database

    Commercialization of fuel ethanol production from lignocellulosic biomass has focused on engineering the glucose-fermenting industrial yeast Saccharomyces cerevisiae to utilize pentose sugars. Since S. cerevisiae naturally metabolizes xylulose, one approach involves introducing xylose isomerase (XI...

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

    PubMed Central

    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

  9. Biochemical characterization of phosphoglucose isomerase and genetic variants from mouse and Drosophila melanogaster.

    PubMed

    Charles, D; Lee, C Y

    1980-01-16

    A simple and unique procedure was developed to purify phosphoglucose isomerase variants from the whole mouse body extracts and Drosophila homogenate. It involved the use of an 8-(6-aminohexyl)-amino-ATP-Sepharose column followed by a preparative isoelectric focusing. In each case, the enzyme in the homogenate was adsorbed by ionic interaction on the ATP-Sepharose column. Substantial purification was achieved by the affinity elution with the substrate-glucose-6-phosphate. Mouse and Drosophila phosphoglucose isomerase as well as the corresponding variants were shown to be dimers of similar molecular weight and to exhibit similar kinetic properties. The isoelectric points for the variants from DBA/2J and C57BL/6J mice were determined to be 8.4 and 8.7 respectively, while they were 6.8 and 6.3 respectively for Drosophila and 4/4 variants. Differential thermal stability was observed for the two mouse variants but not for the Drosophila ones. Amino acid composition analysis was performed for both mouse and Drosophila enzymes. Rabbit antisera for mouse (DBA/2J) and Drosophila (2/2) enzymes were raised. Within each species, complete immunological identity was observed between the variants. The antisera were used to characterize the null mutants of phosphoglucose isomerase identified in the mouse and Drosophila populations. By rocket immunoelectrophoresis, the null allele of the naturally occurring heterozygous null variant of Drosophila was shown to express no cross-reacting materials (CRM).

  10. Chalcone Isomerase from Eubacterium ramulus Catalyzes the Ring Contraction of Flavanonols.

    PubMed

    Braune, Annett; Engst, Wolfram; Elsinghorst, Paul W; Furtmann, Norbert; Bajorath, Jürgen; Gütschow, Michael; Blaut, Michael

    2016-11-01

    The enzyme catalyzing the ring-contracting conversion of the flavanonol taxifolin to the auronol alphitonin in the course of flavonoid degradation by the human intestinal anaerobe Eubacterium ramulus was purified and characterized. It stereospecifically catalyzed the isomerization of (+)-taxifolin but not that of (-)-taxifolin. The Km for (+)-taxifolin was 6.4 ± 0.8 μM, and the Vmax was 108 ± 4 μmol min(-1) (mg protein)(-1) The enzyme also isomerized (+)-dihydrokaempferol, another flavanonol, to maesopsin. Inspection of the encoding gene revealed its complete identity to that of the gene encoding chalcone isomerase (CHI) from E. ramulus Based on the reported X-ray crystal structure of CHI (M. Gall et al., Angew Chem Int Ed 53:1439-1442, 2014, http://dx.doi.org/10.1002/anie.201306952), docking experiments suggest the substrate binding mode of flavanonols and their stereospecific conversion. Mutation of the active-site histidine (His33) to alanine led to a complete loss of flavanonol isomerization by CHI, which indicates that His33 is also essential for this activity. His33 is proposed to mediate the stereospecific abstraction of a proton from the hydroxymethylene carbon of the flavanonol C-ring followed by ring opening and recyclization. A flavanonol-isomerizing enzyme was also identified in the flavonoid-converting bacterium Flavonifractor plautii based on its 50% sequence identity to the CHI from E. ramulus IMPORTANCE: Chalcone isomerase was known to be involved in flavone/flavanone conversion by the human intestinal bacterium E. ramulus Here we demonstrate that this enzyme moreover catalyzes a key step in the breakdown of flavonols/flavanonols. Thus, a single isomerase plays a dual role in the bacterial conversion of dietary bioactive flavonoids. The identification of a corresponding enzyme in the human intestinal bacterium F. plautii suggests a more widespread occurrence of this isomerase in flavonoid-degrading bacteria.

  11. Chemistry of coordinated nitroxyl. Reagent-specific protonations of trans-Re(CO)(2)(NO)(PR(3))(2) (R = Ph, Cy) that give the neutral nitroxyl complexes cis,trans-ReCl(CO)(2)(NH=O)(PR(3))(2) or the cationic hydride complex [trans,trans-ReH(CO)(2)(NO)(PPh(3))(2)(+)][SO(3)CF(3)(-)].

    PubMed

    Southern, J S; Green, M T; Hillhouse, G L; Guzei, I A; Rheingold, A L

    2001-11-05

    The reactions of hydrochloric and triflic acids with the five-coordinate nitrosyl complexes trans-Re(CO)(2)(NO)(PR(3))(2) (2a, R = Ph; 2b, R = Cy) have been investigated. Reaction of anhydrous HCl with 2 results in a formal protonation of the nitrosyl ligand and addition of chloride to the metal, giving the neutral nitroxyl complex cis,trans-ReCl(CO)(2)(NH=O)(PR(3))(2) (3a, R = Ph; 3b, R = Cy). Reaction of Brønsted bases with 3a or 3b results in clean conversion of 3 to 2 when the base is appropriately strong (pK(b) approximately 7). Addition of HOSO(2)CF(3) to solutions of 2a results in protonation at the metal and formation of the cationic rhenium hydride [trans,trans-ReH(CO)(2)(NO)(PPh(3))(2)(+)][SO(3)CF(3)(-)] (4) in 74% yield; the deuteride [trans,trans-Re((2)H)(CO)(2)(NO)(PPh(3))(2)(+)][SO(3)CF(3)(-)] (4-d) was analogously prepared from (2)HOSO(2)CF(3). 4 crystallized from CH(2)Cl(2)/Et(2)O solution in the orthorhombic space group Pnma, with a = 17.2201(2) A, b = 23.6119(3) A, c = 9.2380(2) A, and Z = 4. The least-squares refinement converged to R(F) = 0.039 and R(wF(2)()) = 0.063 for the 4330 unique data with I > 2 sigma(I). The structure of 4 shows that the hydride (Re-H = 1.74 A) occupies the position trans to the linear nitrosyl ligand (Re-N-O = 178.1(4) degrees ) in the pseudooctahedral complex cation. Complex 4 does not react with chloride to give 3a. DFT calculations carried out on free nitroxyl and its model complexes [Re(CO)(5)(NH=O)(+)] (5), [mer,trans-Re(CO)(3)(NH=O)(PH(3))(2)(+)] (6), and cis,trans-ReCl(CO)(2)(NH=O)(PH(3))(2) (7) indicate that coordinated nitroxyl acts as both a sigma-donor and pi-acceptor ligand, consistent with the observed trend for nu(NO) in free HN=O (1563 cm(-1)), [mer,trans-Re(CO)(3)(NH=O)(PPh(3))(2)(+)] (1, 1391 cm(-1)), 3a (1376 cm(-1)), and 3b (1335 cm(-1)).

  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. 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

  14. Secretion of protein disulphide isomerase AGR2 confers tumorigenic properties.

    PubMed

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

    2016-05-30

    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.

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

    PubMed

    Velazquez, Hector A; Hamelberg, Donald

    2015-02-21

    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.

  16. Kemp Eliminase Activity of Ketosteroid Isomerase.

    PubMed

    Lamba, Vandana; Sanchez, Enis; Fanning, Lauren Rose; Howe, Kathryn; Alvarez, Maria Alejandra; Herschlag, Daniel; Forconi, Marcello

    2017-01-31

    Kemp eliminases represent the most successful class of computationally designed enzymes, with rate accelerations of up to 10(9)-fold relative to the rate of the same reaction in aqueous solution. Nevertheless, several other systems such as micelles, catalytic antibodies, and cavitands are known to accelerate the Kemp elimination by several orders of magnitude. We found that the naturally occurring enzyme ketosteroid isomerase (KSI) also catalyzes the Kemp elimination. Surprisingly, mutations of D38, the residue that acts as a general base for its natural substrate, produced variants that catalyze the Kemp elimination up to 7000-fold better than wild-type KSI does, and some of these variants accelerate the Kemp elimination more than the computationally designed Kemp eliminases. Analysis of the D38N general base KSI variant suggests that a different active site carboxylate residue, D99, performs the proton abstraction. Docking simulations and analysis of inhibition by active site binders suggest that the Kemp elimination takes place in the active site of KSI and that KSI uses the same catalytic strategies of the computationally designed enzymes. In agreement with prior observations, our results strengthen the conclusion that significant rate accelerations of the Kemp elimination can be achieved with very few, nonspecific interactions with the substrate if a suitable catalytic base is present in a hydrophobic environment. Computational design can fulfill these requirements, and the design of more complex and precise environments represents the next level of challenges for protein design.

  17. Plant Triose Phosphate Isomerase Isozymes 1

    PubMed Central

    Pichersky, Eran; Gottlieb, Leslie D.

    1984-01-01

    We report the first complete purifications of the cytosolic and plastid isozymes of triose phosphate isomerase (TPI; EC 5.3.1.1) from higher plants including spinach (Spinacia oleracea), lettuce (Lactuca sativa), and celery (Apium graveolens). Both isozymes are composed of two isosubunits with approximate molecular weight of 27,000; in spinach and lettuce the plastid isozyme is 200 to 400 larger than the cytosolic isozyme. The two isozymes, purified from lettuce, had closely similar amino acid compositions with the exception of methionine which was four times more prevalent in the cytosolic isozyme. Partial amino acid sequences from the N-terminus were also obtained for both lettuce TPIs. Nine of the 13 positions sequenced in the two proteins had identical amino acid residues. The partial sequences of the plant proteins showed high similarity to previously sequenced animal TPIs. Immunological studies, using antisera prepared independently against the purified plastid and cytosolic isozymes from spinach, revealed that the cytosolic isozymes from a variety of species formed an immunologically distinct group as did the plastid isozymes. However, both plastid and cytosolic TPIs shared some antigenic determinants. The overall similarity of the two isozymes and the high similarity of their partial amino acid sequences to those of several animals indicate that TPI is a very highly conserved protein. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:16663420

  18. Purification and characterization of two isoforms of isopentenyl-diphosphate isomerase from elicitor-treated Cinchona robusta cells.

    PubMed

    Ramos-Valdivia, A C; van der Heijden, R; Verpoorte, R; Camara, B

    1997-10-01

    In Cinchona robusta (Rubiaceae) cell suspension cultures, the activity of the enzyme isopentenyl-diphosphate isomerase (isopentenyl-POP isomerase) is transiently induced after addition of a homogenate of the phytopathogenic fungus Phytophthora cinnamomi. The enzyme catalyses the interconversion of isopentenyl-POP and dimethylallyl diphosphate (dimethylallyl-POP) and may be involved in the biosynthesis of anthraquinone phytoalexins that accumulate rapidly after elicitation of Cinchona cells. From elicitor-treated C. robusta cells, two isoforms of isopentenyl-POP isomerase have been purified to apparent homogeneity in four chromatographic steps. The purified forms are monomeric enzymes of 34 kDa (isoform I) and 29 kDa (isoform II), with Km values for isopentenyl-POP of 5.1 microM and 1.0 microM, respectively. Both isoforms require Mn2+ or Mg2+ as cofactor, isoform II showing a preference for Mn2+ with maximum activity at 1.5-2 mM. Isoform I was most active in the presence of 0.5-1.5 mM Mg2+ or in the presence of 0.5 mM Mn2+. A pH optimum of 7-7.8 was found for both forms and both were competitively inhibited by geranyl diphosphate (Ki 96 microM for isoform I) and the transition state analogue 2-(dimethylamino)ethyl diphosphate. Rechromatography of purified isoforms did not indicate any interconversion of both forms. Western blot analysis, using antibodies raised against isopentenyl-POP isomerase purified from Capsicum annuum, showed the presence of both isoforms in the crude protein extracts from C. robusta cells. Isoform II was specifically induced by elicitation, non-treated cells contained low activity of this isoform. The possible role of isopentenyl-POP isomerase in the biosynthesis of anthraquinones is discussed.

  19. Thermolabile triose phosphate isomerase in a psychrophilic Clostridium.

    NASA Technical Reports Server (NTRS)

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

    1972-01-01

    It was found that a psychrophilic Clostridium contains a triose phosphate isomerase which is very labile at moderate temperatures. An investigation showed that the optimal growth temperature of the psychrophile was between 15 and 20 deg C. No growth occurred at 25 deg C. The thermostability of the glycolytic enzymes in the cell-free extracts of Clostridium sp. strain 69 was studied. The data obtained show that the triose phosphate isomerase is quite labile at moderate temperatures. The instability of the enzyme is sufficient to explain the low maximum growth temperature of the psychrophile.

  20. Thermolabile triose phosphate isomerase in a psychrophilic Clostridium.

    NASA Technical Reports Server (NTRS)

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

    1972-01-01

    It was found that a psychrophilic Clostridium contains a triose phosphate isomerase which is very labile at moderate temperatures. An investigation showed that the optimal growth temperature of the psychrophile was between 15 and 20 deg C. No growth occurred at 25 deg C. The thermostability of the glycolytic enzymes in the cell-free extracts of Clostridium sp. strain 69 was studied. The data obtained show that the triose phosphate isomerase is quite labile at moderate temperatures. The instability of the enzyme is sufficient to explain the low maximum growth temperature of the psychrophile.

  1. 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.

  2. Structural determinants of product specificity of sucrose isomerases.

    PubMed

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

    2009-06-18

    The healthy sweetener isomaltulose is industrially produced from the conversion of sucrose by the sucrose isomerase SmuA from Protaminobacter rubrum. Crystal structures of SmuA in native and deoxynojirimycin complexed forms completed with modeling studies unravel the characteristics of the isomaltulose synthases catalytic pocket and their substrate binding mode. Comparison with the trehalulose synthase MutB highlights the role of Arg(298) and Arg(306) active site residues and surface charges in controlling product specificity of sucrose isomerases (isomaltulose versus trehalulose). The results provide a rationale for the specific design of optimized enzymes.

  3. Enzymes do what is expected (chalcone isomerase versus chorismate mutase).

    PubMed

    Hur, Sun; Bruice, Thomas C

    2003-02-12

    Madicago sativa chalcone isomerase (CI) catalyzes the isomerization of chalcone to flavanone, whereas E. coli chorismate mutase (CM) catalyzes the pericyclic rearrangement of chorismate to prephenate. Covalent intermediates are not formed in either of the enzyme-catalyzed reactions, K(M) and k(cat) are virtually the same for both enzymes, and the rate constants (k(o)) for the noncatalyzed reactions in water are also the same. This kinetic identity of both the enzymatic and the nonenzymatic reactions is not shared by a similarity in driving forces. The efficiency (DeltaG(o)() - DeltaG(cat)()) for the CI mechanism involves transition-state stabilization through general-acid catalysis and freeing of three water molecules trapped in the E.S species. The contribution to lowering DeltaG(cat)() by an increase in near attack conformer (NAC) formation in E.S as compared to S in water is not so important. In the CM reaction, the standard free energy for NAC formation in water is 8.4 kcal/mol as compared to 0.6 kcal/mol in E.S. Because the value of (DeltaG(o)() - DeltaG(cat)()) is 9 kcal/mol, the greater percentage of NACs accounts for approximately 90% of the kinetic advantage of the CM reaction. There is no discernible transition-state stabilization in the CM reaction. These results are discussed. In anthropomorphic terms, each enzyme has had to do what it must to have a biologically relevant rate of reaction.

  4. Protein disulfide isomerase is essential for viability in Saccharomyces cerevisiae.

    PubMed

    Farquhar, R; Honey, N; Murant, S J; Bossier, P; Schultz, L; Montgomery, D; Ellis, R W; Freedman, R B; Tuite, M F

    1991-12-01

    Protein disulfide isomerase (PDI) is an enzyme involved in the catalysis of disulfide bond formation in secretory and cell-surface proteins. Using an oligodeoxyribonucleotide designed to detect the conserved 'thioredoxin-like' active site of vertebrate PDIs, we have isolated a gene encoding PDI from the lower eukaryote, Saccharomyces cerevisiae. The nucleotide sequence and deduced open reading frame of the cloned gene predict a 530-amino-acid (aa) protein of Mr 59,082 and a pI of 4.1, physical properties characteristic of mammalian PDIs. Furthermore, the aa sequence shows 30-32% identity with mammalian and avian PDI sequences and has a very similar overall organisation, namely the presence of two approx. 100-aa segments, each of which is repeated, with the most significant homologies to mammalian and avian PDIs being in the regions (a, a') that contain the conserved 'thioredoxin-like' active site. The N-terminal region has the characteristics of a cleavable secretory signal sequence and the C-terminal four aa (-His-Asp-Glu-Leu) are consistent with the protein being a component of the S. cerevisiae endoplasmic reticulum. Transformants carrying multiple copies of this gene (designated PDI1) have tenfold higher levels of PDI activity and overproduce a protein of the predicted Mr. The PDI1 gene is unique in the yeast genome and encodes a single 1.8-kb transcript that is not found in stationary phase cells. Disruption of the PDI1 gene is haplo-lethal indicating that the product of this gene is essential for viability.

  5. Molecular identification, immunolocalization, and characterization of Clonorchis sinensis triosephosphate isomerase.

    PubMed

    Zhou, Juanjuan; Liao, Hua; Li, Shan; Zhou, Chenhui; Huang, Yan; Li, Xuerong; Liang, Chi; Yu, Xinbing

    2015-08-01

    Clonorchis sinensis triosephosphate isomerase (CsTIM) is a key regulatory enzyme of glycolysis and gluconeogenesis, which catalyzes the interconversion of glyceraldehyde 3-phosphate to dihydroxyacetone phosphate. In this study, the biochemical characterizations of CsTIM have been examined. A full-length complementary DNA (cDNA; Cs105350) sequence encoding CsTIM was obtained from our C. sinensis cDNA library. The open reading frame of CsTIM contains 759 bp which encodes 252 amino acids. The amino acid sequence of CsTIM shares 60-65% identity with other species. Western blot analysis displayed that recombinant CsTIM (rCsTIM) can be probed by anti-rCsTIM rat serum and anti-C. sinensis excretory/secretory products (anti-CsESPs) rat serum. Quantitative reverse transcription (RT)-PCR and western blotting analysis revealed that CsTIM messenger RNA (mRNA) and protein were differentially expressed in development cycle stages of the parasite, including adult worm, metacercaria, excysted metacercaria, and egg. In addition, immunolocalization assay showed that CsTIM was located in the seminal vesicle, eggs, and testicle. Moreover, rCsTIM exhibited active enzyme activity in catalytic reactions. The Michaelis constant (K m) of rCsTIM was 0.33 mM, when using glyceraldehyde 3-phosphate as the substrate. The optimal temperature and pH of CsTIM were 37 °C and 7.5-9.5, respectively. Collectively, these results suggest that CsTIM is an important protein involved in glycometabolism, and CsTIM possibly take part in many biological functions in the growth and development of C. sinensis.

  6. 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…

  7. 21 CFR 862.1570 - Phosphohexose isomerase test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Phosphohexose isomerase test system. 862.1570 Section 862.1570 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES... muscular dystrophy, liver diseases such as hepatitis or cirrhosis, and metastatic carcinoma. (b...

  8. 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…

  9. L-Ribose isomerase and mannose-6-phosphate isomerase: properties and applications for L-ribose production.

    PubMed

    Xu, Zheng; Sha, Yuanyuan; Liu, Chao; Li, Sha; Liang, Jinfeng; Zhou, Jiahai; Xu, Hong

    2016-11-01

    L-Ribose is a synthetic L-form monosaccharide. It is a building block of many novel nucleotide analog anti-viral drugs. Bio-production of L-ribose relies on a two-step reaction: (i) conversion of L-arabinose to L-ribulose by the catalytic action of L-arabinose isomerase (L-AI) and (ii) conversion of L-ribulose to L-ribose by the catalytic action of L-ribose isomerase (L-RI, EC 5.3.1.B3) or mannose-6-phosphate isomerase (MPI, EC 5.3.1.8, alternately named as phosphomannose isomerase). Between the two enzymes, L-RI is a rare enzyme that was discovered in 1996 by Professor Izumori's group, whereas MPI is an essential enzyme in metabolic pathways in humans and microorganisms. Recent studies have focused on their potentials for industrial production of L-ribose. This review summarizes the applications of L-RI and MPI for L-ribose production.

  10. 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.

  11. 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

  12. Structural insights from a novel invertebrate triosephosphate isomerase from Litopenaeus vannamei.

    PubMed

    Lopez-Zavala, Alonso A; Carrasco-Miranda, Jesus S; Ramirez-Aguirre, Claudia D; López-Hidalgo, Marisol; Benitez-Cardoza, Claudia G; Ochoa-Leyva, Adrian; Cardona-Felix, Cesar S; Diaz-Quezada, Corina; Rudiño-Piñera, Enrique; Sotelo-Mundo, Rogerio R; Brieba, Luis G

    2016-12-01

    Triosephosphate isomerase (TIM; EC 5.3.1.1) is a key enzyme involved in glycolysis and gluconeogenesis. Glycolysis is one of the most regulated metabolic pathways, however little is known about the structural mechanisms for its regulation in non-model organisms, like crustaceans. To understand the structure and function of this enzyme in invertebrates, we obtained the crystal structure of triosephosphate isomerase from the marine Pacific whiteleg shrimp (Litopenaeus vannamei, LvTIM) in complex with its inhibitor 2-phosphogyceric acid (2-PG) at 1.7Å resolution. LvTIM assembles as a homodimer with residues 166-176 covering the active site and residue Glu166 interacting with the inhibitor. We found that LvTIM is the least stable TIM characterized to date, with the lowest range of melting temperatures, and with the lowest activation enthalpy associated with the thermal unfolding process reported. In TIMs dimer stabilization is maintained by an interaction of loop 3 by a set of hydrophobic contacts between subunits. Within these contacts, the side chain of a hydrophobic residue of one subunit fits into a cavity created by a set of hydrophobic residues in the neighboring subunit, via a "ball and socket" interaction. LvTIM presents a Cys47 at the "ball" inter-subunit contact indicating that the character of this residue is responsible for the decrease in dimer stability. Mutational studies show that this residue plays a role in dimer stability but is not a solely determinant for dimer formation.

  13. Type II Isopentenyl Diphosphate Isomerase: Probing the Mechanism with Alkyne/Allene Diphosphate Substrate Analogues†

    PubMed Central

    Sharma, Nagendra K.; Pan, Jian-Jung; Poulter, C. Dale

    2010-01-01

    Isopentenyl diphosphate isomerase (IDI) catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the basic five-carbon building blocks of isoprenoid molecules. Two structurally unrelated classes of IDI are known. Type I IPP isomerase (IDI-1) utilizes a divalent metal in a protonation-deprotonation reaction. In contrast, the type II enzyme (IDI-2) requires reduced flavin, raising the possibility that the reaction catalyzed by IDI-2 involves the net addition/abstraction of a hydrogen atom. As part of our studies of the mechanism of isomerization for IDI-2, we synthesized allene and alkyne substrate analogues for the enzyme. These molecules are predicted to be substantially less reactive toward proton addition than IPP and DMAPP, but have similar reactivities toward hydrogen atom addition. This prediction was verified by calculations of gas phase heats of reaction for addition of a proton and of a hydrogen atom to 1-butyne (3) and 1,2-butadiene (4) to form the 1-buten-2-yl carbocation and radical, respectively, and related affinities for 2-methyl-1-butene (5) and 2-methyl-2-butene (6) using G3MP2B3 and CBS-QB3 protocols. Alkyne 1-OPP and allene 2-OPP were not substrates for Thermus thermophilus IDI-2 or Escherichia coli IDI-1, but instead were competitive inhibitors. The experimental and computational results are consistent with a protonation-deprotonation mechanism for the enzyme-catalyzed isomerization of IPP and DMAPP. PMID:20560533

  14. Sequential oxygenation of linoleic acid in the fungus Gaeumannomyces graminis: stereochemistry of dioxygenase and hydroperoxide isomerase reactions.

    PubMed

    Hamberg, M; Zhang, L Y; Brodowsky, I D; Oliw, E H

    1994-02-15

    Linoleic acid is sequentially oxygenated to (7S,8S)-dihydroxylinoleic acid by dioxygenase and hydroperoxide isomerase activities present in the fungus Gaeumannomyces graminis (Brodowsky, I. D., Hamberg, M., and Oliw, E. H., J. Biol. Chem. 267, 14738-14745 (1992)). Linoleic acids stereospecifically deuterated at C-7 and C-8 were prepared by biological desaturation of the corresponding stearates and used to determine the stereochemistry of the hydrogen abstractions occurring in the dioxygenase- and hydroperoxide isomerase-catalyzed reactions. The dioxygenase reaction was found to involve stereospecific abstraction of the pro-S hydrogen from C-8 followed by antarafacial insertion of dioxygen to produce (8R)-hydroperoxylinoleic acid. The hydroperoxide isomerase reaction consisted of conversion of (8R)-hydroperoxylinoleic acid into (7S,8S)-dihydroxylinoleic acid by stereospecific elimination of the pro-S hydrogen from C-7 and intramolecular suprafacial insertion of oxygen at C-7. Accordingly, during the conversion of linoleic acid into (8R)-hydroperoxylinoleic acid, the absolute configuration of C-8 was inverted, while the conversion of (8R)-hydroperoxylinoleic acid into (7S,8S)-dihydroxylinoleic acid occurred with retention of absolute configuration at C-7.

  15. Isopentenyl diphosphate isomerase: A checkpoint to isoprenoid biosynthesis.

    PubMed

    Berthelot, Karine; Estevez, Yannick; Deffieux, Alain; Peruch, Frédéric

    2012-08-01

    Even if the isopentenyl diphosphate (IPP) isomerases have been discovered in the 50s, it is only in the last decade that the genetical, enzymatical, structural richness and cellular importance of this large family of crucial enzymes has been uncovered. Present in all living kingdoms, they can be classified in two subfamilies: type 1 and type 2 IPP isomerases, which show clearly distinct characteristics. They all perform the regulatory isomerization of isopentenyl diphosphate into dimethylallyl diphosphate, a key rate-limiting step of the terpenoid biosynthesis, via a protonation/deprotonation mechanism. Due to their importance in the isoprenoid metabolism and the increasing interest of industry devoted to terpenoid production, it is foreseen that the biotechnological development of such enzymes should be under intense scrutiny in the near future.

  16. Solubility of glucose isomerase in ammonium sulphate solutions

    NASA Astrophysics Data System (ADS)

    Chayen, N.; Akins, J.; Campbell-Smith, S.; Blow, D. M.

    1988-07-01

    In order to quantify protein crystallization techniques, a method for measuring protein solubility in high salt concentration has been developed. It is based on a sensitive protein concentration assay, using binding to Coomassie blue dye. The protein concentration in a supernatant from which glucose isomerase is crystallising has been studied as a function of time. Equilibrium is established in 3-5 weeks, and the protein concentration remaining in solution is defined as the solubility of the protein. The solubility of glucose isomerase has been determined as a function of ammonium sulphate concentration; its variation with pH in 1.50M ammonium sulphate has also been studied. A remarkable dependence on pH over the range of 5.5 to 6.5 has been observed.

  17. 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

  18. 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

  19. The small subunit 1 of the Arabidopsis isopropylmalate isomerase is required for normal growth and development and the early stages of glucosinolate formation.

    PubMed

    Imhof, Janet; Huber, Florian; Reichelt, Michael; Gershenzon, Jonathan; Wiegreffe, Christoph; Lächler, Kurt; Binder, Stefan

    2014-01-01

    In Arabidopsis thaliana the evolutionary and functional relationship between Leu biosynthesis and the Met chain elongation pathway, the first part of glucosinolate formation, is well documented. Nevertheless the exact functions of some pathway components are still unclear. Isopropylmalate isomerase (IPMI), an enzyme usually involved in Leu biosynthesis, is a heterodimer consisting of a large and a small subunit. While the large protein is encoded by a single gene (isopropylmalate isomerase large subunit1), three genes encode small subunits (isopropylmalate isomerase small subunit1 to 3). We have now analyzed small subunit 1 (isopropylmalate isomerase small subunit1) employing artificial microRNA for a targeted knockdown of the encoding gene. Strong reduction of corresponding mRNA levels to less than 5% of wild-type levels resulted in a severe phenotype with stunted growth, narrow pale leaf blades with green vasculature and abnormal adaxial-abaxial patterning as well as anomalous flower morphology. Supplementation of the knockdown plants with leucine could only partially compensate for the morphological and developmental abnormalities. Detailed metabolite profiling of the knockdown plants revealed changes in the steady state levels of isopropylmalate and glucosinolates as well as their intermediates demonstrating a function of IPMI SSU1 in both leucine biosynthesis and the first cycle of Met chain elongation. Surprisingly the levels of free leucine slightly increased suggesting an imbalanced distribution of leucine within cells and/or within plant tissues.

  20. The Small Subunit 1 of the Arabidopsis Isopropylmalate Isomerase Is Required for Normal Growth and Development and the Early Stages of Glucosinolate Formation

    PubMed Central

    Imhof, Janet; Huber, Florian; Reichelt, Michael; Gershenzon, Jonathan; Wiegreffe, Christoph; Lächler, Kurt; Binder, Stefan

    2014-01-01

    In Arabidopsis thaliana the evolutionary and functional relationship between Leu biosynthesis and the Met chain elongation pathway, the first part of glucosinolate formation, is well documented. Nevertheless the exact functions of some pathway components are still unclear. Isopropylmalate isomerase (IPMI), an enzyme usually involved in Leu biosynthesis, is a heterodimer consisting of a large and a small subunit. While the large protein is encoded by a single gene (ISOPROPYLMALATE ISOMERASE LARGE SUBUNIT1), three genes encode small subunits (ISOPROPYLMALATE ISOMERASE SMALL SUBUNIT1 to 3). We have now analyzed small subunit 1 (ISOPROPYLMALATE ISOMERASE SMALL SUBUNIT1) employing artificial microRNA for a targeted knockdown of the encoding gene. Strong reduction of corresponding mRNA levels to less than 5% of wild-type levels resulted in a severe phenotype with stunted growth, narrow pale leaf blades with green vasculature and abnormal adaxial-abaxial patterning as well as anomalous flower morphology. Supplementation of the knockdown plants with leucine could only partially compensate for the morphological and developmental abnormalities. Detailed metabolite profiling of the knockdown plants revealed changes in the steady state levels of isopropylmalate and glucosinolates as well as their intermediates demonstrating a function of IPMI SSU1 in both leucine biosynthesis and the first cycle of Met chain elongation. Surprisingly the levels of free leucine slightly increased suggesting an imbalanced distribution of leucine within cells and/or within plant tissues. PMID:24608865

  1. 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.

  2. Isoform-specific inhibition of cyclophilins.

    PubMed

    Daum, Sebastian; Schumann, Michael; Mathea, Sebastian; Aumüller, Tobias; Balsley, Molly A; Constant, Stephanie L; de Lacroix, Boris Féaux; Kruska, Fabian; Braun, Manfred; Schiene-Fischer, Cordelia

    2009-07-07

    Cyclophilins belong to the enzyme class of peptidyl prolyl cis-trans isomerases which catalyze the cis-trans isomerization of prolyl bonds in peptides and proteins in different folding states. Cyclophilins have been shown to be involved in a multitude of cellular functions like cell growth, proliferation, and motility. Among the 20 human cyclophilin isoenzymes, the two most abundant members of the cyclophilin family, CypA and CypB, exhibit specific cellular functions in several inflammatory diseases, cancer development, and HCV replication. A small-molecule inhibitor on the basis of aryl 1-indanylketones has now been shown to discriminate between CypA and CypB in vitro. CypA binding of this inhibitor has been characterized by fluorescence anisotropy- and isothermal titration calorimetry-based cyclosporin competition assays. Inhibition of CypA- but not CypB-mediated chemotaxis of mouse CD4(+) T cells by the inhibitor provided biological proof of discrimination in vivo.

  3. Hmo1p, a high mobility group 1/2 homolog, genetically and physically interacts with the yeast FKBP12 prolyl isomerase.

    PubMed Central

    Dolinski, K J; Heitman, J

    1999-01-01

    The immunosuppressive drugs FK506 and rapamycin bind to the cellular protein FKBP12, and the resulting FKBP12-drug complexes inhibit signal transduction. FKBP12 is a ubiquitous, highly conserved, abundant enzyme that catalyzes a rate-limiting step in protein folding: peptidyl-prolyl cis-trans isomerization. However, FKBP12 is dispensible for viability in both yeast and mice, and therefore does not play an essential role in protein folding. The functions of FKBP12 may involve interactions with a number of partner proteins, and a few proteins that interact with FKBP12 in the absence of FK506 or rapamycin have been identified, including the ryanodine receptor, aspartokinase, and the type II TGF-beta receptor; however, none of these are conserved from yeast to humans. To identify other targets and functions of FKBP12, we have screened for mutations that are synthetically lethal with an FKBP12 mutation in yeast. We find that mutations in HMO1, which encodes a high mobility group 1/2 homolog, are synthetically lethal with mutations in the yeast FPR1 gene encoding FKBP12. Deltahmo1 and Deltafpr1 mutants share two phenotypes: an increased rate of plasmid loss and slow growth. In addition, Hmo1p and FKBP12 physically interact in FKBP12 affinity chromatography experiments, and two-hybrid experiments suggest that FKBP12 regulates Hmo1p-Hmo1p or Hmo1p-DNA interactions. Because HMG1/2 proteins are conserved from yeast to humans, our findings suggest that FKBP12-HMG1/2 interactions could represent the first conserved function of FKBP12 other than mediating FK506 and rapamycin actions. PMID:10049913

  4. Metabolic fate of unsaturated glucuronic/iduronic acids from glycosaminoglycans: molecular identification and structure determination of streptococcal isomerase and dehydrogenase.

    PubMed

    Maruyama, Yukie; Oiki, Sayoko; Takase, Ryuichi; Mikami, Bunzo; Murata, Kousaku; Hashimoto, Wataru

    2015-03-06

    Glycosaminoglycans in mammalian extracellular matrices are degraded to their constituents, unsaturated uronic (glucuronic/iduronic) acids and amino sugars, through successive reactions of bacterial polysaccharide lyase and unsaturated glucuronyl hydrolase. Genes coding for glycosaminoglycan-acting lyase, unsaturated glucuronyl hydrolase, and the phosphotransferase system are assembled into a cluster in the genome of pathogenic bacteria, such as streptococci and clostridia. Here, we studied the streptococcal metabolic pathway of unsaturated uronic acids and the structure/function relationship of its relevant isomerase and dehydrogenase. Two proteins (gbs1892 and gbs1891) of Streptococcus agalactiae strain NEM316 were overexpressed in Escherichia coli, purified, and characterized. 4-Deoxy-l-threo-5-hexosulose-uronate (Dhu) nonenzymatically generated from unsaturated uronic acids was converted to 2-keto-3-deoxy-d-gluconate via 3-deoxy-d-glycero-2,5-hexodiulosonate through successive reactions of gbs1892 isomerase (DhuI) and gbs1891 NADH-dependent reductase/dehydrogenase (DhuD). DhuI and DhuD enzymatically corresponded to 4-deoxy-l-threo-5-hexosulose-uronate ketol-isomerase (KduI) and 2-keto-3-deoxy-d-gluconate dehydrogenase (KduD), respectively, involved in pectin metabolism, although no or low sequence identity was observed between DhuI and KduI or between DhuD and KduD, respectively. Genes for DhuI and DhuD were found to be included in the streptococcal genetic cluster, whereas KduI and KduD are encoded in clostridia. Tertiary and quaternary structures of DhuI and DhuD were determined by x-ray crystallography. Distinct from KduI β-barrels, DhuI adopts an α/β/α-barrel structure as a basic scaffold similar to that of ribose 5-phosphate isomerase. The structure of DhuD is unable to accommodate the substrate/cofactor, suggesting that conformational changes are essential to trigger enzyme catalysis. This is the first report on the bacterial metabolism of

  5. Escherichia coli arabinose isomerase and Staphylococcus aureus tagatose-6-phosphate isomerase: which is a better template for directed evolution of non-natural substrate isomerization?

    PubMed

    Kim, Hye Jung; Uhm, Tae Guk; Kim, Seong Bo; Kim, Pil

    2010-06-01

    Metallic and non-metallic isomerases can be used to produce commercially important monosaccharides. To determine which category of isomerase is more suitable as a template for directed evolution to improve enzymes for galactose isomerization, L-arabinose isomerase from Escherichia coli (ECAI; E.C. 5.3.1.4) and tagatose-6-phosphate isomerase from Staphylococcus aureus (SATI; E.C. 5.3.1.26) were chosen as models of a metallic and non-metallic isomerase, respectively. Random mutations were introduced into the genes encoding ECAI and SATI at the same rate, resulting in the generation of 515 mutants of each isomerase. The isomerization activity of each of the mutants toward a non-natural substrate (galactose) was then measured. With an average mutation rate of 0.2 mutations/kb, 47.5% of the mutated ECAIs showed an increase in activity compared with wild-type ECAI, and the remaining 52.5% showed a decrease in activity. Among the mutated SATIs, 58.6% showed an increase in activity, whereas 41.4% showed a decrease in activity. Mutant clones showing a significant change in relative activity were sequenced and specific increases in activity were measured. The maximum increase in activity achieved by mutation of ECAI was 130%, and that for SATI was 190%. Based on these results, the characteristics of the different isomerases are discussed in terms of their usefulness for directed evolution of non-natural substrate isomerization.

  6. Cyclophilin D Is Involved in the Regulation of Autophagy and Affects the Lifespan of P. anserina in Response to Mitochondrial Oxidative Stress

    PubMed Central

    Kramer, Piet; Jung, Alexander T.; Hamann, Andrea; Osiewacz, Heinz D.

    2016-01-01

    The mitochondrial permeability transition pore plays a key role in programmed cell death and the induction of autophagy. Opening of the pore is regulated by the mitochondrial peptidyl prolyl-cis, trans-isomerase cyclophilin D (CYPD). Previously it was shown in the aging model organism Podospora anserina that PaCYPD abundance increases during aging and that PaCypD overexpressors are characterized by accelerated aging. Here, we describe a role of PaCYPD in the regulation of autophagy. We found that the accelerated aging phenotype observed in a strain overexpressing PaCypD is not metacaspase-dependent but is accompanied by an increase of general autophagy and mitophagy, the selective autophagic degradation of mitochondria. It thus is linked to what has been defined as “autophagic cell death” or “type II” programmed cell death. Moreover, we found that the previously demonstrated age-related induction of autophagy in wild-type aging depends on the presence of PaCYPD. Deletion of PaCypD leads to a decrease in autophagy in later stages of age and under paraquat-mediated oxidative stress. Finally, we report that PaCYPD is also required for mitohormesis, the beneficial effect of mild mitochondrial stress. Thus, PaCYPD plays a key role in the context-dependent regulation of pathways leading to pro-survival and pro-death effects of autophagy. PMID:27683587

  7. 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. © 2016 Federation of European Biochemical Societies.

  8. Dual activity of quinolinate synthase: triose phosphate isomerase and dehydration activities play together to form quinolinate.

    PubMed

    Reichmann, Debora; Couté, Yohann; Ollagnier de Choudens, Sandrine

    2015-10-27

    Quinolinate synthase (NadA) is an Fe4S4 cluster-containing dehydrating enzyme involved in the synthesis of quinolinic acid (QA), the universal precursor of the essential coenzyme nicotinamide adenine dinucleotide. The reaction catalyzed by NadA is not well understood, and two mechanisms have been proposed in the literature that differ in the nature of the molecule (DHAP or G-3P) that condenses with iminoaspartate (IA) to form QA. In this article, using biochemical approaches, we demonstrate that DHAP is the triose that condenses with IA to form QA. The capacity of NadA to use G-3P is due to its previously unknown triose phosphate isomerase activity.

  9. Identification of the critical residues responsible for differential reactivation of the triosephosphate isomerases of two trypanosomes.

    PubMed

    Rodríguez-Bolaños, Monica; Cabrera, Nallely; Perez-Montfort, Ruy

    2016-10-01

    The reactivation of triosephosphate isomerase (TIM) from unfolded monomers induced by guanidine hydrochloride involves different amino acids of its sequence in different stages of protein refolding. We describe a systematic mutagenesis method to find critical residues for certain physico-chemical properties of a protein. The two similar TIMs of Trypanosoma brucei and Trypanosoma cruzi have different reactivation velocities and efficiencies. We used a small number of chimeric enzymes, additive mutants and planned site-directed mutants to produce an enzyme from T. brucei with 13 mutations in its sequence, which reactivates fast and efficiently like wild-type (WT) TIM from T. cruzi, and another enzyme from T. cruzi, with 13 slightly altered mutations, which reactivated slowly and inefficiently like the WT TIM of T. brucei Our method is a shorter alternative to random mutagenesis, saturation mutagenesis or directed evolution to find multiple amino acids critical for certain properties of proteins.

  10. Identification of the critical residues responsible for differential reactivation of the triosephosphate isomerases of two trypanosomes

    PubMed Central

    Rodríguez-Bolaños, Monica; Cabrera, Nallely

    2016-01-01

    The reactivation of triosephosphate isomerase (TIM) from unfolded monomers induced by guanidine hydrochloride involves different amino acids of its sequence in different stages of protein refolding. We describe a systematic mutagenesis method to find critical residues for certain physico-chemical properties of a protein. The two similar TIMs of Trypanosoma brucei and Trypanosoma cruzi have different reactivation velocities and efficiencies. We used a small number of chimeric enzymes, additive mutants and planned site-directed mutants to produce an enzyme from T. brucei with 13 mutations in its sequence, which reactivates fast and efficiently like wild-type (WT) TIM from T. cruzi, and another enzyme from T. cruzi, with 13 slightly altered mutations, which reactivated slowly and inefficiently like the WT TIM of T. brucei. Our method is a shorter alternative to random mutagenesis, saturation mutagenesis or directed evolution to find multiple amino acids critical for certain properties of proteins. PMID:27733588

  11. Thermodynamics of Enzyme-Catalyzed Reactions: Part 5. Isomerases and Ligases

    NASA Astrophysics Data System (ADS)

    Goldberg, Robert N.; Tewari, Yadu B.

    1995-11-01

    Equilibrium constants and enthalpy changes for reactions catalyzed by the isomerase and ligase classes of enzymes have been compiled. For each reaction the following information is given: the reference for the data; the reaction studied; the name of the enzyme used and its Enzyme Commission number; the method of measurement; the conditions of measurement (temperature, pH, ionic strength, and the buffer(s) and cofactor(s) used); the data and an evaluation of it; and, sometimes, commentary on the data and on any corrections which have been applied to it or any calculations for which the data have been used. The data from 176 references have been examined and evaluated. Chemical Abstract Service registry numbers are given for the substances involved in these various reactions. There is a cross reference between the substances and the Enzyme Commission numbers of the enzymes used to catalyze the reactions in which the substances participate.

  12. xylA cloning and sequencing and biochemical characterization of xylose isomerase from Thermotoga neapolitana.

    PubMed Central

    Vieille, C; Hess, J M; Kelly, R M; Zeikus, J G

    1995-01-01

    The xylA gene coding for xylose isomerase from the hyperthermophile Thermotoga neapolitana 5068 was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a polypeptide of 444 residues with a calculated molecular weight of 50,892. The native enzyme was a homotetramer with a molecular weight of 200,000. This xylose isomerase was a member of the family II enzymes (these differ from family I isomerases by the presence of approximately 50 additional residues at the amino terminus). The enzyme was extremely thermostable, with optimal activity above 95 degrees C. The xylose isomerase showed maximum activity at pH 7.1, but it had high relative activity over a broad pH range. The catalytic efficiency (kcat/Km) of the enzyme was essentially constant between 60 and 90 degrees C, and the catalytic efficiency decreased between 90 and 98 degrees C primarily because of a large increase in Km. The T. neapolitana xylose isomerase had a higher turnover number and a lower Km for glucose than other family II xylose isomerases. Comparisons with other xylose isomerases showed that the catalytic and cation binding regions were well conserved. Comparison of different xylose isomerase sequences showed that numbers of asparagine and glutamine residues decreased with increasing enzyme thermostability, presumably as a thermophilic strategy for diminishing the potential for chemical denaturation through deamidation at elevated temperatures. PMID:7646024

  13. Thermal-unfolding reaction of triosephosphate isomerase from Trypanosoma cruzi.

    PubMed

    Mixcoha-Hernández, Edgar; Moreno-Vargas, Liliana M; Rojo-Domínguez, Arturo; Benítez-Cardoza, Claudia G

    2007-10-01

    Thermal denaturation of triosephosphate isomerase from Trypanosoma cruzi was studied by circular dicrhoism and fluorescence spectroscopies. The unfolding transition was found to be highly irreversible even at the very early stages of the reaction. Kinetic studies, allowed us to identify consecutive reactions. Firstly, only the tryptophan environment is altered. Next, changes on the secondary structure and hydrophobic surface exposure measured by 1-anilino-8-naphthalenesulfonate (ANS) binding were observed. Further conformational changes imply additional modifications on the secondary and tertiary structures and release of the hydrophobic dye leading to the formation of the unfolded state that is prone to aggregate.

  14. GPI Mount Scopus--a variant of glucosephosphate isomerase deficiency.

    PubMed

    Shalev, O; Shalev, R S; Forman, L; Beutler, E

    1993-10-01

    Glucosephosphate isomerase (GPI) deficiency is an unusual cause of hereditary nonspherocytic hemolytic anemia. The disease, inherited as an autosomal recessive disorder, is most often manifested by symptoms and signs of chronic hemolysis, ameliorated by splenectomy. We recently diagnosed GPI deficiency in a 23-year-old Ashkenazi Jewish man who displayed the typical clinical course of this disorder. The biophysical characteristics of the GPI variant are slow electrophoretic mobility, presence of only one of the two bands normally present, and extreme thermolability. To the best of our knowledge, this is the first report of GPI deficiency in a patient of Jewish descent, and we propose to designate this enzyme variant "GPI Mount Scopus".

  15. The immunosuppressant SR 31747 blocks cell proliferation by inhibiting a steroid isomerase in Saccharomyces cerevisiae.

    PubMed Central

    Silve, S; Leplatois, P; Josse, A; Dupuy, P H; Lanau, C; Kaghad, M; Dhers, C; Picard, C; Rahier, A; Taton, M; Le Fur, G; Caput, D; Ferrara, P; Loison, G

    1996-01-01

    SR 31747 is a novel immunosuppressant agent that arrests cell proliferation in the yeast Saccharomyces cerevisiae, SR 31747-treated cells accumulate the same aberrant sterols as those found in a mutant impaired in delta 8- delta 7-sterol isomerase. Sterol isomerase activity is also inhibited by SR 31747 in in vitro assays. Overexpression of the sterol isomerase-encoding gene, ERG2, confers enhanced SR resistance. Cells growing anaerobically on ergosterol-containing medium are not sensitive to SR. Disruption of the sterol isomerase-encoding gene is lethal in cells growing in the absence of exogenous ergosterol, except in SR-resistant mutants lacking either the SUR4 or the FEN1 gene product. The results suggest that sterol isomerase is the target of SR 31747 and that both the SUR4 and FEN1 gene products are required to mediate the proliferation arrest induced by ergosterol depletion. PMID:8649379

  16. Geometry matters: inverse cytotoxic relationship for cis/trans-Ru(ii) polypyridyl complexes from cis/trans-[PtCl2(NH3)2].

    PubMed

    Wachter, Erin; Zamora, Ana; Heidary, David K; Ruiz, José; Glazer, Edith C

    2016-08-09

    Two thermally activated ruthenium(ii) polypyridyl complexes, cis-Ru(bpy)2Cl2 and trans-Ru(qpy)Cl2 were investigated to determine the impact of the geometric arrangement of the exchangable ligands on the potential of the compounds to act as chemotherapeutics. In contrast to the geometry requirements for cisplatin, trans-Ru(qpy)Cl2 was 7.1-9.5× more cytotoxic than cis-Ru(bpy)2Cl2. This discovery could open up a new area of metal-based chemotherapeutic research.

  17. 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.

  18. 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

  19. Arabidopsis Responds to Alternaria alternata Volatiles by Triggering Plastid Phosphoglucose Isomerase-Independent Mechanisms1[OPEN

    PubMed Central

    Sánchez-López, Ángela María; Bahaji, Abdellatif; De Diego, Nuria; Baslam, Marouane; Li, Jun; Almagro, Goizeder; García-Gómez, Pablo; Ricarte-Bermejo, Adriana; Novák, Ondřej; Spíchal, Lukáš; Ciordia, Sergio; Mena, María Carmen

    2016-01-01

    Volatile compounds (VCs) emitted by phylogenetically diverse microorganisms (including plant pathogens and microbes that do not normally interact mutualistically with plants) promote photosynthesis, growth, and the accumulation of high levels of starch in leaves through cytokinin (CK)-regulated processes. In Arabidopsis (Arabidopsis thaliana) plants not exposed to VCs, plastidic phosphoglucose isomerase (pPGI) acts as an important determinant of photosynthesis and growth, likely as a consequence of its involvement in the synthesis of plastidic CKs in roots. Moreover, this enzyme plays an important role in connecting the Calvin-Benson cycle with the starch biosynthetic pathway in leaves. To elucidate the mechanisms involved in the responses of plants to microbial VCs and to investigate the extent of pPGI involvement, we characterized pPGI-null pgi1-2 Arabidopsis plants cultured in the presence or absence of VCs emitted by Alternaria alternata. We found that volatile emissions from this fungal phytopathogen promote growth, photosynthesis, and the accumulation of plastidic CKs in pgi1-2 leaves. Notably, the mesophyll cells of pgi1-2 leaves accumulated exceptionally high levels of starch following VC exposure. Proteomic analyses revealed that VCs promote global changes in the expression of proteins involved in photosynthesis, starch metabolism, and growth that can account for the observed responses in pgi1-2 plants. The overall data show that Arabidopsis plants can respond to VCs emitted by phytopathogenic microorganisms by triggering pPGI-independent mechanisms. PMID:27663407

  20. QM/MM modelling of ketosteroid isomerase reactivity indicates that active site closure is integral to catalysis.

    PubMed

    van der Kamp, Marc W; Chaudret, Robin; Mulholland, Adrian J

    2013-07-01

    Ketosteroid isomerase (Δ⁵-3-keto steroid isomerase or steroid Δ-isomerase) is a highly efficient enzyme at the centre of current debates on enzyme catalysis. We have modelled the reaction mechanism of the isomerization of 3-oxo-Δ⁵-steroids into their Δ⁴-conjugated isomers using high-level combined quantum mechanics/molecular mechanics (QM/MM) methods, and semi-empirical QM/MM molecular dynamics simulations. Energy profiles were obtained at various levels of QM theory (AM1, B3LYP and SCS-MP2). The high-level QM/MM profile is consistent with experimental data. QM/MM dynamics simulations indicate that active site closure and desolvation of the catalytic Asp38 occur before or during formation of dienolate intermediates. These changes have a significant effect on the reaction barrier. A low barrier to reaction is found only when the active site is closed, poising it for catalysis. This conformational change is thus integral to the whole process. The effects on the barrier are apparently largely due to changes in solvation. The combination of high-level QM/MM energy profiles and QM/MM dynamics simulation shows that the reaction involves active site closure, desolvation of the catalytic base, efficient isomerization and re-opening of the active site. These changes highlight the transition between the ligand binding/releasing form and the catalytic form of the enzyme. The results demonstrate that electrostatic interactions (as a consequence of pre-organization of the active site) are crucial for stabilization during the chemical reaction step, but closure of the active site is essential for efficient catalysis to occur.

  1. Involvement of polyphosphate kinase in virulence and stress tolerance of uropathogenic Proteus mirabilis.

    PubMed

    Peng, Liang; Jiang, Qiao; Pan, Jia-Yun; Deng, Cong; Yu, Jing-Yi; Wu, Xiao-Man; Huang, Sheng-He; Deng, Xiao-Yan

    2016-04-01

    Proteus mirabilis (P. mirabilis), a gram-negative enteric bacterium, frequently causes urinary tract infections. Many virulence factors of uropathogenic P. mirabilis have been identified, including urease, flagella, hemolysin and fimbriae. However, the functions of polyphosphate kinase (PPK), which are related to the pathogenicity of many bacteria, remain entirely unknown in P. mirabilis. In this study, a ppk gene encoding the PPK insertional mutant in P. mirabilis strain HI4320 was constructed, and its biological functions were examined. The results of survival studies demonstrated that the ppk mutant was deficient in resistance to oxidative, hyperosmotic and heat stress. The swarming and biofilm formation abilities of P. mirabilis were also attenuated after the ppk interruption. In vitro and in vivo experiments suggested that ppk was required for P. mirabilis to invade the bladder. The negative phenotypes of the ppk mutant could be restored by ppk gene complementation. Furthermore, two-dimensional gel electrophoresis and liquid chromatography-mass spectrometry were used to analyze the proteomes of the wild-type strain and the ppk mutant. Compared with the wild-type strain, seven proteins including TonB-dependent receptor, universal stress protein G, major mannose-resistant/Proteus-like fimbrial protein (MR/P fimbriae), heat shock protein, flagellar capping protein, putative membrane protein and multidrug efflux protein were down-regulated, and four proteins including exported peptidase, repressor protein for FtsI, FKBP-type peptidyl-prolyl cis-trans isomerase and phosphotransferase were up-regulated in the ppk mutant. As a whole, these results indicate that PPK is an important regulator and plays a crucial role in stress tolerance and virulence in uropathogenic P. mirabilis.

  2. [Cloning of Escherichia coli K12 xylose isomerase (glucose isomerase) and studying the enzymatic properties of its expression product].

    PubMed

    Rozanov, A S; Zagrebel'nyĭ, S N; Beklemishchev, A B

    2009-01-01

    The coding region of Escherichia coli K12 xylose (glucose) isomerase gene was inserted into the pRAC expression vector and cloned in E. coli BL21 (DE3) cells. After induction of expression of the cloned gene, the proportion of recombinant xylose isomerase accounted for 40% of the total protein content. As a result of one-stage purification by affinity chromatography, a protein preparation of 90% purity was obtained. The recombinant enzyme catalyzed the isomerization of glucose to fructose and exhibited maximum activity (0.8 U/mg) at 45 degrees C and pH 6.8. The enzyme required Mg2+ ions as a cofactor. When Mg2+ and Co2+ ions were simultaneously present in the reaction medium, the enzyme activity increased by 15-20%. Complete replacement of Mg2+ with Co2+ decreased the enzyme activity. In the presence of Ca2+ at concentrations comparable to the concentration of Mg2+, the enzyme was not inhibited, although published data reported inhibition of similar enzymes by Ca2+. The recombinant enzyme exhibited a very low thermostability: it underwent a slow inactivation when incubated at 45 degrees C and was completely inactivated after incubation at 65 degrees C for 1 h.

  3. 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.

  4. Solution structure of 3-oxo-delta5-steroid isomerase.

    PubMed

    Wu, Z R; Ebrahimian, S; Zawrotny, M E; Thornburg, L D; Perez-Alvarado, G C; Brothers, P; Pollack, R M; Summers, M F

    1997-04-18

    The three-dimensional structure of the enzyme 3-oxo-delta5-steroid isomerase (E.C. 5.3.3.1), a 28-kilodalton symmetrical dimer, was solved by multidimensional heteronuclear magnetic resonance spectroscopy. The two independently folded monomers pack together by means of extensive hydrophobic and electrostatic interactions. Each monomer comprises three alpha helices and a six-strand mixed beta-pleated sheet arranged to form a deep hydrophobic cavity. Catalytically important residues Tyr14 (general acid) and Asp38 (general base) are located near the bottom of the cavity and positioned as expected from mechanistic hypotheses. An unexpected acid group (Asp99) is also located in the active site adjacent to Tyr14, and kinetic and binding studies of the Asp99 to Ala mutant demonstrate that Asp99 contributes to catalysis by stabilizing the intermediate.

  5. Prolyl isomerase Pin1 regulates the osteogenic activity of Osterix.

    PubMed

    Lee, Sung Ho; Jeong, Hyung Min; Han, Younho; Cheong, Heesun; Kang, Bok Yun; Lee, Kwang Youl

    2015-01-15

    Osterix is an essential transcription factor for osteoblast differentiation and bone formation. The mechanism of regulation of Osterix by post-translational modification remains unknown. Peptidyl-prolyl isomerase 1 (Pin1) catalyzes the isomerization of pSer/Thr-Pro bonds and induces a conformational change in its substrates, subsequently regulating diverse cellular processes. In this study, we demonstrated that Pin1 interacts with Osterix and influences its protein stability and transcriptional activity. This regulation is likely due to the suppression of poly-ubiquitination-mediated proteasomal degradation of Osterix. Collectively, our data demonstrate that Pin1 is a novel regulator of Osterix and may play an essential role in the regulation of osteogenic differentiation.

  6. 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.

  7. Three phenotypes of glucosephosphate isomerase in sheep: improved staining recipe.

    PubMed

    Manwell, C; Baker, C M; Graydon, R J

    1985-01-01

    Contrary to results published recently, we observe three, rather than two, phenotypes for the enzyme glucosephosphate isomerase (EC 5.3.1.9) from sheep. The phenotypic electrophoretic patterns conform to the patterns observed for this dimeric enzyme in other species. Genotype frequencies in a flock of Southdowns do not deviate significantly from those predicted under the assumption of the Hardy-Weinberg equilibrium. A remarkable observation is that the electrophoretically distinct phenotypes of GPI are largely or entirely obliterated by the addition of 1-10 mmol/l MgCl2 to the electrophoretic buffers. Modification of the usual staining recipe for GPI result in greater resolution and shorter staining times.

  8. Noncatalytic Role of the FKBP52 Peptidyl-Prolyl Isomerase Domain in the Regulation of Steroid Hormone Signaling▿

    PubMed Central

    Riggs, Daniel L.; Cox, Marc B.; Tardif, Heather L.; Hessling, Martin; Buchner, Johannes; Smith, David F.

    2007-01-01

    Hormone-dependent transactivation by several of the steroid hormone receptors is potentiated by the Hsp90-associated cochaperone FKBP52, although not by the closely related FKBP51. Here we analyze the mechanisms of potentiation and the functional differences between FKBP51 and FKBP52. While both have peptidyl-prolyl isomerase activity, this is not required for potentiation, as mutations abolishing isomerase activity did not affect potentiation. Genetic selection in Saccharomyces cerevisiae for gain of potentiation activity in a library of randomly mutated FKBP51 genes identified a single residue at position 119 in the N-terminal FK1 domain as being a critical difference between these two proteins. In both the yeast model and mammalian cells, the FKBP51 mutation L119P, which is located in a hairpin loop overhanging the catalytic pocket and introduces the proline found in FKBP52, conferred significant potentiation activity, whereas the converse P119L mutation in FKBP52 decreased potentiation. A second residue in this loop, A116, also influences potentiation levels; in fact, the FKBP51-A116V L119P double mutant potentiated hormone signaling as well as wild-type FKBP52 did. These results suggest that the FK1 domain, and in particular the loop overhanging the catalytic pocket, is critically involved in receptor interactions and receptor activity. PMID:17938211

  9. Noncatalytic role of the FKBP52 peptidyl-prolyl isomerase domain in the regulation of steroid hormone signaling.

    PubMed

    Riggs, Daniel L; Cox, Marc B; Tardif, Heather L; Hessling, Martin; Buchner, Johannes; Smith, David F

    2007-12-01

    Hormone-dependent transactivation by several of the steroid hormone receptors is potentiated by the Hsp90-associated cochaperone FKBP52, although not by the closely related FKBP51. Here we analyze the mechanisms of potentiation and the functional differences between FKBP51 and FKBP52. While both have peptidyl-prolyl isomerase activity, this is not required for potentiation, as mutations abolishing isomerase activity did not affect potentiation. Genetic selection in Saccharomyces cerevisiae for gain of potentiation activity in a library of randomly mutated FKBP51 genes identified a single residue at position 119 in the N-terminal FK1 domain as being a critical difference between these two proteins. In both the yeast model and mammalian cells, the FKBP51 mutation L119P, which is located in a hairpin loop overhanging the catalytic pocket and introduces the proline found in FKBP52, conferred significant potentiation activity, whereas the converse P119L mutation in FKBP52 decreased potentiation. A second residue in this loop, A116, also influences potentiation levels; in fact, the FKBP51-A116V L119P double mutant potentiated hormone signaling as well as wild-type FKBP52 did. These results suggest that the FK1 domain, and in particular the loop overhanging the catalytic pocket, is critically involved in receptor interactions and receptor activity.

  10. Transmutation of human glutathione transferase A2-2 with peroxidase activity into an efficient steroid isomerase.

    PubMed

    Pettersson, Par L; Johansson, Ann-Sofie; Mannervik, Bengt

    2002-08-16

    A major goal in protein engineering is the tailor-making of enzymes for specified chemical reactions. Successful attempts have frequently been based on directed molecular evolution involving libraries of random mutants in which variants with desired properties were identified. For the engineering of enzymes with novel functions, it would be of great value if the necessary changes of the active site could be predicted and implemented. Such attempts based on the comparison of similar structures with different substrate selectivities have previously met with limited success. However, the present work shows that the knowledge-based redesign restricted to substrate-binding residues in human glutathione transferase A2-2 can introduce high steroid double-bond isomerase activity into the enzyme originally characterized by glutathione peroxidase activity. Both the catalytic center activity (k(cat)) and catalytic efficiency (k(cat)/K(m)) match the values of the naturally evolved glutathione transferase A3-3, the most active steroid isomerase known in human tissues. The substrate selectivity of the mutated glutathione transferase was changed 7000-fold by five point mutations. This example demonstrates the functional plasticity of the glutathione transferase scaffold as well as the potential of rational active-site directed mutagenesis as a complement to DNA shuffling and other stochastic methods for the redesign of proteins with novel functions.

  11. Expression of xylA genes encoding xylose isomerases from Escherichia coli and Streptomyces coelicolor in the methylotrophic yeast Hansenula polymorpha.

    PubMed

    Voronovsky, Andriy Y; Ryabova, Olena B; Verba, Olena V; Ishchuk, Olena P; Dmytruk, Kostyantyn V; Sibirny, Andriy A

    2005-11-01

    The thermotolerant methylotrophic yeast Hansenula polymorpha is able to ferment xylose to ethanol at high temperatures. H. polymorpha xylose reductase and xylitol dehydrogenase are involved during the first steps of this fermentation. In this article, expression of bacterial xylA genes coding for xylose isomerases from Escherichia coli or Streptomyces coelicolor in the yeast H. polymorpha was shown. The expression was achieved by integration of the xylA genes driven by the promoter of the H. polymorpha glyceraldehyde-3-phosphate dehydrogenase gene ( HpGAP) into the H. polymorpha genome. Expression of the bacterial xylose isomerase genes restored the ability of the H. polymorpha Deltaxyl1 mutant to grow in a medium with xylose as the sole carbon source. This mutant has a deletion of the XYL1 gene encoding xylose reductase and is not able to grow in the xylose medium. The H. polymorpha Deltaxyl1(xylA) transformants displayed xylose isomerase activities, which were near 20% of that of the bacterial host strain. The transformants did not differ from the yeast wild-type strain with respect to ethanol production in xylose medium.

  12. New Role of Flavin as a General Acid-Base Catalyst with No Redox Function in Type 2 Isopentenyl-diphosphate Isomerase*S⃞

    PubMed Central

    Unno, Hideaki; Yamashita, Satoshi; Ikeda, Yosuke; Sekiguchi, Shin-ya; Yoshida, Norie; Yoshimura, Tohru; Kusunoki, Masami; Nakayama, Toru; Nishino, Tokuzo; Hemmi, Hisashi

    2009-01-01

    Using FMN and a reducing agent such as NAD(P)H, type 2 isopentenyl-diphosphate isomerase catalyzes isomerization between isopentenyl diphosphate and dimethylallyl diphosphate, both of which are elemental units for the biosynthesis of highly diverse isoprenoid compounds. Although the flavin cofactor is expected to be integrally involved in catalysis, its exact role remains controversial. Here we report the crystal structures of the substrate-free and complex forms of type 2 isopentenyl-diphosphate isomerase from the thermoacidophilic archaeon Sulfolobus shibatae, not only in the oxidized state but also in the reduced state. Based on the active-site structures of the reduced FMN-substrate-enzyme ternary complexes, which are in the active state, and on the data from site-directed mutagenesis at highly conserved charged or polar amino acid residues around the active site, we demonstrate that only reduced FMN, not amino acid residues, can catalyze proton addition/elimination required for the isomerase reaction. This discovery is the first evidence for this long suspected, but previously unobserved, role of flavins just as a general acid-base catalyst without playing any redox roles, and thereby expands the known functions of these versatile coenzymes. PMID:19158086

  13. New role of flavin as a general acid-base catalyst with no redox function in type 2 isopentenyl-diphosphate isomerase.

    PubMed

    Unno, Hideaki; Yamashita, Satoshi; Ikeda, Yosuke; Sekiguchi, Shin-Ya; Yoshida, Norie; Yoshimura, Tohru; Kusunoki, Masami; Nakayama, Toru; Nishino, Tokuzo; Hemmi, Hisashi

    2009-04-03

    Using FMN and a reducing agent such as NAD(P)H, type 2 isopentenyl-diphosphate isomerase catalyzes isomerization between isopentenyl diphosphate and dimethylallyl diphosphate, both of which are elemental units for the biosynthesis of highly diverse isoprenoid compounds. Although the flavin cofactor is expected to be integrally involved in catalysis, its exact role remains controversial. Here we report the crystal structures of the substrate-free and complex forms of type 2 isopentenyl-diphosphate isomerase from the thermoacidophilic archaeon Sulfolobus shibatae, not only in the oxidized state but also in the reduced state. Based on the active-site structures of the reduced FMN-substrate-enzyme ternary complexes, which are in the active state, and on the data from site-directed mutagenesis at highly conserved charged or polar amino acid residues around the active site, we demonstrate that only reduced FMN, not amino acid residues, can catalyze proton addition/elimination required for the isomerase reaction. This discovery is the first evidence for this long suspected, but previously unobserved, role of flavins just as a general acid-base catalyst without playing any redox roles, and thereby expands the known functions of these versatile coenzymes.

  14. 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.

  15. Cumene peroxide and Fe(2+)-ascorbate-induced lipid peroxidation and effect of phosphoglucose isomerase.

    PubMed

    Agadjanyan, Z S; Dugin, S F; Dmitriev, L F

    2006-09-01

    Malondialdehyde (MDA) is one of cytotoxic aldehydes produced in cells as a result of lipid peroxidation and further MDA metabolism in cytoplasm is not known. In our experiments the liver fraction 10,000 g containing phosphoglucose isomerase and enzymes of the glyoxalase system was used and obtained experimental data shows that in this fraction there is an aggregate of reactions taking place both in membranes (lipid peroxidation) and outside membranes. MDA accumulation is relatively slow because MDA is a substrate of aldehyde isomerase (MDA <--> methylglyoxal). The well known enzyme phosphoglucose isomerase acts as an aldehyde isomerase (Michaelis constant for this enzyme Km = 133 +/- 8 microM). MDA conversion to methylglyoxal and further to neutral product D-lactate (with GSH as a cofactor) occurs in cytoplasm and D-lactate should be regarded as the end product of two different parametabolic reactions: lipid peroxidation or protein glycation.

  16. A single and two step isomerization process for d-tagatose and l-ribose bioproduction using l-arabinose isomerase and d-lyxose isomerase.

    PubMed

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

    2017-02-01

    l-ribose and d-tagatose are biochemically synthesized using sugar isomerases. The l-arabinose isomerase gene from Shigella flexneri (Sf-AI) was cloned and expressed in Escherichia coli BL-21. Sf-AI was applied for the bioproduction of d-tagatose from d-galactose. l-ribose synthesis was performed by two step isomerization using Sf-AI and d-lyxose/ribose isomerase from Cohnella laevoribosii. The overall 22.3% and 25% conversion rate were observed for d-tagatose and l-ribose production from d-galactose and l-arabinose respectively. In the present manuscript, synthesis of rare sugars from naturally available sugars is discussed along with the biochemical characterization of Sf-AI and its efficiency.

  17. Functional analysis of type 1 isopentenyl diphosphate isomerase from Halobacterium sp. NRC-1.

    PubMed

    Hoshino, Takeshi; Eguchi, Tadashi

    2007-10-01

    Here we report the characterization of the type-1 isopentenyl diphosphate isomerase derived from Halobacterium sp. NRC-1. The expressed purified enzyme showed maximum isomerase activity in the presence of 1 M NaCl at 37 degrees C at pH 6.0. This type-1 enzyme appears to be the first for which the Co2+ ion is required for activity.

  18. 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.

  19. The Posttranslocational Chaperone Lipoprotein PrsA Is Involved in both Glycopeptide and Oxacillin Resistance in Staphylococcus aureus

    PubMed Central

    Jousselin, Ambre; Renzoni, Adriana; Andrey, Diego O.; Monod, Antoinette; Lew, Daniel P.

    2012-01-01

    Understanding in detail the factors which permit Staphylococcus aureus to counteract cell wall-active antibiotics is a prerequisite to elaborating effective strategies to prolong the usefulness of these drugs and define new targets for pharmacological intervention. Methicillin-resistant S. aureus (MRSA) strains are major pathogens of hospital-acquired and community-acquired infections and are most often treated with glycopeptides (vancomycin and teicoplanin) because of their resistance to most penicillins and a limited arsenal of clinically proven alternatives. In this study, we examined PrsA, a lipid-anchored protein of the parvulin PPIase family (peptidyl-prolyl cis/trans isomerase) found ubiquitously in all Gram-positive species, in which it assists posttranslocational folding at the outer surface of the cytoplasmic membrane. We show by both genetic and biochemical assays that prsA is directly regulated by the VraRS two-component sentinel system of cell wall stress. Disruption of prsA is tolerated by S. aureus, and its loss results in no detectable overt macroscopic changes in cell wall architecture or growth rate under nonstressed growth conditions. Disruption of prsA leads, however, to notable alterations in the sensitivity to glycopeptides and dramatically decreases the resistance of COL (MRSA) to oxacillin. Quantitative transcriptional analysis reveals that prsA and vraR are coordinately upregulated in a panel of stable laboratory and clinical glycopeptide-intermediate S. aureus (GISA) strains compared to their susceptible parents. Collectively, our results point to a role for prsA as a facultative facilitator of protein secretion or extracellular folding and provide a framework for understanding why prsA is a key element of the VraRS-mediated cell wall stress response. PMID:22526301

  20. Molecular phylogenetics and comparative modeling of HEN1, a methyltransferase involved in plant microRNA biogenesis

    PubMed Central

    Tkaczuk, Karolina L; Obarska, Agnieszka; Bujnicki, Janusz M

    2006-01-01

    Background Recently, HEN1 protein from Arabidopsis thaliana was discovered as an essential enzyme in plant microRNA (miRNA) biogenesis. HEN1 transfers a methyl group from S-adenosylmethionine to the 2'-OH or 3'-OH group of the last nucleotide of miRNA/miRNA* duplexes produced by the nuclease Dicer. Previously it was found that HEN1 possesses a Rossmann-fold methyltransferase (RFM) domain and a long N-terminal extension including a putative double-stranded RNA-binding motif (DSRM). However, little is known about the details of the structure and the mechanism of action of this enzyme, and about its phylogenetic origin. Results Extensive database searches were carried out to identify orthologs and close paralogs of HEN1. Based on the multiple sequence alignment a phylogenetic tree of the HEN1 family was constructed. The fold-recognition approach was used to identify related methyltransferases with experimentally solved structures and to guide the homology modeling of the HEN1 catalytic domain. Additionally, we identified a La-like predicted RNA binding domain located C-terminally to the DSRM domain and a domain with a peptide prolyl cis/trans isomerase (PPIase) fold, but without the conserved PPIase active site, located N-terminally to the catalytic domain. Conclusion The bioinformatics analysis revealed that the catalytic domain of HEN1 is not closely related to any known RNA:2'-OH methyltransferases (e.g. to the RrmJ/fibrillarin superfamily), but rather to small-molecule methyltransferases. The structural model was used as a platform to identify the putative active site and substrate-binding residues of HEN and to propose its mechanism of action. PMID:16433904

  1. Molecular characterization and expression profiling of the protein disulfide isomerase gene family in Brachypodium distachyon L.

    PubMed

    Zhu, Chong; Luo, Nana; He, Miao; Chen, Guanxing; Zhu, Jiantang; Yin, Guangjun; Li, Xiaohui; Hu, Yingkao; Li, Jiarui; Yan, Yueming

    2014-01-01

    Protein disulfide isomerases (PDI) are involved in catalyzing protein disulfide bonding and isomerization in the endoplasmic reticulum and functions as a chaperone to inhibit the aggregation of misfolded proteins. Brachypodium distachyon is a widely used model plant for temperate grass species such as wheat and barley. In this work, we report the first molecular characterization, phylogenies, and expression profiles of PDI and PDI-like (PDIL) genes in B. distachyon in different tissues under various abiotic stresses. Eleven PDI and PDIL genes in the B. distachyon genome by in silico identification were evenly distributed across all five chromosomes. The plant PDI family has three conserved motifs that are involved in catalyzing protein disulfide bonding and isomerization, but a different exon/intron structural organization showed a high degree of structural differentiation. Two pairs of genes (BdPDIL4-1 and BdPDIL4-2; BdPDIL7-1 and BdPDIL7-2) contained segmental duplications, indicating each pair originated from one progenitor. Promoter analysis showed that Brachypodium PDI family members contained important cis-acting regulatory elements involved in seed storage protein synthesis and diverse stress response. All Brachypodium PDI genes investigated were ubiquitously expressed in different organs, but differentiation in expression levels among different genes and organs was clear. BdPDIL1-1 and BdPDIL5-1 were expressed abundantly in developing grains, suggesting that they have important roles in synthesis and accumulation of seed storage proteins. Diverse treatments (drought, salt, ABA, and H2O2) induced up- and down-regulated expression of Brachypodium PDI genes in seedling leaves. Interestingly, BdPDIL1-1 displayed significantly up-regulated expression following all abiotic stress treatments, indicating that it could be involved in multiple stress responses. Our results provide new insights into the structural and functional characteristics of the plant PDI gene

  2. Inhibitors of protein disulfide isomerase suppress apoptosis induced by misfolded proteins

    PubMed Central

    Hoffstrom, Benjamin G.; Kaplan, Anna; Letso, Reka; Schmid, Ralf; Turmel, Gregory J.; Lo, Donald C.; Stockwell, Brent R.

    2010-01-01

    A hallmark of many neurodegenerative diseases is accumulation of misfolded proteins within neurons, leading to cellular dysfunction and cell death. Although several mechanisms have been proposed to link protein misfolding to cellular toxicity, the connection remains enigmatic. Here, we report a cell death pathway involving protein disulfide isomerase (PDI), a protein chaperone that catalyzes isomerization, reduction, and oxidation of disulfides. Through a small-molecule-screening approach, we discovered five structurally distinct compounds that prevent apoptosis induced by mutant huntingtin protein. Using modified Huisgen cycloaddition chemistry, we then identified PDI as the molecular target of these small molecules. Expression of polyglutamine-expanded huntingtin exon 1 in PC12 cells caused PDI to accumulate at mitochondrial-associated-ER-membranes and trigger apoptotic cell death, via mitochondrial outer membrane permeabilization. Inhibiting PDI in rat brain cells suppressed the toxicity of mutant huntingtin exon1 and Aβ peptides processed from the amyloid precursor protein. This pro-apoptotic function of PDI provides a new mechanism linking protein misfolding and apoptotic cell death. PMID:21079601

  3. Protein disulfide isomerase externalization in endothelial cells follows classical and unconventional routes.

    PubMed

    Araujo, Thaís L S; Zeidler, Julianna D; Oliveira, Percíllia V S; Dias, Matheus H; Armelin, Hugo A; Laurindo, Francisco R M

    2017-02-01

    Extracellular protein disulfide isomerase (PDIA1) pool mediates thrombosis and vascular remodeling, however its externalization mechanisms remain unclear. We performed systematic pharmacological screening of secretory pathways affecting extracellular PDIA1 in endothelial cells (EC). We identified cell-surface (csPDIA1) and secreted non-particulated PDIA1 pools in EC. Such Golgi bypass also occurred for secreted PDIA1 in EC at baseline or after PMA, thrombin or ATP stimulation. Inhibitors of Type I, II and III unconventional routes, secretory lysosomes and recycling endosomes, including syntaxin-12 deletion, did not impair EC PDIA1 externalization. This suggests predominantly Golgi-independent unconventional secretory route(s), which were GRASP55-independent. Also, these data reinforce a vesicular-type traffic for PDIA1. We further showed that PDIA1 traffic is ATP-independent, while actin or tubulin cytoskeletal disruption markedly increased EC PDIA1 secretion. Clathrin inhibition enhanced extracellular soluble PDIA1, suggesting dynamic cycling. Externalized PDIA1 represents <2% of intracellular PDIA1. PDIA1 was robustly secreted by physiological levels of arterial laminar shear in EC and supported alpha 5 integrin thiol oxidation. Such results help clarify signaling and homeostatic mechanisms involved in multiple (patho)physiological extracellular PDIA1 functions. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Human Eosinophil Leukocytes Express Protein Disulfide Isomerase in Secretory Granules and Vesicles: Ultrastructural Studies.

    PubMed

    Dias, Felipe F; Amaral, Kátia B; Carmo, Lívia A S; Shamri, Revital; Dvorak, Ann M; Weller, Peter F; Melo, Rossana C N

    2014-06-01

    Protein disulfide isomerase (PDI) has fundamental roles in the oxidative folding of proteins in the endoplasmic reticulum (ER) of eukaryotic cells. The study of this molecule has been attracting considerable attention due to its association with other cell functions and human diseases. In leukocytes, such as neutrophils, PDI is involved with cell adhesion, signaling and inflammation. However, the expression of PDI in other leukocytes, such as eosinophils, important cells in inflammatory, allergic and immunomodulatory responses, remains to be defined. Here we used different approaches to investigate PDI expression within human eosinophils. Western blotting and flow cytometry demonstrated high PDI expression in both unstimulated and CCL11/eotaxin-1-stimulated eosinophils, with similar levels in both conditions. By using an immunogold electron microscopy technique that combines better epitope preservation and secondary Fab-fragments of antibodies linked to 1.4-nm gold particles for optimal access to microdomains, we identified different intracellular sites for PDI. In addition to predictable strong PDI labeling at the nuclear envelope, other unanticipated sites, such as secretory granules, lipid bodies and vesicles, including large transport vesicles (eosinophil sombrero vesicles), were also labeled. Thus, we provide the first identification of PDI in human eosinophils, suggesting that this molecule may have additional/specific functions in these leukocytes.

  5. Mapping Soluble Guanylyl Cyclase and Protein Disulfide Isomerase Regions of Interaction.

    PubMed

    Heckler, Erin J; Kholodovych, Vladyslav; Jain, Mohit; Liu, Tong; Li, Hong; Beuve, Annie

    2015-01-01

    Soluble guanylyl cyclase (sGC) is a heterodimeric nitric oxide (NO) receptor that produces cyclic GMP. This signaling mechanism is a key component in the cardiovascular system. NO binds to heme in the β subunit and stimulates the catalytic conversion of GTP to cGMP several hundred fold. Several endogenous factors have been identified that modulate sGC function in vitro and in vivo. In previous work, we determined that protein disulfide isomerase (PDI) interacts with sGC in a redox-dependent manner in vitro and that PDI inhibited NO-stimulated activity in cells. To our knowledge, this was the first report of a physical interaction between sGC and a thiol-redox protein. To characterize this interaction between sGC and PDI, we first identified peptide linkages between sGC and PDI, using a lysine cross-linking reagent and recently developed mass spectrometry analysis. Together with Flag-immunoprecipitation using sGC domain deletions, wild-type (WT) and mutated PDI, regions of sGC involved in this interaction were identified. The observed data were further explored with computational modeling to gain insight into the interaction mechanism between sGC and oxidized PDI. Our results indicate that PDI interacts preferentially with the catalytic domain of sGC, thus providing a mechanism for PDI inhibition of sGC. A model in which PDI interacts with either the α or the β catalytic domain is proposed.

  6. Diversifying selection underlies the origin of allozyme polymorphism at the phosphoglucose isomerase locus in Tigriopus californicus.

    PubMed

    Schoville, Sean D; Flowers, Jonathan M; Burton, Ronald S

    2012-01-01

    The marine copepod Tigriopus californicus lives in intertidal rock pools along the Pacific coast, where it exhibits strong, temporally stable population genetic structure. Previous allozyme surveys have found high frequency private alleles among neighboring subpopulations, indicating that there is limited genetic exchange between populations. Here we evaluate the factors responsible for the diversification and maintenance of alleles at the phosphoglucose isomerase (Pgi) locus by evaluating patterns of nucleotide variation underlying previously identified allozyme polymorphism. Copepods were sampled from eleven sites throughout California and Baja California, revealing deep genetic structure among populations as well as genetic variability within populations. Evidence of recombination is limited to the sample from Pescadero and there is no support for linkage disequilibrium across the Pgi locus. Neutrality tests and codon-based models of substitution suggest the action of natural selection due to elevated non-synonymous substitutions at a small number of sites in Pgi. Two sites are identified as the charge-changing residues underlying allozyme polymorphisms in T. californicus. A reanalysis of allozyme variation at several focal populations, spanning a period of 26 years and over 200 generations, shows that Pgi alleles are maintained without notable frequency changes. Our data suggest that diversifying selection accounted for the origin of Pgi allozymes, while McDonald-Kreitman tests and the temporal stability of private allozyme alleles suggests that balancing selection may be involved in the maintenance of amino acid polymorphisms within populations.

  7. Diversifying Selection Underlies the Origin of Allozyme Polymorphism at the Phosphoglucose Isomerase Locus in Tigriopus californicus

    PubMed Central

    Schoville, Sean D.; Flowers, Jonathan M.; Burton, Ronald S.

    2012-01-01

    The marine copepod Tigriopus californicus lives in intertidal rock pools along the Pacific coast, where it exhibits strong, temporally stable population genetic structure. Previous allozyme surveys have found high frequency private alleles among neighboring subpopulations, indicating that there is limited genetic exchange between populations. Here we evaluate the factors responsible for the diversification and maintenance of alleles at the phosphoglucose isomerase (Pgi) locus by evaluating patterns of nucleotide variation underlying previously identified allozyme polymorphism. Copepods were sampled from eleven sites throughout California and Baja California, revealing deep genetic structure among populations as well as genetic variability within populations. Evidence of recombination is limited to the sample from Pescadero and there is no support for linkage disequilibrium across the Pgi locus. Neutrality tests and codon-based models of substitution suggest the action of natural selection due to elevated non-synonymous substitutions at a small number of sites in Pgi. Two sites are identified as the charge-changing residues underlying allozyme polymorphisms in T. californicus. A reanalysis of allozyme variation at several focal populations, spanning a period of 26 years and over 200 generations, shows that Pgi alleles are maintained without notable frequency changes. Our data suggest that diversifying selection accounted for the origin of Pgi allozymes, while McDonald-Kreitman tests and the temporal stability of private allozyme alleles suggests that balancing selection may be involved in the maintenance of amino acid polymorphisms within populations. PMID:22768211

  8. Reduction of PDC1 expression in S. cerevisiae with xylose isomerase on xylose medium.

    PubMed

    Kim, Dong Min; Choi, Seung-Hyun; Ko, Byung Sam; Jeong, Gwon-Young; Jang, Han-Bit; Han, Jae-Gun; Jeong, Kyung-Hwan; Lee, Hyeon-Yong; Won, Yonggwan; Kim, Il-Chul

    2012-01-01

    Ethanol production using hemicelluloses has recently become a focus of many researchers. In order to promote D: -xylose fermentation, we cloned the bacterial xylA gene encoding for xylose isomerase with 434 amino acid residues from Agrobacterium tumefaciens, and successfully expressed it in Saccharomyces cerevisiae, a non-xylose assimilating yeast. The recombinant strain S. cerevisiae W303-1A/pAGROXI successfully colonized a minimal medium containing D: -xylose as a sole carbon source and was capable of growth in minimal medium containing 2% xylose via aerobic shake cultivation. Although the recombinant strain assimilates D: -xylose, its ethanol productivity is quite low during fermentation with D: -xylose alone. In order to ascertain the key enzyme in ethanol production from D: -xylose, we checked the expression levels of the gene clusters involved in the xylose assimilating pathway. Among the genes classified into four groups by their expression patterns, the mRNA level of pyruvate decarboxylase (PDC1) was reduced dramatically in xylose media. This reduced expression of PDC1, an enzyme which converts pyruvate to acetaldehyde, may cause low ethanol productivity in xylose medium. Thus, the enhancement of PDC1 gene expression may provide us with a useful tool for the fermentation of ethanol from hemicellulose.

  9. Cis-trans isomerizations of beta-carotene and lycopene: a theoretical study.

    PubMed

    Guo, Wen-Hsin; Tu, Cheng-Yi; Hu, Ching-Han

    2008-09-25

    The all-trans to mono-cis isomerizations of polyenes and two C40H56 carotenes, beta-carotene and lycopene, at the ground singlet (S0) and triplet (T1) states are studied by means of quantum chemistry computations. At the S0 state of polyenes containing n acetylene units (Pn), we find that the energy barrier of the central C=C rotation decreases with n. In contrast, however, at the T 1 state, the rotational barrier increases with n. For the C40H56 carotenes, the rotational barriers of lycopene are lower than those of their beta-carotene counterparts. This difference renders the rotational rates of lycopene to be 1-2 orders of magnitude higher than those of beta-carotene at room temperature. For both these carotenes, the barrier is lowest for the rotation toward the 13-cis isomer. The relative abundances are in the following order: all-trans > 9-cis > 13-cis > 15-cis. Although the 5-cis isomer of lycopene has the lowest energy among the cis isomers, its formation from the all-trans form is restricted, owing to a very large rotational barrier. The possible physiological implications of this study are discussed.

  10. Impact of water on the cis-trans photoisomerization of hydroxychalcones.

    PubMed

    Leydet, Yoann; Batat, Pinar; Jonusauskas, Gediminas; Denisov, Sergey; Lima, João Carlos; Parola, A Jorge; McClenaghan, Nathan D; Pina, Fernando

    2013-05-23

    The photochromism of a 2-hydroxychalcone has been studied in CH3CN and H2O/CH3OH (1/1, v/v), as well as in analogous deuterated solvents using steady-state (UV-vis absorption, (1)H and (13)C NMR) and time-resolved (ultrafast transient absorption and nanosecond flow flash photolysis) spectroscopies. Whereas the irradiation of trans-chalcone (Ct) under neutral pH conditions leads to the formation of the same final chromene derivative (B) in both media, two distinct photochemical mechanisms are proposed in agreement with thermodynamic and kinetic properties of the chemical reaction network at the ground state. Following light excitation, the first steps are identical in acetonitrile and aqueous solution: the Franck-Condon excited state rapidly populates the trans-chalcone singlet excited state (1)Ct* (LE), which evolves into a twisted state (1)P*. This excited state is directly responsible for the photochemistry in acetonitrile in the nanosecond time scale (16 ns) leading to the formation of cis-chalcone (Cc) through a simple isomerization process. The resulting cis-chalcone evolves into the chromene B through a tautomerization process in the ground state (τ = 10 ms). Unlike in acetonitrile, in H2O/CH3OH (1/1, v/v), the P* state becomes unstable and evolves into a new state attributed to the tautomer (1)Q*. This state directly evolves into B in one photochemical step through a consecutive ultrafast tautomerization process followed by electrocyclization. This last case represents a new hypothesis in the photochromism of 2-hydroxychalcone derivatives.

  11. Modelling of the cis-trans partitioning in the photoisomerizations of cyanines and stilbene derivatives

    NASA Astrophysics Data System (ADS)

    Caselli, M.; Momicchioli, F.; Ponterini, G.

    1993-12-01

    In the course of photoisomerization, polymethine cyanines as well as stilbene and its derivates decay from the S 1 potential energy minimum, corresponding to the perpendicular geometry, to yield either cis or trans ground-state molecules. The fraction of cis isomers obtained, α, spans a larger range of values for symmetric cyanines than for stilbene derivatives. It is argued that such different behaviour for the two classes of compounds should be traceable to the electronically different nature of their S 1 perp species. Making use of radiationless transition theory results, it is shown the relative location of the S 1 minimum and S 0 maximum along the internal rotation coordinate is crucial to the evaluation of α: even small differences between these critical twisting angles, which are more reasonably expected for polymethine cyanines than for stilbene-like compounds, may cause strong deviations from equipartitioning (α=0.5).

  12. IRIS Toxicological Review of cis- & trans-1,2-Dichloroethylene (Interagency Science Consultation Draft)

    EPA Science Inventory

    On September 24, 2009, the Toxicological Review of cis- and trans-1,2-dichloroethylene and the charge to external peer reviewers were released for external peer review and public comment. The Toxicological Review and charge were reviewed internally by EPA and by other federal ag...

  13. IRIS Toxicological Review of cis- & trans-1,2-Dichloroethylene (Interagency Science Discussion Draft)

    EPA Science Inventory

    EPA is releasing the draft report, Toxicological Review of cis-1,2-Dichloroethylene and trans-1,2-Dichloroethylene, that was distributed to Federal agencies and White House Offices for comment during the Science Discussion step of the IRIS Toxicological Review of Cis-& Trans-1,2-Dichloroethylene (External Review Draft)

    EPA Science Inventory

    EPA is conducting a peer review of the scientific basis supporting the human health hazard and dose-response assessment of cis- and trans-1,2-dichloroethylene that will appear in the Integrated Risk Information System (IRIS) database.

  14. Proline cis-trans isomerization is influenced by local lysine acetylation-deacetylation

    PubMed Central

    Howe, Françoise S.; Mellor, Jane

    2014-01-01

    Acetylation of lysine residues has several characterised functions in chromatin. These include neutralization of the lysine’s positive charge to directly influence histone tail-DNA/internucleosomal interactions or indirect effects via bromodomain-containing effector proteins. Recently, we described a novel function of lysine acetylation to influence proline isomerization and thus local protein conformation. We found that acetylation of lysine 14 in the histone H3 N-terminal tail (H3K14ac), an intrinsically disordered domain, increased the proportion of neighbouring proline 16 (H3P16) in the trans conformation. This conformation of the tail was associated with reduced tri-methylation on histone H3 lysine 4 (H3K4me3) due to both decreased methylation by the Set1 methyltransferase (with the me3-specific subunit Spp1) and increased demethylation by the demethylase Jhd2. Interestingly, H3K4me3 on individual genes was differentially affected by substitution of H3K14 or H3P16, with ribosomal protein genes losing the least H3K4me3 and environmental stress-induced genes losing the most. PMID:28357218

  15. Separate cis-trans Pathways Post-transcriptionally Regulate Murine CD154 (CD40 Ligand) Expression

    PubMed Central

    Hamilton, B. JoNell; Wang, Xiao-Wei; Collins, Jane; Bloch, Donald; Bergeron, Alan; Henry, Brian; Terry, Benjamin M.; Zan, Moe; Mouland, Andrew J.; Rigby, William F. C.

    2008-01-01

    We report a role for CA repeats in the 3′-untranslated region (3′-UTR) in regulating CD154 expression. Human CD154 is encoded by an unstable mRNA; this instability is conferred in cis by a portion of its 3′-UTR that includes a polypyrimidine-rich region and CA dinucleotide repeat. We demonstrate similar instability activity with the murine CD154 3′-UTR. This instability element mapped solely to a conserved 100-base CU-rich region alone, which we call a CU-rich response element. Surprisingly, the CA dinucleotide-rich region also regulated reporter expression but at the level of translation. This activity was associated with poly(A) tail shortening and regulated by heterogeneous nuclear ribonucleoprotein L levels. We conclude that the CD154 3′-UTR contains dual cis-acting elements, one of which defines a novel function for exonic CA dinucleotide repeats. These findings suggest a mechanism for the association of 3′-UTR CA-rich response element polymorphisms with CD154 overexpression and the subsequent risk of autoimmune disease. PMID:18640985

  16. Fluorescence of carotenoids. Effect of oxygenation and cis/trans isomerization

    NASA Astrophysics Data System (ADS)

    Jørgensen, Kevin; Stapelfeldt, Henrik; Skibsted, Leif H.

    1992-03-01

    C 40 carotenoids fall, with respect to fluorescence in homogeneous solution, into two distinct groups depending on the presence of a CO group in the molecule. Excitation spectra agree with absorption spectra for the carbonyl derivatives astaxanthin and canthaxanthin. In contrast, zeaxanthin and isomers of β-carotene have a twentyfold increase in fluorescence quantum yield for excitation around 350 nm compared to excitation near the absorption maximum (at approximatively 430 nm). These differences are interpreted in terms of the role of non-emitting 1(n, π*) states related to the CO group in facilitating non-radiative deactivation of higher 1(π, π*) states.

  17. Communication: One-photon phase control of cis-trans isomerization in retinal

    SciTech Connect

    Arango, Carlos A.; Brumer, Paul

    2013-02-21

    We computationally demonstrate the one-photon phase control of retinal isomerization under conditions of low laser intensity. The calculations, utilizing the multiconfigurational time dependent Hartree method, include coupling between the two modes that are active in isomerization and the background molecular vibrational environment. Noting previously unsuccessful computations highlights the significance of this result.

  18. PBOND: web server for the prediction of proline and non-proline cis/trans isomerization.

    PubMed

    Exarchos, Konstantinos P; Exarchos, Themis P; Papaloukas, Costas; Troganis, Anastassios N; Fotiadis, Dimitrios I

    2009-09-01

    PBOND is a web server that predicts the conformation of the peptide bond between any two amino acids. PBOND classifies the peptide bonds into one out of four classes, namely cis imide (cis-Pro), cis amide (cis-nonPro), trans imide (trans-Pro) and trans amide (trans-nonPro). Moreover, for every prediction a reliability index is computed. The underlying structure of the server consists of three stages: (1) feature extraction, (2) feature selection and (3) peptide bond classification. PBOND can handle both single sequences as well as multiple sequences for batch processing. The predictions can either be directly downloaded from the web site or returned via e-mail. The PBOND web server is freely available at http://195.251.198.21/pbond.html.

  19. The role of intersection topography in bond selectivity of cis-trans photoisomerization

    PubMed Central

    Ben-Nun, M.; Molnar, F.; Schulten, K.; Martínez, Todd J.

    2002-01-01

    Ab initio methods are used to characterize the ground and first excited state of the chromophore in the rhodopsin family of proteins: retinal protonated Schiff base. Retinal protonated Schiff base has five double bonds capable of undergoing isomerization. Upon absorption of light, the chromophore isomerizes and the character of the photoproducts (e.g., 13-cis and 11-cis) depends on the environment, protein vs. solution. Our ab initio calculations show that, in the absence of any specific interactions with the environment (e.g., discrete ordered charges in a protein), energetic considerations cannot explain the observed bond selectivity. We instead attribute the origin of bond selectivity to the shape (topography) of the potential energy surfaces in the vicinity of points of true degeneracy (conical intersections) between the ground and first excited electronic states. This provides a molecular example where a competition between two distinct but nearly isoenergetic photochemical reaction pathways is resolved by a topographical difference between two conical intersections. PMID:11854479

  1. IRIS Toxicological Review of cis- & trans-1,2-Dichloroethylene (Interagency Science Discussion Draft)

    EPA Science Inventory

    EPA is releasing the draft report, Toxicological Review of cis-1,2-Dichloroethylene and trans-1,2-Dichloroethylene, that was distributed to Federal agencies and White House Offices for comment during the Science Discussion step of the IRIS Toxicological Review of cis- & trans-1,2-Dichloroethylene (Interagency Science Consultation Draft)

    EPA Science Inventory

    On September 24, 2009, the Toxicological Review of cis- and trans-1,2-dichloroethylene and the charge to external peer reviewers were released for external peer review and public comment. The Toxicological Review and charge were reviewed internally by EPA and by other federal ag...

  2. IRIS Toxicological Review of Cis-& Trans-1,2-Dichloroethylene (External Review Draft)

    EPA Science Inventory

    EPA is conducting a peer review of the scientific basis supporting the human health hazard and dose-response assessment of cis- and trans-1,2-dichloroethylene that will appear in the Integrated Risk Information System (IRIS) database.

  3. Comparing long-range corrected functionals in the cis-trans isomerisation of the retinal chromophore

    NASA Astrophysics Data System (ADS)

    Rostov, Ivan V.; Kobayashi, Rika; Amos, Roger D.

    2012-10-01

    Earlier results for the 11-cis to all-trans isomerisation of the retinal chromophore after photoexcitation, studied using time-dependent density functional theory with the hybrid CAM-B3LYP functional, are compared with new results using other long-range corrected DFT functionals. The TDDFT S0 and S1 minimum energy paths have been compared with the approximate coupled-cluster method RI-CC2. All calculations were consistent in producing an additional avoided crossing minimum on the S 1 minimum energy path lying approximately halfway between the 11-cis and all-trans S1 minima. In this minimum on the S1 potential energy surface, the retinal chromophore has inverted bond order in its carbon chain and lower energy than it has in both the 11-cis and all-trans S1 minima.

  4. Proline cis-trans isomerization in staphylococcal nuclease: multi-substrate free energy perturbation calculations.

    PubMed Central

    Hodel, A.; Rice, L. M.; Simonson, T.; Fox, R. O.; Brünger, A. T.

    1995-01-01

    Staphylococcal nuclease A exists in two folded forms that differ in the isomerization state of the Lys 116-Pro 117 peptide bond. The dominant form (90% occupancy) adopts a cis peptide bond, which is observed in the crystal structure. NMR studies show that the relatively small difference in free energy between the cis and trans forms (delta Gcis-->trans approximately 1.2 kcal/mol) results from large and nearly compensating differences in enthalpy and entropy (delta Hcis-->trans approximately delta TScis-->trans approximately 10 kcal/mol). There is evidence from X-ray crystal structures that the structural differences between the cis and the trans forms of nuclease are confined to the conformation of residues 112-117, a solvated protein loop. Here, we obtain a thermodynamic and structural description of the conformational equilibrium of this protein loop through an exhaustive conformational search that identified several substates followed by free energy simulations between the substrates. By partitioning the search into conformational substates, we overcame the multiple minima problem in this particular case and obtained precise and reproducible free energy values. The protein and water environment was implicitly modeled by appropriately chosen nonbonded terms between the explicitly treated loop and the rest of the protein. These simulations correctly predicted a small free energy difference between the cis and trans forms composed of larger, compensating differences in enthalpy and entropy. The structural predictions of these simulations were qualitatively consistent with known X-ray structures of nuclease variants and yield a model of the unknown minor trans conformation. PMID:7613463

  5. Assembly and disassembly of the Golgi complex: two processes arranged in a cis-trans direction

    PubMed Central

    1992-01-01

    We have studied the disassembly and assembly of two morphologically and functionally distinct parts of the Golgi complex, the cis/middle and trans cisterna/trans network compartments. For this purpose we have followed the redistribution of three cis/middle- (GMPc-1, GMPc-2, MG 160) and two trans- (GMPt-1 and GMPt-2) Golgi membrane proteins during and after treatment of normal rat kidney (NRK) cells with brefeldin A (BFA). BFA induced complete disassembly of the cis/middle- and trans- Golgi complex and translocation of GMPc and GMPt to the ER. Cells treated for short times (3 min) with BFA showed extensive disorganization of both cis/middle- and trans-Golgi complexes. However, complete disorganization of the trans part required much longer incubations with the drug. Upon removal of BFA the Golgi complex was reassembled by a process consisting of three steps: (a) exist of cis/middle proteins from the ER and their accumulation into vesicular structures scattered throughout the cytoplasm; (b) gradual relocation and accumulation of the trans proteins in the vesicles containing the cis/middle proteins; and (c) assembly of the cisternae, and reconstruction of the Golgi complex within an area located in the vicinity of the centrosome from which the ER was excluded. Reconstruction of the cis/middle-Golgi complex occurred under temperature conditions inhibitory of the reorganization of the trans- Golgi complex, and was dependent on microtubules. Reconstruction of the trans-Golgi complex, disrupted with nocodazole after selective fusion of the cis/middle-Golgi complex with the ER, occurred after the release of cis/middle-Golgi proteins from the ER and the assembly of the cis/middle cisternae. PMID:1730750

  6. The carbon mainframe structure of cis-trans-1,3-difluoroacetone

    NASA Astrophysics Data System (ADS)

    Grubbs, G. S.

    2017-01-01

    The carbon-13 backbone structure of 1,3-difluoroacetone has been obtained and reported for the first time. This was achieved through the collection of singly substituted 13C isotopologue rotational spectra at each carbon position. The rotational constants for each isotopologue have been determined and are reported for the first time. Kraitchman coordinates and second moment analysis verify the structure determined by previous literature studies and are presented.

  7. Communication: One-photon phase control of cis-trans isomerization in retinal

    NASA Astrophysics Data System (ADS)

    Arango, Carlos A.; Brumer, Paul

    2013-02-01

    We computationally demonstrate the one-photon phase control of retinal isomerization under conditions of low laser intensity. The calculations, utilizing the multiconfigurational time dependent Hartree method, include coupling between the two modes that are active in isomerization and the background molecular vibrational environment. Noting previously unsuccessful computations highlights the significance of this result.

  8. Giardial triosephosphate isomerase as possible target of the cytotoxic effect of omeprazole in Giardia lamblia.

    PubMed

    Reyes-Vivas, Horacio; de la Mora-de la Mora, Ignacio; Castillo-Villanueva, Adriana; Yépez-Mulia, Lilian; Hernández-Alcántara, Gloria; Figueroa-Salazar, Rosalia; García-Torres, Itzhel; Gómez-Manzo, Saúl; Méndez, Sara T; Vanoye-Carlo, América; Marcial-Quino, Jaime; Torres-Arroyo, Angélica; Oria-Hernández, Jesús; Gutiérrez-Castrellón, Pedro; Enríquez-Flores, Sergio; López-Velázquez, Gabriel

    2014-12-01

    Giardiasis is highly prevalent in the developing world, and treatment failures with the standard drugs are common. This work deals with the proposal of omeprazole as a novel antigiardial drug, focusing on a giardial glycolytic enzyme used to follow the cytotoxic effect at the molecular level. We used recombinant technology and enzyme inactivation to demonstrate the capacity of omeprazole to inactivate giardial triosephosphate isomerase, with no adverse effects on its human counterpart. To establish the specific target in the enzyme, we used single mutants of every cysteine residue in triosephosphate isomerase. The effect on cellular triosephosphate isomerase was evaluated by following the remnant enzyme activity on trophozoites treated with omeprazole. The interaction of omeprazole with giardial proteins was analyzed by fluorescence spectroscopy. The susceptibility to omeprazole of drug-susceptible and drug-resistant strains of Giardia lamblia was evaluated to demonstrate its potential as a novel antigiardial drug. Our results demonstrate that omeprazole inhibits giardial triosephosphate isomerase in a species-specific manner through interaction with cysteine at position 222. Omeprazole enters the cytoplasmic compartment of the trophozoites and inhibits cellular triosephosphate isomerase activity in a dose-dependent manner. Such inhibition takes place concomitantly with the cytotoxic effect caused by omeprazole on trophozoites. G. lamblia triosephosphate isomerase (GlTIM) is a cytoplasmic protein which can help analyses of how omeprazole works against the proteins of this parasite and in the effort to understand its mechanism of cytotoxicity. Our results demonstrate the mechanism of giardial triosephosphate isomerase inhibition by omeprazole and show that this drug is effective in vitro against drug-resistant and drug-susceptible strains of G. lamblia. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  9. Role of LRAT on the retinoid isomerase activity and membrane association of Rpe65.

    PubMed

    Jin, Minghao; Yuan, Quan; Li, Songhua; Travis, Gabriel H

    2007-07-20

    Absorption of a photon by a vertebrate opsin pigment induces 11-cis to all-trans isomerization of its retinaldehyde chromophore. Restoration of light sensitivity to the bleached opsin requires chemical re-isomerization of the chromophore via an enzyme pathway called the visual cycle. The retinoid isomerase in this pathway is Rpe65, a membrane-associated protein in the retinal pigment epithelium (RPE) with no predicted membrane-spanning segments. It has been suggested that Rpe65 is S-palmitoylated by lecithin:retinol acyl transferase (LRAT) on Cys(231), Cys(329), and Cys(330), and that this palmitoylation is required for isomerase activity and the association of Rpe65 with membranes. Here we show that the affinity of Rpe65 for membranes is similar in wild-type and lrat(-/-) mice. The isomerase activity of Rpe65 is also similar in both strains when all-trans-retinyl palmitate is used as substrate. With all-trans-retinol substrate, isomerase activity is present in wild-type but undetectable in RPE homogenates from lrat(-/-) mice. Substitution of Cys(231), Cys(329), and Cys(330) with Ser or Ala did not affect the affinity of Rpe65 for membranes. Further, these Cys residues are not palmitoylated in Rpe65 by mass spectrometric analysis. Global inhibition of protein palmitoylation by 2-bromopalmitate did not affect the solubility or isomerase activity of Rpe65. Finally, we show that soluble and membrane-associated Rpe65 possesses similar isomerase specific activities. These results indicate that LRAT is not required for isomerase activity beyond synthesis of retinyl-ester substrate, and that the association of Rpe65 with membranes is neither dependent upon LRAT nor the result of S-palmitoylation. The affinity of Rpe65 for membranes is probably an intrinsic feature of this protein.

  10. Giardial Triosephosphate Isomerase as Possible Target of the Cytotoxic Effect of Omeprazole in Giardia lamblia

    PubMed Central

    Reyes-Vivas, Horacio; de la Mora-de la Mora, Ignacio; Castillo-Villanueva, Adriana; Yépez-Mulia, Lilian; Hernández-Alcántara, Gloria; Figueroa-Salazar, Rosalia; García-Torres, Itzhel; Gómez-Manzo, Saúl; Méndez, Sara T.; Vanoye-Carlo, América; Marcial-Quino, Jaime; Torres-Arroyo, Angélica; Oria-Hernández, Jesús; Gutiérrez-Castrellón, Pedro; Enríquez-Flores, Sergio

    2014-01-01

    Giardiasis is highly prevalent in the developing world, and treatment failures with the standard drugs are common. This work deals with the proposal of omeprazole as a novel antigiardial drug, focusing on a giardial glycolytic enzyme used to follow the cytotoxic effect at the molecular level. We used recombinant technology and enzyme inactivation to demonstrate the capacity of omeprazole to inactivate giardial triosephosphate isomerase, with no adverse effects on its human counterpart. To establish the specific target in the enzyme, we used single mutants of every cysteine residue in triosephosphate isomerase. The effect on cellular triosephosphate isomerase was evaluated by following the remnant enzyme activity on trophozoites treated with omeprazole. The interaction of omeprazole with giardial proteins was analyzed by fluorescence spectroscopy. The susceptibility to omeprazole of drug-susceptible and drug-resistant strains of Giardia lamblia was evaluated to demonstrate its potential as a novel antigiardial drug. Our results demonstrate that omeprazole inhibits giardial triosephosphate isomerase in a species-specific manner through interaction with cysteine at position 222. Omeprazole enters the cytoplasmic compartment of the trophozoites and inhibits cellular triosephosphate isomerase activity in a dose-dependent manner. Such inhibition takes place concomitantly with the cytotoxic effect caused by omeprazole on trophozoites. G. lamblia triosephosphate isomerase (GlTIM) is a cytoplasmic protein which can help analyses of how omeprazole works against the proteins of this parasite and in the effort to understand its mechanism of cytotoxicity. Our results demonstrate the mechanism of giardial triosephosphate isomerase inhibition by omeprazole and show that this drug is effective in vitro against drug-resistant and drug-susceptible strains of G. lamblia. PMID:25223993

  11. 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

  12. Solubility and crystallization of xylose isomerase from Streptomyces rubiginosus

    NASA Astrophysics Data System (ADS)

    Vuolanto, Antti; Uotila, Sinikka; Leisola, Matti; Visuri, Kalevi

    2003-10-01

    We have studied the crystallization and crystal solubility of xylose isomerase (XI) from Streptomyces rubiginosus. In this paper, we show a rational approach for developing a large-scale crystallization process for XI. Firstly, we measured the crystal solubility in salt solutions with respect to salt concentration, temperature and pH. In ammonium sulfate the solubility of XI decreased logarithmically when increasing the salt concentration. Surprisingly, the XI crystals had a solubility minimum at low concentration of magnesium sulfate. The solubility of XI in 0.17 M magnesium sulfate was less than 0.5 g l -1. The solubility of XI increased logarithmically when increasing the temperature. We also found a solubility minimum around pH 7. This is far from the isoelectric point of XI (pH 3.95). Secondly, based on the solubility study, we developed a large-scale crystallization process for XI. In a simple and economical cooling crystallization of XI from 0.17 M magnesium sulfate solution, the recovery of crystalline active enzyme was over 95%. Moreover, we developed a process for production of uniform crystals and produced homogenous crystals with average crystal sizes between 12 and 360 μm.

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

    DOE PAGES

    Kiser, Philip D.; Zhang, Jianye; Badiee, Mohsen; ...

    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

  14. 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.

  15. 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.

  16. 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.

  17. Molecular simulations of solute transport in xylose isomerase crystals.

    PubMed

    Malek, Kourosh; Coppens, Marc-Olivier

    2008-02-07

    Cross-linked enzyme crystals (CLECs) enclose an extensive regular matrix of chiral solvent-filled nanopores, via which ions and solutes travel in and out. Several cross-linked enzyme crystals have recently been used for chiral separation and as biocatalysts. We studied the dynamics of solute transport in orthorhombic d-xylose isomerase (XI) crystals by means of Brownian dynamics (BD) and molecular dynamics (MD) simulations, which show how the protein residues influence the dynamics of solute molecules in confined regions inside the lattice. In the BD simulations, coarse-grained beads represent solutes of different sizes. The diffusion of S-phenylglycine molecules inside XI crystals is investigated by long-time MD simulations. The computed diffusion coefficients within a crystal are found to be orders of magnitude lower than in bulk water. The simulation results are compared to the recent experimental studies of diffusion and reaction inside XI crystals. The insights obtained from simulations allow us to understand the nature of solute-protein interactions and transport phenomena in CLECs, which is useful for the design of novel nanoporous biocatalysts and bioseparations based on CLECs.

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

    PubMed

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

    2015-06-01

    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 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.

  19. 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.

  1. Microbial sucrose isomerases: producing organisms, genes and enzymes.

    PubMed

    Goulter, Ken C; Hashimi, Saeed M; Birch, Robert G

    2012-01-05

    Sucrose isomerase (SI) activity is used industrially for the conversion of sucrose into isomers, particularly isomaltulose or trehalulose, which have properties advantageous over sucrose for some food uses. All of the known microbial SIs are TIM barrel proteins that convert sucrose without need for any cofactors, with varying kinetics and product specificities. The current analysis was undertaken to bridge key gaps between the information in patents and scientific publications about the microbes and enzymes useful for sucrose isomer production. This analysis shows that microbial SIs can be considered in 5 structural classes with corresponding functional distinctions that broadly align with the taxonomic differences between producing organisms. The most widely used bacterial strain for industrial production of isomaltulose, widely referred to as "Protaminobacter rubrum" CBS 574.77, is identified as Serratia plymuthica. The strain producing the most structurally divergent SI, with a high product specificity for trehalulose, widely referred to as "Pseudomonas mesoacidophila" MX-45, is identified as Rhizobium sp. Each tested SI-producer is shown to have a single SI gene and enzyme, so the properties reported previously for the isolated proteins can reasonably be associated with the products of the genes subsequently cloned from the same isolates and SI classes. Some natural isolates with potent SI activity do not catabolize the isomer under usual production conditions. The results indicate that their industrial potential may be further enhanced by selection for variants that do not catabolize the sucrose substrate.

  2. Structure and inactivation of triosephosphate isomerase from Entamoeba histolytica.

    PubMed

    Rodríguez-Romero, Adela; Hernández-Santoyo, Alejandra; del Pozo Yauner, Luis; Kornhauser, Adrián; Fernández-Velasco, D Alejandro

    2002-09-27

    Triosephosphate isomerase (TIM) has been proposed as a target for drug design. TIMs from several parasites have a cysteine residue at the dimer interface, whose derivatization with thiol-specific reagents induces enzyme inactivation and aggregation. TIMs lacking this residue, such as human TIM, are less affected. TIM from Entamoeba histolytica (EhTIM) has the interface cysteine residue and presents more than ten insertions when compared with the enzyme from other pathogens. To gain further insight into the role that interface residues play in the stability and reactivity of these enzymes, we determined the high-resolution structure and characterized the effect of methylmethane thiosulfonate (MMTS) on the activity and conformational properties of EhTIM. The structure of this enzyme was determined at 1.5A resolution using molecular replacement, observing that the dimer is not symmetric. EhTIM is completely inactivated by MMTS, and dissociated into stable monomers that possess considerable secondary structure. Structural and spectroscopic analysis of EhTIM and comparison with TIMs from other pathogens reveal that conformational rearrangements of the interface after dissociation, as well as intramonomeric contacts formed by the inserted residues, may contribute to the unusual stability of the derivatized EhTIM monomer.

  3. 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.

  4. Specificity and kinetics of triose phosphate isomerase from chicken muscle

    PubMed Central

    Putman, Sylvia J.; Coulson, A. F. W.; Farley, I. R. T.; Riddleston, B.; Knowles, J. R.

    1972-01-01

    The isolation of crystalline triose phosphate isomerase from chicken breast muscle is described. The values of kcat. and Km for the reaction in each direction were determined from experiments over wide substrate-concentration ranges, and the reactions were shown to obey simple Michaelis–Menten kinetics. With d-glyceraldehyde 3-phosphate as substrate, kcat. is 2.56×105min−1 and Km is 0.47mm; with dihydroxyacetone phosphate as substrate, kcat. is 2.59×104min−1 and Km is 0.97mm. The enzyme-catalysed exchange of the methyl hydrogen atoms of the `virtual substrate' monohydroxyacetone phosphate with solvent 2H2O or 3H2O was shown. This exchange is about 104-fold slower than the corresponding exchange of the C-3 hydrogen of dihydroxyacetone phosphate. The other deoxy substrate, 3-hydroxypropionaldehyde phosphate, was synthesized, but is too unstable in aqueous solution for analogous proton-exchange reactions to be studied. PMID:4643318

  5. Expression and Localization of Plant Protein Disulfide Isomerase.

    PubMed Central

    Shorrosh, B. S.; Subramaniam, J.; Schubert, K. R.; Dixon, R. A.

    1993-01-01

    A cDNA clone encoding a putative protein disulfide isomerase (PDI, EC 5.3.4.1) from alfalfa (Medicago sativa L.) was expressed in Escherichia coli cells, and an antiserum was raised against the expressed PDI-active protein. The antiserum recognized a protein of approximately 60 kD in extracts from alfalfa, soybean, and tobacco roots and stems. Levels of this protein remained relatively constant on exposure of alfalfa cell suspension cultures to the protein glycosylation inhibitor tunicamycin, whereas a slightly lower molecular mass form, also detected by the antiserum, was induced by this treatment. A lower molecular mass form of PDI was also observed in roots of alfalfa seedlings during the first 5 weeks after germination. PDI levels increased in developing soybean seeds up to 17 d after fertilization and then declined. Tissue print immunoblots revealed highest levels of PDI protein in the cambial tissues of soybean stems and petioles and in epidermal, subepidermal, cortical, and pith tissues of stems of alfalfa and tobacco. Immunogold electron microscopy confirmed the localization of PDI to the endoplasmic reticulum in soybean root nodules. PMID:12231974

  6. The flexibility and dynamics of protein disulfide isomerase

    PubMed Central

    Wells, Stephen A.; Emilio Jimenez‐Roldan, J.; Bhattacharyya, Moitrayee; Vishweshwara, Saraswathi; Freedman, Robert B.

    2016-01-01

    ABSTRACT We have studied the mobility of the multidomain folding catalyst, protein disulfide isomerase (PDI), by a coarse‐graining approach based on flexibility. We analyze our simulations of yeast PDI (yPDI) using measures of backbone movement, relative positions and orientations of domains, and distances between functional sites. We find that there is interdomain flexibility at every interdomain junction but these show very different characteristics. The extent of interdomain flexibility is such that yPDI's two active sites can approach much more closely than is found in crystal structures—and indeed hinge motion to bring these sites into proximity is the lowest energy normal mode of motion of the protein. The flexibility predicted for yPDI (based on one structure) includes the other known conformation of yPDI and is consistent with (i) the mobility observed experimentally for mammalian PDI and (ii) molecular dynamics. We also observe intradomain flexibility and clear differences between the domains in their propensity for internal motion. Our results suggest that PDI flexibility enables it to interact with many different partner molecules of widely different sizes and shapes, and highlights considerable similarities of yPDI and mammalian PDI. Proteins 2016; 84:1776–1785. © 2016 Wiley Periodicals, Inc. PMID:27616289

  7. Crystal structure of triosephosphate isomerase from Trypanosoma cruzi in hexane

    PubMed Central

    Gao, Xiu-Gong; Maldonado, Ernesto; Pérez-Montfort, Ruy; Garza-Ramos, Georgina; de Gómez-Puyou, Marietta Tuena; Gómez-Puyou, Armando; Rodríguez-Romero, Adela

    1999-01-01

    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-Å 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 Å 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. PMID:10468562

  8. Immobilization of Recombinant Glucose Isomerase for Efficient Production of High Fructose Corn Syrup.

    PubMed

    Jin, Li-Qun; Xu, Qi; Liu, Zhi-Qiang; Jia, Dong-Xu; Liao, Cheng-Jun; Chen, De-Shui; Zheng, Yu-Guo

    2017-03-11

    Glucose isomerase is the important enzyme for the production of high fructose corn syrup (HFCS). One-step production of HFCS containing more than 55% fructose (HFCS-55) is receiving much attention for its industrial applications. In this work, the Escherichia coli harboring glucose isomerase mutant TEGI-W139F/V186T was immobilized for efficient production of HFCS-55. The immobilization conditions were optimized, and the maximum enzyme activity recovery of 92% was obtained. The immobilized glucose isomerase showed higher pH, temperature, and operational stabilities with a K m value of 272 mM and maximum reaction rate of 23.8 mM min(-1). The fructose concentration still retained above 55% after the immobilized glucose isomerase was reused for 10 cycles, and more than 85% of its initial activity was reserved even after 15 recycles of usage at temperature of 90 °C. The results highlighted the immobilized glucose isomerase as a potential biocatalyst for HFCS-55 production.

  9. Characterization of divergent pseudo-sucrose isomerase from Azotobacter vinelandii: Deciphering the absence of sucrose isomerase activity.

    PubMed

    Jung, Jong-Hyun; Kim, Min-Ji; Jeong, Woo-Soo; Seo, Dong-Ho; Ha, Suk-Jin; Kim, Young Wan; Park, Cheon-Seok

    2017-01-29

    Among members of the glycoside hydrolase (GH) family, sucrose isomerase (SIase) and oligo-1,6-glucosidase (O16G) are evolutionarily closely related even though their activities show different specificities. A gene (Avin_08330) encoding a putative SIase (AZOG: Azotobacterglucocosidase) from the nitrogen-fixing bacterium Azotobacter vinelandii is a type of pseudo-SIase harboring the "RLDRD" motif, a SIase-specific region in 329-333. However, neither sucrose isomerization nor hydrolysis activities were observed in recombinant AZOG (rAZOG). The rAZOG showed similar substrate specificity to Bacillus O16G as it catalyzes the hydrolysis of isomaltulose and isomaltose, which contain α-1,6-glycosidic linkages. Interestingly, rAZOG could generate isomaltose from the small substrate methyl-α-glucoside (MαG) via intermolecular transglycosylation. In addition, sucrose isomers isomaltulose and trehalulose were produced when 250 mM fructose was added to the MαG reaction mixture. The conserved regions I and II of AZOG are shared with many O16Gs, while regions III and IV are very similar to those of SIases. Strikingly, a shuffled AZOG, in which the N-terminal region of SIase containing conserved regions I and II was exchanged with the original enzyme, exhibited a production of sucrose isomers. This study demonstrates an evolutionary relationship between SIase and O16G and suggests some of the main regions that determine the specificity of SIase and O16G. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. 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

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Phosphomannose isomerase in all plants... ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS PROCEDURES AND REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.527 Phosphomannose isomerase in all plants...

  11. 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

    ... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Phosphomannose isomerase in all plants... ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS PROCEDURES AND REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.527 Phosphomannose isomerase in all plants...

  12. Functional expression of a bacterial xylose isomerase in Saccharomyces cerevisiae.

    PubMed

    Brat, Dawid; Boles, Eckhard; Wiedemann, Beate

    2009-04-01

    In industrial fermentation processes, the yeast Saccharomyces cerevisiae is commonly used for ethanol production. However, it lacks the ability to ferment pentose sugars like d-xylose and l-arabinose. Heterologous expression of a xylose isomerase (XI) would enable yeast cells to metabolize xylose. However, many attempts to express a prokaryotic XI with high activity in S. cerevisiae have failed so far. We have screened nucleic acid databases for sequences encoding putative XIs and finally were able to clone and successfully express a highly active new kind of XI from the anaerobic bacterium Clostridium phytofermentans in S. cerevisiae. Heterologous expression of this enzyme confers on the yeast cells the ability to metabolize d-xylose and to use it as the sole carbon and energy source. The new enzyme has low sequence similarities to the XIs from Piromyces sp. strain E2 and Thermus thermophilus, which were the only two XIs previously functionally expressed in S. cerevisiae. The activity and kinetic parameters of the new enzyme are comparable to those of the Piromyces XI. Importantly, the new enzyme is far less inhibited by xylitol, which accrues as a side product during xylose fermentation. Furthermore, expression of the gene could be improved by adapting its codon usage to that of the highly expressed glycolytic genes of S. cerevisiae. Expression of the bacterial XI in an industrially employed yeast strain enabled it to grow on xylose and to ferment xylose to ethanol. Thus, our findings provide an excellent starting point for further improvement of xylose fermentation in industrial yeast strains.

  13. Evolutionary genomics of Colias Phosphoglucose Isomerase (PGI) introns.

    PubMed

    Wang, Baiqing; Mason Depasse, J; Watt, Ward B

    2012-02-01

    Little is known of intron sequences' variation in cases where eukaryotic gene coding regions undergo strong balancing selection. Phosphoglucose isomerase, PGI, of Colias butterflies offers such a case. Its 11 introns include many point mutations, insertions, and deletions. This variation changes with intron position and length, and may leave little evidence of homology within introns except for their first and last few basepairs. Intron position is conserved between PGIs of Colias and the silkmoth, but no intron sequence homology remains. % GC content and length are functional properties of introns which can affect whole-gene transcription; we find a relationship between these properties which may indicate selection on transcription speed. Intragenic recombination is active in these introns, as in coding sequences. The small extent of linkage disequilibrium (LD) in the introns decays over a few hundred basepairs. Subsequences of Colias introns match subsequences of other introns, untranslated regions of cDNAs, and insect-related transposons and pathogens, showing that a diverse pool of sequence fragments is the source of intron contents via turnover due to deletion, recombination, and transposition. Like Colias PGI's coding sequences, the introns evolve reticulately with little phylogenetic signal. Exceptions are coding-region allele clades defined by multiple amino acid variants in strong LD, whose introns are closely related but less so than their exons. Similarity of GC content between introns and flanking exons, lack of small introns despite mutational bias toward deletion, and findings already mentioned suggest constraining selection on introns, possibly balancing transcription performance against advantages of higher recombination rate conferred by intron length.

  14. 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

  15. Oxidation of kinetically trapped thiols by protein disulfide isomerase.

    PubMed

    Walker, K W; Gilbert, H F

    1995-10-17

    The formation of a stabilized structure during oxidative protein folding can severely retard disulfide formation if the structure must be disrupted to gain access to buried cysteines. These kinetic traps can slow protein folding and disulfide bond formation to the extent that unassisted folding is too slow to be kinetically competent in the cell. Protein disulfide isomerase (PDI) facilitates the oxidation of a kinetically trapped state of RTEM-1 beta-lactamase in which two cysteines that form the single disulfide bond in the native protein are buried and approximately 500-fold less reactive than exposed cysteines. Under second-order conditions, PDI-dependent oxidation of reduced, folded beta-lactamase is 500-fold faster than GSSG-dependent oxidation. The rate difference observed between PDI and GSSG can be accounted for by the 520-fold higher kinetic reactivity of PDI as an oxidant. Noncovalent interactions between PDI (35 microM) and beta-lactamase increase the reactivity or unfolding of beta-lactamase in the steady-state by less than 3-fold. At high concentrations of PDI or alkylating agents, the reaction of beta-lactamase cysteines approaches a constant rate, limited by the spontaneous unfolding of the protein (kunfold = 0.024 +/- 0.005 min-1). PDI does not substantially increase the rate of beta-lactamase unfolding; however, once beta-lactamase spontaneously unfolds, PDI at concentrations greater than 44 +/- 4 microM, oxidizes the unfolded substrate before it can refold (kfold = 1.5 +/- 0.2 min-1).(ABSTRACT TRUNCATED AT 250 WORDS)

  16. 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)

  17. Patagonfibrase modifies protein expression of tissue factor and protein disulfide isomerase in rat skin.

    PubMed

    Peichoto, María Elisa; Santoro, Marcelo Larami

    2016-09-01

    Patagonfibrase is a hemorrhagic metalloproteinase isolated from the venom of the South American rear-fanged snake Philodryas patagoniensis, and is an important contributor to local lesions inflicted by this species. The tissue factor (TF)-factor VIIa complex, besides triggering the coagulation cascade, has been demonstrated to be involved in inflammatory events. Our aim was to determine whether patagonfibrase affects the expression of TF and protein disulfide isomerase (PDI), an enzyme that controls TF biological activity, at the site of patagonfibrase injection, and thus if they may play a role in hemostatic and inflammatory events induced by snake venoms. Patagonfibrase (60 μg/kg) was administered s.c. to rats, and after 3 h blood was collected to evaluate hemostasis parameters, and skin fragments close to the site of injection were taken to assess TF and PDI expression. Patagonfibrase did not alter blood cell counts, plasma fibrinogen levels, or levels of TF activity in plasma. However, by semiquantitative Western blotting, patagonfibrase increased TF expression by 2-fold, and decreased PDI expression by 3-fold in skin samples. In agreement, by immunohistochemical analyses, prominent TF expression was observed in the subcutaneous tissue. Thus, patagonfibrase affects the local expression of TF and PDI without inducing any systemic hemostatic disturbance, although that they may be involved in the local inflammatory events induced by hemorrhagic metalloproteinases. Once antivenom therapy is not totally effective to treat the local injury induced by snake venoms, modulation of the activity and expression of TF and/or PDI might become a strategy for treating snake envenomation.

  18. Iron Binding Properties of Recombinant Class A Protein Disulfide Isomerase from Arabidopsis thaliana.

    PubMed

    Remelli, William; Santabarbara, Stefano; Carbonera, Donatella; Bonomi, Francesco; Ceriotti, Aldo; Casazza, Anna Paola

    2017-04-07

    The protein disulfide isomerase (PDI) family comprises a wide set of enzymes mainly involved in thiol-disulfide exchange reactions in the endoplasmic reticulum. Class A PDIs (PDI-A) constitute the smallest members of the family, consisting of a single thioredoxin (TRX) module without any additional domains. To date, their catalytic activity and cellular function are still poorly understood. To gain insight into the role of higher-plant class A PDIs, the biochemical properties of rAtPDI-A, the recombinant form of Arabidopsis thaliana PDI-A, have been investigated. As expressed, rAtPDI-A has only little oxidoreductase activity, but it appears to be capable of binding an iron-sulfur (Fe-S) cluster, most likely a [2Fe-2S] center, at the interface between two protein monomers. A mutational survey of all cysteine residues of rAtPDI-A indicates that only the second and third cysteines of the CXXXCKHC stretch, containing the putative catalytic site CKHC, are primarily involved in cluster coordination. A key role is also played by the lysine residue. Its substitution with glycine, which restores the canonical PDI active site CGHC, does not influence the oxidoreductase activity of the protein, which remains marginal, but strongly affects the binding of the cluster. It is therefore proposed that the unexpected ability of rAtPDI-A to accommodate an Fe-S cluster is due to its very unique CKHC motif, which is conserved in all higher-plant class A PDIs, differentiating them from all other members of the PDI family.

  19. High production of D-tagatose, a potential sugar substitute, using immobilized L-arabinose isomerase.

    PubMed

    Kim, P; Yoon, S H; Roh, H J; Choi, J H

    2001-01-01

    An L-arabinose isomerase of Escherichia coli was immobilized using covalent binding to agarose to produce D-tagatose, a bulking sweetener that can be economically used as a sugar substitute. The immobilized L-arabinose isomerase stably produced an average of 7.5 g-tagatose/L.day for 7 days with a productivity exceeding that of the free enzyme (0.47 vs 0.30 mg/U.day). Using a scaled-up immobilized enzyme system, 99.9 g-tagatose/L was produced from galactose with 20% equilibrium in 48 h. The process was repeated two more times with production of 104.1 and 103.5 g-tagatose/L. D-Tagatose production using an immobilized L-arabinose isomerase has a high potential for commercial application.

  20. Directed evolution of xylose isomerase for improved xylose catabolism and fermentation in the yeast Saccharomyces cerevisiae.

    PubMed

    Lee, Sun-Mi; Jellison, Taylor; Alper, Hal S

    2012-08-01

    The heterologous expression of a highly functional xylose isomerase pathway in Saccharomyces cerevisiae would have significant advantages for ethanol yield, since the pathway bypasses cofactor requirements found in the traditionally used oxidoreductase pathways. However, nearly all reported xylose isomerase-based pathways in S. cerevisiae suffer from poor ethanol productivity, low xylose consumption rates, and poor cell growth compared with an oxidoreductase pathway and, additionally, often require adaptive strain evolution. Here, we report on the directed evolution of the Piromyces sp. xylose isomerase (encoded by xylA) for use in yeast. After three rounds of mutagenesis and growth-based screening, we isolated a variant containing six mutations (E15D, E114G, E129D, T142S, A177T, and V433I) that exhibited a 77% increase in enzymatic activity. When expressed in a minimally engineered yeast host containing a gre3 knockout and tal1 and XKS1 overexpression, the strain expressing this mutant enzyme improved its aerobic growth rate by 61-fold and both ethanol production and xylose consumption rates by nearly 8-fold. Moreover, the mutant enzyme enabled ethanol production by these yeasts under oxygen-limited fermentation conditions, unlike the wild-type enzyme. Under microaerobic conditions, the ethanol production rates of the strain expressing the mutant xylose isomerase were considerably higher than previously reported values for yeast harboring a xylose isomerase pathway and were also comparable to those of the strains harboring an oxidoreductase pathway. Consequently, this study shows the potential to evolve a xylose isomerase pathway for more efficient xylose utilization.

  1. 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

  2. Co-expression of sulphydryl oxidase and protein disulphide isomerase in Escherichia coli allows for production of soluble CRM197.

    PubMed

    Roth, R; van Zyl, P; Tsekoa, T; Stoychev, S; Mamputha, S; Buthelezi, S; Crampton, M

    2017-05-01

    To investigate the production of soluble cross-reacting material 197 (CRM197 ) in Escherichia coli, a safe and effective T-cell-dependent protein carrier for polysaccharides used in the manufacture and application of multivalent conjugate vaccines. The use of co-expression of a sulphydryl oxidase (SOX) and protein disulphide isomerase for the production of soluble CRM197 in E. coli is described. CRM197 contains two disulphide bonds, which are normally unable to form in the reducing environment of the E. coli cytoplasm. It was found that co-expression yielded soluble CRM197 , at a production rate ~10% of the production of insoluble CRM197 , in equivalent small-scale cultures. Structural analysis of the purified CRM197 compared to CRM197 commercially produced in cultures of recombinant Pseudomonas fluorescens indicated that the E. coli soluble protein compares favourably on all structural levels. SOX and protein disulphide isomerase are enzymes involved in the formation of intra-protein disulphide bonds, and can influence the tertiary structure of the protein being produced, resulting in increased solubility due to the correct folding of the protein. Their use enabled the production of soluble untagged CRM197 in E. coli, which was previously unachievable. Previous literature reports have shown that CRM197 can be expressed in E. coli, though only in an insoluble form, or in soluble form as a fusion protein. It is currently commercially produced in cultures of recombinant P. fluorescens. The use of a widely used, well-characterized expression host such as E. coli, rather than P. fluorescens broadens the applicability of the production technology, and the production system described here is worthy of further investigation for scaled up manufacture of CRM197 . © 2017 The Society for Applied Microbiology.

  3. Method for the assay of glucose isomerase activity in complex fermentation mixtures

    SciTech Connect

    Boguslawski, G.; Bertch, S.W.

    1980-10-01

    A method for the determination of glucose isomerase activity is described. The method employs D-sorbitol dehydrogenase for conversion of fructose, formed in the glucose isomerase reaction, to sorbitol, with the concomitant oxidation of reduced nicotinamide adenine dinucleotide. The assay technique is simple, sensitive, and accurate. The few interferences by some sugars and components of a complex fermentation medium are easily corrected for. The method compares favorably with such alternative procedures as the cysteine--H/sub 2/SO/sub 4/ or hydrochloric acid methods of fructose determination.

  4. Interruption of the phosphoglucose isomerase gene results in glucose auxotrophy in Mycobacterium smegmatis.

    PubMed Central

    Tuckman, D; Donnelly, R J; Zhao, F X; Jacobs, W R; Connell, N D

    1997-01-01

    Two glycerol utilization mutants of Mycobacterium smegmatis that were unable to utilize most carbon sources except glucose were isolated. Supplementation of these media with small amounts of glucose restored growth in the mutants; these strains are therefore glucose auxotrophs. The mutant phenotype is complemented by the gene encoding phosphoglucose isomerase (pgi), and direct measurement of enzyme activities in the mutants suggests that this gene product is absent in the auxotrophic strains. Mapping of the mutant allele by Southern analysis demonstrates the presence of a 1-kb deletion extending into the coding sequence of pgi. The possible roles of phosphoglucose isomerase in mycobacterial cell wall synthesis and metabolic regulation are discussed. PMID:9098072

  5. Interruption of the phosphoglucose isomerase gene results in glucose auxotrophy in Mycobacterium smegmatis.

    PubMed

    Tuckman, D; Donnelly, R J; Zhao, F X; Jacobs, W R; Connell, N D

    1997-04-01

    Two glycerol utilization mutants of Mycobacterium smegmatis that were unable to utilize most carbon sources except glucose were isolated. Supplementation of these media with small amounts of glucose restored growth in the mutants; these strains are therefore glucose auxotrophs. The mutant phenotype is complemented by the gene encoding phosphoglucose isomerase (pgi), and direct measurement of enzyme activities in the mutants suggests that this gene product is absent in the auxotrophic strains. Mapping of the mutant allele by Southern analysis demonstrates the presence of a 1-kb deletion extending into the coding sequence of pgi. The possible roles of phosphoglucose isomerase in mycobacterial cell wall synthesis and metabolic regulation are discussed.

  6. 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

  7. Disentangling mechanisms involved in collagen pyridinoline cross-linking: The immunophilin FKBP65 is critical for dimerization of lysyl hydroxylase 2

    PubMed Central

    Gjaltema, Rutger A. F.; van der Stoel, Miesje M.; Boersema, Miriam; Bank, Ruud A.

    2016-01-01

    Collagens are subjected to extensive posttranslational modifications, such as lysine hydroxylation. Bruck syndrome (BS) is a connective tissue disorder characterized at the molecular level by a loss of telopeptide lysine hydroxylation, resulting in reduced collagen pyridinoline cross-linking. BS results from mutations in the genes coding for lysyl hydroxylase (LH) 2 or peptidyl-prolyl cis-trans isomerase (PPIase) FKBP65. Given that the immunophilin FKBP65 does not exhibit LH activity, it is likely that LH2 activity is somehow dependent on FKPB65. In this report, we provide insights regarding the interplay between LH2 and FKBP65. We found that FKBP65 forms complexes with LH2 splice variants LH2A and LH2B but not with LH1 and LH3. Ablating the catalytic activity of FKBP65 or LH2 did not affect complex formation. Both depletion of FKBP65 and inhibition of FKBP65 PPIase activity reduced the dimeric (active) form of LH2 but did not affect the binding of monomeric (inactive) LH2 to procollagen Iα1. Furthermore, we show that LH2A and LH2B cannot form heterodimers with each other but are able to form heterodimers with LH1 and LH3. Collectively, our results indicate that FKBP65 is linked to pyridinoline cross-linking by specifically mediating the dimerization of LH2. Moreover, FKBP65 does not interact with LH1 and LH3, explaining why in BS triple-helical hydroxylysines are not affected. Our results provide a mechanistic link between FKBP65 and the loss of pyridinolines and may hold the key to future treatments for diseases related to collagen cross-linking anomalies, such as fibrosis and cancer. PMID:27298363

  8. A metal-mediated hydride shift mechanism for xylose isomerase based on the 1.6 A Streptomyces rubiginosus structures with xylitol and D-xylose.

    PubMed

    Whitlow, M; Howard, A J; Finzel, B C; Poulos, T L; Winborne, E; Gilliland, G L

    1991-01-01

    The crystal structure of recombinant Streptomyces rubiginosus D-xylose isomerase (D-xylose keto-isomerase, EC 5.3.1.5) solved by the multiple isomorphous replacement technique has been refined to R = 0.16 at 1.64 A resolution. As observed in an earlier study at 4.0 A (Carrell et al., J. Biol. Chem. 259: 3230-3236, 1984), xylose isomerase is a tetramer composed of four identical subunits. The monomer consists of an eight-stranded parallel beta-barrel surrounded by eight helices with an extended C-terminal tail that provides extensive contacts with a neighboring monomer. The active site pocket is defined by an opening in the barrel whose entrance is lined with hydrophobic residues while the bottom of the pocket consists mainly of glutamate, aspartate, and histidine residues coordinated to two manganese ions. The structures of the enzyme in the presence of MnCl2, the inhibitor xylitol, and the substrate D-xylose in the presence and absence of MnCl2 have also been refined to R = 0.14 at 1.60 A, R = 0.15 at 1.71 A, R = 0.15 at 1.60 A, and R = 0.14 at 1.60 A, respectively. Both the ring oxygen of the cyclic alpha-D-xylose and its C1 hydroxyl are within hydrogen bonding distance of NE2 of His-54 in the structure crystallized in the presence of D-xylose. Both the inhibitor, xylitol, and the extended form of the substrate, D-xylose, bind such that the C2 and C4 OH groups interact with one of the two divalent cations found in the active site and the C1 OH with the other cation. The remainder of the OH groups hydrogen bond with neighboring amino acid side chains. A detailed mechanism for D-xylose isomerase is proposed. Upon binding of cyclic alpha-D-xylose to xylose isomerase, His-54 acts as the catalytic base in a ring opening reaction. The ring opening step is followed by binding of D-xylose, involving two divalent cations, in an extended conformation. The isomerization of D-xylose to D-xylulose involves a metal-mediated 1,2-hydride shift. The final step in the mechanism is

  9. Diagnostic value of glucose-6-phosphate isomerase in rheumatoid arthritis.

    PubMed

    Fan, Lie Ying; Zong, Ming; Wang, Qiang; Yang, Lin; Sun, Li Shan; Ye, Qin; Ding, Yuan Yuan; Ma, Jian Wei

    2010-12-14

    Although glucose-6-phosphate isomerase (G6PI), anti-G6PI antibodies and G6PI-containing immune complexes (G6PI-CIC) have proved high expression in patients with rheumatoid arthritis (RA), comprehensive evaluation of the G6PI-derived markers, G6PI antigen, anti-G6PI Abs, G6PI-CIC and G6PI mRNA, in the diagnosis of RA remains necessary. We measured G6PI antigen, anti-G6PI Abs, C1q/G6PI-CIC as well as anti-cyclic citrullinated peptide antibodies (anti-CCP Abs) in serum and concomitantly synovial fluid (SF) by ELISA in RA, other rheumatic diseases and healthy controls. The G6PI mRNA expression in peripheral blood mononuclear cells (PBMCs) was assessed with real-time PCR. As compared with non-RA patients, RA patients had increased levels of G6PI antigen, anti-G6PI Abs, C1q/G6PI-CIC and G6PI mRNA expression in sera or PBMCs, and increased levels of G6PI and C1q/G6PI-CIC in SF. The serum G6PI levels in RA patients positively correlated with anti-G6PI Abs, C1q/G6PI-CIC, G6PI mRNA, anti-CCP Abs, RF, CRP and ESR, respectively. The area under curve analyses demonstrated that serum G6PI had the best discriminating power for RA and active RA followed by C1q/G6PI-CIC, anti-G6PI Abs and G6PI mRNA. The simultaneous use of serum G6PI and anti-CCP Abs assays in the form of either of them tested positive gave improved sensitivities of 88.1% for RA and 95.8% for active RA. Despite the elevated expression of all G6PI-derived markers in RA, the serum G6PI has the best discriminating power among the four G6PI-derived markers. The serum G6PI determination either alone or in combination with anti-CCP Abs improves the diagnosis of RA. Copyright © 2010 Elsevier B.V. All rights reserved.

  10. Prevalent overexpression of prolyl isomerase Pin1 in human cancers.

    PubMed

    Bao, Lere; Kimzey, Amy; Sauter, Guido; Sowadski, Janusz M; Lu, Kun Ping; Wang, Da-Gong

    2004-05-01

    Phosphorylation of proteins on serine or threonine residues preceding proline (pSer/Thr-Pro) is a major regulatory mechanism in cell proliferation and transformation. Interestingly, the pSer/Thr-Pro motifs in proteins exist in two distinct cis and trans conformations, whose conversion rate is normally reduced on phosphorylation, but is catalyzed specifically by the prolyl isomerase Pin1. Pin1 can catalytically induce conformational changes in proteins after phosphorylation, thereby having profound effects on catalytic activity, dephosphorylation, protein-protein interactions, subcellular location, and/or turnover of certain phosphorylated proteins. Recently, it has been shown that Pin1 is overexpressed in human breast cancer cell lines and cancer tissues and plays a critical role in the transformation of mammary epithelial cells by activating multiple oncogenic pathways. Furthermore, Pin1 expression is an excellent independent prognostic marker in prostate cancer. However, little is known about Pin1 expression in other human normal and cancerous tissues. In the present study, we quantified Pin1 expression in 2041 human tumor samples and 609 normal tissue samples as well as normal and transformed human cell lines. We found that Pin1 was usually expressed at very low levels in most normal tissues and its expression was normally associated with cell proliferation, with high Pin1 levels being found only in a few cell types. However, Pin1 was strikingly overexpressed in many different human cancers. Most tumors (38 of 60 tumor types) have Pin1 overexpression in more than 10% of the cases, as compared with the corresponding normal controls, which included prostate, lung, ovary, cervical, brain tumors, and melanoma. Consistent with these findings, Pin1 expression in human cancer cell lines was also higher than that in the normal cell lines examined. These results indicate that Pin1 overexpression is a prevalent and specific event in human cancers. Given previous findings

  11. Cytosolic Triosephosphate Isomerase from Arabidopsis thaliana Is Reversibly Modified by Glutathione on Cysteines 127 and 218

    PubMed Central

    Dumont, Sébastien; Bykova, Natalia V.; Pelletier, Guillaume; Dorion, Sonia; Rivoal, Jean

    2016-01-01

    In plant cells, an increase in cellular oxidants can have multiple effects, including the promotion of mixed disulfide bonds between glutathione and some proteins (S-glutathionylation). The present study focuses on the cytosolic isoform of the glycolytic enzyme triosephosphate isomerase (cTPI) from Arabidopsis thaliana and its reversible modification by glutathione. We used purified recombinant cTPI to demonstrate the enzyme sensitivity to inhibition by N-ethylmaleimide, hydrogen peroxide and diamide. Treatment of cTPI with diamide in the presence of reduced glutathione (GSH) led to a virtually complete inhibition of its enzymatic activity by S-glutathionylation. Recombinant cTPI was also sensitive to the oxidized form of glutathione (GSSG) in the micromolar range. Activity of cTPI was restored after reversion of S-glutathionylation by two purified recombinant A. thaliana cytosolic glutaredoxins (GRXs). GRXs-mediated deglutathionylation of cTPI was dependent on a GSH-regenerating system. Analysis of cTPI by mass spectrometry after S-glutathionylation by GSSG revealed that two Cys residues (Cys127 and Cys218) were modified by glutathione. The role of these two residues was assessed using site-directed mutagenesis. Mutation of Cys127 and Cys218 to Ser separately or together caused different levels of decrease in enzyme activity, loss of stability, as well as alteration of intrinsic fluorescence, underlining the importance of these Cys residues in protein conformation. Comparison of wild-type and mutant proteins modified with biotinyl glutathione ethyl ester (BioGEE) showed partial binding with single mutants and total loss of binding with the double mutant, demonstrating that both Cys residues were significantly S-glutathionylated. cTPI modification with BioGEE was reversed using DTT. Our study provides the first identification of the amino acid residues involved in cTPI S-glutathionylation and supports the hypothesis that this reversible modification could be part

  12. Phycoerythrin-specific bilin lyase-isomerase controls blue-green chromatic acclimation in marine Synechococcus.

    PubMed

    Shukla, Animesh; Biswas, Avijit; Blot, Nicolas; Partensky, Frédéric; Karty, Jonathan A; Hammad, Loubna A; Garczarek, Laurence; Gutu, Andrian; Schluchter, Wendy M; Kehoe, David M

    2012-12-04

    The marine cyanobacterium Synechococcus is the second most abundant phytoplanktonic organism in the world's oceans. The ubiquity of this genus is in large part due to its use of a diverse set of photosynthetic light-harvesting pigments called phycobiliproteins, which allow it to efficiently exploit a wide range of light colors. Here we uncover a pivotal molecular mechanism underpinning a widespread response among marine Synechococcus cells known as "type IV chromatic acclimation" (CA4). During this process, the pigmentation of the two main phycobiliproteins of this organism, phycoerythrins I and II, is reversibly modified to match changes in the ambient light color so as to maximize photon capture for photosynthesis. CA4 involves the replacement of three molecules of the green light-absorbing chromophore phycoerythrobilin with an equivalent number of the blue light-absorbing chromophore phycourobilin when cells are shifted from green to blue light, and the reverse after a shift from blue to green light. We have identified and characterized MpeZ, an enzyme critical for CA4 in marine Synechococcus. MpeZ attaches phycoerythrobilin to cysteine-83 of the α-subunit of phycoerythrin II and isomerizes it to phycourobilin. mpeZ RNA is six times more abundant in blue light, suggesting that its proper regulation is critical for CA4. Furthermore, mpeZ mutants fail to normally acclimate in blue light. These findings provide insights into the molecular mechanisms controlling an ecologically important photosynthetic process and identify a unique class of phycoerythrin lyase/isomerases, which will further expand the already widespread use of phycoerythrin in biotechnology and cell biology applications.

  13. Testing Electrostatic Complementarity in Enzyme Catalysis: Hydrogen Bonding in the Ketosteroid Isomerase Oxyanion Hole

    PubMed Central

    Kraut, Daniel A; Sigala, Paul A; Pybus, Brandon; Liu, Corey W; Ringe, Dagmar; Petsko, Gregory A

    2006-01-01

    A longstanding proposal in enzymology is that enzymes are electrostatically and geometrically complementary to the transition states of the reactions they catalyze and that this complementarity contributes to catalysis. Experimental evaluation of this contribution, however, has been difficult. We have systematically dissected the potential contribution to catalysis from electrostatic complementarity in ketosteroid isomerase. Phenolates, analogs of the transition state and reaction intermediate, bind and accept two hydrogen bonds in an active site oxyanion hole. The binding of substituted phenolates of constant molecular shape but increasing p K a models the charge accumulation in the oxyanion hole during the enzymatic reaction. As charge localization increases, the NMR chemical shifts of protons involved in oxyanion hole hydrogen bonds increase by 0.50–0.76 ppm/p K a unit, suggesting a bond shortening of ˜0.02 Å/p K a unit. Nevertheless, there is little change in binding affinity across a series of substituted phenolates (ΔΔG = −0.2 kcal/mol/p K a unit). The small effect of increased charge localization on affinity occurs despite the shortening of the hydrogen bonds and a large favorable change in binding enthalpy (ΔΔH = −2.0 kcal/mol/p K a unit). This shallow dependence of binding affinity suggests that electrostatic complementarity in the oxyanion hole makes at most a modest contribution to catalysis of ˜300-fold. We propose that geometrical complementarity between the oxyanion hole hydrogen-bond donors and the transition state oxyanion provides a significant catalytic contribution, and suggest that KSI, like other enzymes, achieves its catalytic prowess through a combination of modest contributions from several mechanisms rather than from a single dominant contribution. PMID:16602823

  14. Relationship of isopentenyl diphosphate (IDP) isomerase activity to isoprene emission of oak leaves.

    PubMed

    Brüggemann, Nicolas; Schnitzler, Jörg-Peter

    2002-10-01

    Oaks emit large amounts of isoprene, a compound that plays an important role in tropospheric chemistry. Isopentenyl diphosphate isomerase (IDI, E.C. 5.3.3.2) catalyzes the isomerization of isopentenyl diphosphate (IDP) to dimethylallyl diphosphate (DMADP), and in isoprene-emitting plants, isoprene synthase (IS) converts the DMADP to isoprene. To study the role of IDI in isoprene biosynthesis of oak leaves, we compared IDI and IS activities in pedunculate oak (Quercus robur L.) and pubescent oak (Quercus pubescens Willd.) with the isoprene emission rates of these species. We developed a non-radioactive enzyme assay to detect IDI activity in crude leaf extracts of Q. robur. The substrate dependency of IDI activity showed biphasic kinetics with Michaelis constants (K(m)(IDP)) of 0.7 +/- 0.2 micro M for a high-affinity phase and 39.5 +/- 6.9 micro M for a low-affinity phase, potentially attributable to different IDI isoforms. Under standard assay conditions, the temperature optimum for IDI activity was about 42 degrees C, but IDI activity was detectable up to 60 degrees C. A sharp pH optimum appeared around pH 7, with 20 mM Mg(2+) also required for IDI activity. Neither IDI activity nor IS activity showed diurnal variation in Q. robur leaves. The sum of IDI activities showed a significant linear correlation with IS activity in both Q. robur and Q. pubescens leaves, and both enzyme activities showed a linear relationship to isoprene emission factors in leaves of these oak species, indicating the possible involvement of IDI in isoprene biosynthesis by oak leaves.

  15. Testing electrostatic complementarity in enzyme catalysis: hydrogen bonding in the ketosteroid isomerase oxyanion hole.

    PubMed

    Kraut, Daniel A; Sigala, Paul A; Pybus, Brandon; Liu, Corey W; Ringe, Dagmar; Petsko, Gregory A; Herschlag, Daniel

    2006-04-01

    A longstanding proposal in enzymology is that enzymes are electrostatically and geometrically complementary to the transition states of the reactions they catalyze and that this complementarity contributes to catalysis. Experimental evaluation of this contribution, however, has been difficult. We have systematically dissected the potential contribution to catalysis from electrostatic complementarity in ketosteroid isomerase. Phenolates, analogs of the transition state and reaction intermediate, bind and accept two hydrogen bonds in an active site oxyanion hole. The binding of substituted phenolates of constant molecular shape but increasing pK(a) models the charge accumulation in the oxyanion hole during the enzymatic reaction. As charge localization increases, the NMR chemical shifts of protons involved in oxyanion hole hydrogen bonds increase by 0.50-0.76 ppm/pK(a) unit, suggesting a bond shortening of 0.02 A/pK(a) unit. Nevertheless, there is little change in binding affinity across a series of substituted phenolates (DeltaDeltaG = -0.2 kcal/mol/pK(a) unit). The small effect of increased charge localization on affinity occurs despite the shortening of the hydrogen bonds and a large favorable change in binding enthalpy (DeltaDeltaH = -2.0 kcal/mol/pK(a) unit). This shallow dependence of binding affinity suggests that electrostatic complementarity in the oxyanion hole makes at most a modest contribution to catalysis of 300-fold. We propose that geometrical complementarity between the oxyanion hole hydrogen-bond donors and the transition state oxyanion provides a significant catalytic contribution, and suggest that KSI, like other enzymes, achieves its catalytic prowess through a combination of modest contributions from several mechanisms rather than from a single dominant contribution.

  16. Peri/Epicellular Protein Disulfide Isomerase Sustains Vascular Lumen Caliber Through an Anticonstrictive Remodeling Effect.

    PubMed

    Tanaka, Leonardo Y; Araújo, Haniel A; Hironaka, Gustavo K; Araujo, Thaís L S; Takimura, Celso K; Rodriguez, Andres I; Casagrande, Annelise S; Gutierrez, Paulo S; Lemos-Neto, Pedro Alves; Laurindo, Francisco R M

    2016-03-01

    Whole-vessel remodeling critically determines lumen caliber in vascular (patho)physiology, and it is reportedly redox-dependent. We hypothesized that the cell-surface pool of the endoplasmic reticulum redox chaperone protein disulfide isomerase-A1 (peri/epicellular=pecPDI), which is known to support thrombosis, also regulates disease-associated vascular architecture. In human coronary atheromas, PDI expression inversely correlated with constrictive remodeling and plaque stability. In a rabbit iliac artery overdistension model, there was unusually high PDI upregulation (≈25-fold versus basal, 14 days postinjury), involving both intracellular and pecPDI. PecPDI neutralization with distinct anti-PDI antibodies did not enhance endoplasmic reticulum stress or apoptosis. In vivo pecPDI neutralization with PDI antibody-containing perivascular gel from days 12 to 14 post injury promoted 25% decrease in the maximally dilated arteriographic vascular caliber. There was corresponding whole-vessel circumference loss using optical coherence tomography without change in neointima, which indicates constrictive remodeling. This was accompanied by decreased hydrogen peroxide generation. Constrictive remodeling was corroborated by marked changes in collagen organization, that is, switching from circumferential to radial fiber orientation and to a more rigid fiber type. The cytoskeleton architecture was also disrupted; there was a loss of stress fiber coherent organization and a switch from thin to medium thickness actin fibers, all leading to impaired viscoelastic ductility. Total and PDI-associated expressions of β1-integrin, and levels of reduced cell-surface β1-integrin, were diminished after PDI antibody treatment, implicating β1-integrin as a likely pecPDI target during vessel repair. Indeed, focal adhesion kinase phosphorylation, a downstream β1-integrin effector, was decreased by PDI antibody. Thus, the upregulated pecPDI pool tunes matrix/cytoskeleton reshaping to

  17. Small molecule-induced oxidation of protein disulfide isomerase is neuroprotective

    PubMed Central

    Kaplan, Anna; Gaschler, Michael M.; Dunn, Denise E.; Colligan, Ryan; Brown, Lewis M.; Palmer, Arthur G.; Lo, Donald C.; Stockwell, Brent R.

    2015-01-01

    Protein disulfide isomerase (PDI) is a chaperone protein in the endoplasmic reticulum that is up-regulated in mouse models of, and brains of patients with, neurodegenerative diseases involving protein misfolding. PDI’s role in these diseases, however, is not fully understood. Here, we report the discovery of a reversible, neuroprotective lead optimized compound (LOC)14, that acts as a modulator of PDI. LOC14 was identified using a high-throughput screen of ∼10,000 lead-optimized compounds for potent rescue of viability of PC12 cells expressing mutant huntingtin protein, followed by an evaluation of compounds on PDI reductase activity in an in vitro screen. Isothermal titration calorimetry and fluorescence experiments revealed that binding to PDI was reversible with a Kd of 62 nM, suggesting LOC14 to be the most potent PDI inhibitor reported to date. Using 2D heteronuclear single quantum correlation NMR experiments, we were able to map the binding site of LOC14 as being adjacent to the active site and to observe that binding of LOC14 forces PDI to adopt an oxidized conformation. Furthermore, we found that LOC14-induced oxidation of PDI has a neuroprotective effect not only in cell culture, but also in corticostriatal brain slice cultures. LOC14 exhibited high stability in mouse liver microsomes and blood plasma, low intrinsic microsome clearance, and low plasma-protein binding. These results suggest that LOC14 is a promising lead compound to evaluate the potential therapeutic effects of modulating PDI in animal models of disease. PMID:25848045

  18. Blastomyces Virulence Adhesin-1 Protein Binding to Glycosaminoglycans Is Enhanced by Protein Disulfide Isomerase

    PubMed Central

    Beaussart, Audrey; Brandhorst, Tristan

    2015-01-01

    ABSTRACT Blastomyces adhesin-1 (BAD-1) protein mediates the virulence of the yeast Blastomyces dermatitidis, in part by binding host lung tissue, the extracellular matrix, and cellular receptors via glycosaminoglycans (GAGs), such as heparan sulfate. The tandem repeats that make up over 90% of BAD-1 appear in their native state to be tightly folded into an inactive conformation, but recent work has shown that they become activated and adhesive upon reduction of a disulfide linkage. Here, atomic force microscopy (AFM) of a single BAD-1 molecule interacting with immobilized heparin revealed that binding is enhanced upon treatment with protein disulfide isomerase and dithiothreitol (PDI/DTT). PDI/DTT treatment of BAD-1 induced a plateau effect in atomic force signatures that was consistent with sequential rupture of tandem binding domains. Inhibition of PDI in murine macrophages blunted BAD-1 binding to heparin in vitro. Based on AFM, we found that a short Cardin-Weintraub sequence paired with a WxxWxxW sequence in the first, degenerate repeat at the N terminus of BAD-1 was sufficient to initiate heparin binding. Removal of half of the 41 BAD-1 tandem repeats led to weaker adhesion, illustrating their role in enhanced binding. Mass spectroscopy of the tandem repeat revealed that the PDI-induced interaction with heparin is characterized by ruptured disulfide bonds and that cysteine thiols remain reduced. Further binding studies showed direct involvement of thiols in heparin ligation. Thus, we propose that the N-terminal domain of BAD-1 governs the initial association with host GAGs and that proximity to GAG-associated host PDI catalyzes activation of additional binding motifs conserved within the tandem repeats, leading to enhanced avidity and availability of reduced thiols. PMID:26396244

  19. Purification and characterization of prostaglandin-H E-isomerase, a sigma-class glutathione S-transferase, from Ascaridia galli.

    PubMed Central

    Meyer, D J; Muimo, R; Thomas, M; Coates, D; Isaac, R E

    1996-01-01

    Comparison of partial primary sequences of sigma-class glutathione S-transferases (GSH) of parasitic helminths and a GSH-dependent prostaglandin (PG)-H D-isomerase of rat immune accessory cells suggested that some of the helminth enzymes may also be involved in PG biosynthesis [Meyer and Thomas (1995) Biochem. J. 311, 739-742]. A soluble GSH transferase of the parasitic nematode Ascaridia galli has now been purified which shows high activity and specificity in the GSH-dependent isomerization of PGH to PGE, comparable to that of the rat spleen enzyme in its isomerization of PGH to PGD, and similarly stimulates the activity of prostaglandin H synthase. The enzyme subunit is structurally related to the rat spleen enzyme and sigma-class GSH transferases of helminths according to the partial primary sequence. The data support the hypothesis that some sigma-class GSH transferases of helminth parasites are involved in PG biosynthesis which, in the case of PGE, is likely to be associated with the subversion or suppression of host immunity. A PG-H E-isomerase of comparable specificity and activity has not previously been isolated. PMID:8546687

  20. Positive selection sites in tertiary structure of Leguminosae chalcone isomerase 1.

    PubMed

    Wang, R K; Zhan, S F; Zhao, T J; Zhou, X L; Wang, C E

    2015-03-20

    Isoflavonoids and the related synthesis enzyme, chalcone isomerase 1 (CHI1), are unique in the Leguminosae, with diverse biological functions. Among the Leguminosae, the soybean is an important oil, protein crop, and model plant. In this study, we aimed to detect the generation pattern of Leguminosae CHI1. Genome-wide sequence analysis of CHI in 3 Leguminosae and 3 other closely related model plants was performed; the expression levels of soybean chalcone isomerases were also analyzed. By comparing positively selected sites and their protein structures, we retrieved the evolution patterns for Leguminosae CHI1. A total of 28 CHI and 7 FAP3 (CHI4) genes were identified and separated into 4 clades: CHI1, CHI2, CHI3, and FAP3. Soybean genes belonging to the same chalcone isomerase subfamily had similar expression patterns. CHI1, the unique chalcone isomerase subfamily in Leguminosae, showed signs of significant positive selection as well as special expression characteristics, indicating an accelerated evolution throughout its divergence. Eight sites were identified as undergoing positive selection with high confidence. When mapped onto the tertiary structure of CHI1, these 8 sites were observed surrounding the enzyme substrate only; some of them connected to the catalytic core of CHI. Thus, we inferred that the generation of Leguminosae CHI1 is dependent on the positively selected amino acids surrounding its catalytic substrate. In other words, the evolution of CHI1 was driven by specific selection or processing conditions within the substrate.

  1. Triosephosphate isomerase and filamin C share common epitopes as novel allergens of Procambarus clarkii

    USDA-ARS?s Scientific Manuscript database

    Triosephosphate isomerase (TIM) is a key enzyme in glycolysis and has been identified as an allergen in saltwater products. In this study, TIM with a molecular mass of 28 kDa was purified from the freshwater crayfish (Procambarus clarkii) muscle. A 90-kDa protein that showed IgG/IgE cross-reactivity...

  2. Identification of Triosephosphate Isomerase as a Novel Allergen in Octopus fangsiao

    USDA-ARS?s Scientific Manuscript database

    A 28 kDa-protein was purified from octopus (Octopus fangsiao) and identified to be triosephosphate isomerase (TIM). The purified TIM is a glycoprotein with 1.7% carbohydrates and the isoelectric point is 7.6. TIM aggregated after heating above 45 °C, and the secondary structure was altered in extre...

  3. Grb7 Protein Stability Modulated by Pin1 in Association with Cell Cycle Progression

    PubMed Central

    Tai, Yu-Ling; Tung, Li-Hsuan; Lin, Yu-Chi; Lu, Pei-Jung; Chu, Pei-Yu; Wang, Ming-Yang; Huang, Wei-Pang; Chen, Ko-Chien; Lee, Hsinyu; Shen, Tang-Long

    2016-01-01

    Growth factor receptor bound protein-7 (Grb7) is a multi-domain adaptor protein that is co-opted by numerous tyrosine kinases involved in various cellular signaling and functions. The molecular mechanisms underlying the regulation of Grb7 remain unclear. Here, we revealed a novel negative post-translational regulation of Grb7 by the peptidyl-prolyl cis/trans isomerase, Pin1. Our data show that phosphorylation of Grb7 protein on the Ser194-Pro motif by c-Jun N-terminal kinase facilitates its binding with the WW domain of Pin1. Subsequently, Grb7 is degraded by the ubiquitin- and proteasome-dependent proteolytic pathway. Indeed, we found that Pin1 exerts its peptidyl-prolyl cis/trans isomerase activity in the modulation of Grb7 protein stability in regulation of cell cycle progression at the G2-M phase. This study illustrates a novel regulatory mechanism in modulating Grb7-mediated signaling, which may take part in pathophysiological consequences. PMID:27658202

  4. Characterization of the highly efficient sucrose isomerase from Pantoea dispersa UQ68J and cloning of the sucrose isomerase gene.

    PubMed

    Wu, Luguang; Birch, Robert G

    2005-03-01

    Sucrose isomerase (SI) genes from Pantoea dispersa UQ68J, Klebsiella planticola UQ14S, and Erwinia rhapontici WAC2928 were cloned and expressed in Escherichia coli. The predicted products of the UQ14S and WAC2928 genes were similar to known SIs. The UQ68J SI differed substantially, and it showed the highest isomaltulose-producing efficiency in E. coli cells. The purified recombinant WAC2928 SI was unstable, whereas purified UQ68J and UQ14S SIs were very stable. UQ68J SI activity was optimal at pH 5 and 30 to 35 degrees C, and it produced a high ratio of isomaltulose to trehalulose (>22:1) across its pH and temperature ranges for activity (pH 4 to 7 and 20 to 50 degrees C). In contrast, UQ14S SI showed optimal activity at pH 6 and 35 degrees C and produced a lower ratio of isomaltulose to trehalulose (<8:1) across its pH and temperature ranges for activity. UQ68J SI had much higher catalytic efficiency; the Km was 39.9 mM, the Vmax was 638 U mg(-1), and the Kcat/Km was 1.79 x 10(4) M(-1) s(-1), compared to a Km of 76.0 mM, a Vmax of 423 U mg(-1), and a Kcat/Km of 0.62 x 10(4) M(-1) s(-1) for UQ14S SI. UQ68J SI also showed no apparent reverse reaction producing glucose, fructose, or trehalulose from isomaltulose. These properties of the P. dispersa UQ68J enzyme are exceptional among purified SIs, and they indicate likely differences in the mechanism at the enzyme active site. They may favor the production of isomaltulose as an inhibitor of competing microbes in high-sucrose environments, and they are likely to be highly beneficial for industrial production of isomaltulose.

  5. Characterization of the Highly Efficient Sucrose Isomerase from Pantoea dispersa UQ68J and Cloning of the Sucrose Isomerase Gene

    PubMed Central

    Wu, Luguang; Birch, Robert G.

    2005-01-01

    Sucrose isomerase (SI) genes from Pantoea dispersa UQ68J, Klebsiella planticola UQ14S, and Erwinia rhapontici WAC2928 were cloned and expressed in Escherichia coli. The predicted products of the UQ14S and WAC2928 genes were similar to known SIs. The UQ68J SI differed substantially, and it showed the highest isomaltulose-producing efficiency in E. coli cells. The purified recombinant WAC2928 SI was unstable, whereas purified UQ68J and UQ14S SIs were very stable. UQ68J SI activity was optimal at pH 5 and 30 to 35°C, and it produced a high ratio of isomaltulose to trehalulose (>22:1) across its pH and temperature ranges for activity (pH 4 to 7 and 20 to 50°C). In contrast, UQ14S SI showed optimal activity at pH 6 and 35°C and produced a lower ratio of isomaltulose to trehalulose (<8:1) across its pH and temperature ranges for activity. UQ68J SI had much higher catalytic efficiency; the Km was 39.9 mM, the Vmax was 638 U mg−1, and the Kcat/Km was 1.79 × 104 M−1 s−1, compared to a Km of 76.0 mM, a Vmax of 423 U mg−1, and a Kcat/Km of 0.62 × 104 M−1 s−1 for UQ14S SI. UQ68J SI also showed no apparent reverse reaction producing glucose, fructose, or trehalulose from isomaltulose. These properties of the P. dispersa UQ68J enzyme are exceptional among purified SIs, and they indicate likely differences in the mechanism at the enzyme active site. They may favor the production of isomaltulose as an inhibitor of competing microbes in high-sucrose environments, and they are likely to be highly beneficial for industrial production of isomaltulose. PMID:15746363

  6. 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

  7. 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

  8. In silico analysis suggests that PH0702 and PH0208 encode for methylthioribose-1-phosphate isomerase and ribose-1,5-bisphosphate isomerase, respectively, rather than aIF2Bβ and aIF2Bδ.

    PubMed

    Gogoi, Prerana; Srivastava, Ambuj; Jayaprakash, Prajisha; Jeyakanthan, Jeyaraman; Kanaujia, Shankar Prasad

    2016-01-01

    The overall process of protein biosynthesis across all domains of life is similar; however, detailed insights reveal a range of differences in the proteins involved. For decades, the process of protein translation in archaea has been considered to be closer to eukaryotes than to bacteria. In archaea, however, several homologues of eukaryotic proteins involved in translation initiation have not yet been identified; one of them being the initiation factor eIF2B consisting of five subunits (α, β, γ, δ and ε). Three open reading frames (PH0440, PH0702 and PH0208) in Pyrococcus horikoshii have been proposed to encode for the α-, β- and δ-subunits of aIF2B, respectively. The crystal structure of PH0440 shows similarity toward the α-subunit of eIF2B. However, the capability of PH0702 and PH0208 to function as the β- and δ-subunits of eIF2B, respectively, remains uncertain. In this study, we have taken up the task of annotating PH0702 and PH0208 using bioinformatics methods. The phylogenetic analysis of protein sequences belonging to IF2B-like family along with PH0702 and PH0208 revealed that PH0702 belonged to methylthioribose-1-phosphate isomerase (MTNA) group of proteins, whereas, PH0208 was found to be clustered in the group of ribose-1,5-bisphosphate isomerase (R15PI) proteins. A careful analysis of protein sequences and structures available for eIF2B, MTNA and R15PI confirms that PH0702 and PH0208 contain residues essential for the enzymatic activity of MTNA and R15PI, respectively. Additionally, the protein PH0208 comprises of the residues required for the dimer formation which is essential for the biological activity of R15PI. This prompted us to examine all eIF2B-like proteins from archaea and to annotate their function. The results reveal that majority of these proteins are homologues of the α-subunit of eIF2B, even though they lack the residues essential for their functional activity. A better understanding of the mechanism of GTP exchange during

  9. The prolyl isomerase domain of PpiD from Escherichia coli shows a parvulin fold but is devoid of catalytic activity

    PubMed Central

    Weininger, Ulrich; Jakob, Roman P; Kovermann, Michael; Balbach, Jochen; Schmid, Franz X

    2010-01-01

    PpiD is a periplasmic folding helper protein of Escherichia coli. It consists of an N-terminal helix that anchors PpiD in the inner membrane near the SecYEG translocon, followed by three periplasmic domains. The second domain (residues 264–357) shows homology to parvulin-like prolyl isomerases. This domain is a well folded, stable protein and follows a simple two-state folding mechanism. In its solution structure, as determined by NMR spectroscopy, it resembles most closely the first parvulin domain of the SurA protein, which resides in the periplasm of E. coli as well. A previously reported prolyl isomerase activity of PpiD could not be reproduced when using improved protease-free peptide assays or assays with refolding proteins as substrates. The parvulin domain of PpiD interacts, however, with a proline-containing tetrapeptide, and the binding site, as identified by NMR resonance shift analysis, colocalized with the catalytic sites of other parvulins. In its structure, the parvulin domain of PpiD resembles most closely the inactive first parvulin domain of SurA, which is part of the chaperone unit of this protein and presumably involved in substrate recognition. PMID:19866485

  10. Coexpression of β-D-galactosidase and L-arabinose isomerase in the production of D-tagatose: a functional sweetener.

    PubMed

    Zhan, Yijing; Xu, Zheng; Li, Sha; Liu, Xiaoliu; Xu, Lu; Feng, Xiaohai; Xu, Hong

    2014-03-19

    The functional sweetener, d-tagatose, is commonly transformed from galactose by l-arabinose isomerase. To make use of a much cheaper starting material, lactose, hydrolization, and isomerization are required to take place collaboratively. Therefore, a single-step method involving β-d-galactosidase was explored for d-tagatose production. The two vital genes, β-d-galactosidase gene (lacZ) and l-arabinose isomerase mutant gene (araA') were extracted separately from Escherichia coli strains and incorporated into E. coli simultaneously. This gave us E. coli-ZY, a recombinant producing strain capable of coexpressing the two key enzymes. The resulted cells exhibited maximum d-tagatose producing activity at 34 °C and pH 6.5 and in the presence of borate, 10 mM Fe(2+), and 1 mM Mn(2+). Further monitoring showed that the recombinant cells could hydrolyze more than 95% lactose and convert 43% d-galactose into d-tagatose. This research has verified the feasibility of single-step d-tagatose fermentation, thereby laying down the foundation for industrial usage of lactose.

  11. Triosephosphate isomerase: 15N and 13C chemical shift assignments and conformational change upon ligand binding by magic-angle spinning solid-state NMR spectroscopy.

    PubMed

    Xu, Yimin; Lorieau, Justin; McDermott, Ann E

    2010-03-19

    Microcrystalline uniformly (13)C,(15)N-enriched yeast triosephosphate isomerase (TIM) is sequentially assigned by high-resolution solid-state NMR (SSNMR). Assignments are based on intraresidue and interresidue correlations, using dipolar polarization transfer methods, and guided by solution NMR assignments of the same protein. We obtained information on most of the active-site residues involved in chemistry, including some that were not reported in a previous solution NMR study, such as the side-chain carbons of His95. Chemical shift differences comparing the microcrystalline environment to the aqueous environment appear to be mainly due to crystal packing interactions. Site-specific perturbations of the enzyme's chemical shifts upon ligand binding are studied by SSNMR for the first time. These changes monitor proteinwide conformational adjustment upon ligand binding, including many of the sites probed by solution NMR and X-ray studies. Changes in Gln119, Ala163, and Gly210 were observed in our SSNMR studies, but were not reported in solution NMR studies (chicken or yeast). These studies identify a number of new sites with particularly clear markers for ligand binding, paving the way for future studies of triosephosphate isomerase dynamics and mechanism.

  12. Molecular characterization and analysis of a novel protein disulfide isomerase-like protein of Eimeria tenella.

    PubMed

    Han, Hongyu; Dong, Hui; Zhu, Shunhai; Zhao, Qiping; Jiang, Lianlian; Wang, Yange; Li, Liujia; Wu, Youlin; Huang, Bing

    2014-01-01

    Protein disulfide isomerase (PDI) and PDI-like proteins are members of the thioredoxin superfamily. They contain thioredoxin-like domains and catalyze the physiological oxidation, reduction and isomerization of protein disulfide bonds, which are involved in cell function and development in prokaryotes and eukaryotes. In this study, EtPDIL, a novel PDI-like gene of Eimeria tenella, was cloned using rapid amplification of cDNA ends (RACE) according to the expressed sequence tag (EST). The EtPDIL cDNA contained 1129 nucleotides encoding 216 amino acids. The deduced EtPDIL protein belonged to thioredoxin-like superfamily and had a single predicted thioredoxin domain with a non-classical thioredoxin-like motif (SXXC). BLAST analysis showed that the EtPDIL protein was 55-59% identical to PDI-like proteins of other apicomplexan parasites. The transcript and protein levels of EtPDIL at different development stages were investigated by real-time quantitative PCR and western blot. The messenger RNA and protein levels of EtPDIL were higher in sporulated oocysts than in unsporulated oocysts, sporozoites or merozoites. Protein expression was barely detectable in unsporulated oocysts. Western blots showed that rabbit antiserum against recombinant EtPDIL recognized only a native 24 kDa protein from parasites. Immunolocalization with EtPDIL antibody showed that EtPDIL had a disperse distribution in the cytoplasm of whole sporozoites and merozoites. After sporozoites were incubated in complete medium, EtPDIL protein concentrated at the anterior of the sporozoites and appeared on the surface of parasites. Specific staining was more intense and mainly located on the parasite surface after merozoites released from mature schizonts invaded DF-1 cells. After development of parasites in DF-1 cells, staining intensified in trophozoites, immature schizonts and mature schizonts. Antibody inhibition of EtPDIL function reduced the ability of E. tenella to invade DF-1 cells. These results

  13. Analysis of the arabinose-5-phosphate isomerase of Bacteroides fragilis provides insight into regulation of single-domain arabinose phosphate isomerases.

    PubMed

    Cech, David; Wang, Pan Fen; Holler, Tod P; Woodard, Ronald W

    2014-08-01

    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. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  14. 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

  15. Characterization of a mutated Geobacillus stearothermophilus L-arabinose isomerase that increases the production rate of D-tagatose.

    PubMed

    Kim, H-J; Kim, J-H; Oh, H-J; Oh, D-K

    2006-07-01

    Characterization of a mutated Geobacillus stearothermophilus L-arabinose isomerase used to increase the production rate of D-tagatose. A mutated gene was obtained by an error-prone polymerase chain reaction using L-arabinose isomerase gene from G. stearothermophilus as a template and the gene was expressed in Escherichia coli. The expressed mutated L-arabinose isomerase exhibited the change of three amino acids (Met322-->Val, Ser393-->Thr, and Val408-->Ala), compared with the wild-type enzyme and was then purified to homogeneity. The mutated enzyme had a maximum galactose isomerization activity at pH 8.0, 65 degrees C, and 1.0 mM Co2+, while the wild-type enzyme had a maximum activity at pH 8.0, 60 degrees C, and 1.0-mM Mn2+. The mutated L-arabinose isomerase exhibited increases in D-galactose isomerization activity, optimum temperature, catalytic efficiency (kcat/Km) for D-galactose, and the production rate of D-tagatose from D-galactose. The mutated L-arabinose isomerase from G. stearothermophilus is valuable for the commercial production of D-tagatose. This work contributes knowledge on the characterization of a mutated L-arabinose isomerase, and allows an increased production rate for D-tagatose from D-galactose using the mutated enzyme.

  16. The Hsp90-binding peptidylprolyl isomerase FKBP52 potentiates glucocorticoid signaling in vivo.

    PubMed

    Riggs, Daniel L; Roberts, Patricia J; Chirillo, Samantha C; Cheung-Flynn, Joyce; Prapapanich, Viravan; Ratajczak, Thomas; Gaber, Richard; Picard, Didier; Smith, David F

    2003-03-03

    Hsp90 is required for the normal activity of steroid receptors, and in steroid receptor complexes it is typically bound to one of the immunophilin-related co-chaperones: the peptidylprolyl isomerases FKBP51, FKBP52 or CyP40, or the protein phosphatase PP5. The physiological roles of the immunophilins in regulating steroid receptor function have not been well defined, and so we examined in vivo the influences of immunophilins on hormone-dependent gene activation in the Saccharomyces cerevisiae model for glucocorticoid receptor (GR) function. FKBP52 selectively potentiates hormone-dependent reporter gene activation by as much as 20-fold at limiting hormone concentrations, and this potentiation is readily blocked by co-expression of the closely related FKBP51. The mechanism for potentiation is an increase in GR hormone-binding affinity that requires both the Hsp90-binding ability and the prolyl isomerase activity of FKBP52.

  17. The Hsp90-binding peptidylprolyl isomerase FKBP52 potentiates glucocorticoid signaling in vivo

    PubMed Central

    Riggs, Daniel L.; Roberts, Patricia J.; Chirillo, Samantha C.; Cheung-Flynn, Joyce; Prapapanich, Viravan; Ratajczak, Thomas; Gaber, Richard; Picard, Didier; Smith, David F.

    2003-01-01

    Hsp90 is required for the normal activity of steroid receptors, and in steroid receptor complexes it is typically bound to one of the immunophilin-related co-chaperones: the peptidylprolyl isomerases FKBP51, FKBP52 or CyP40, or the protein phosphatase PP5. The physiological roles of the immunophilins in regulating steroid receptor function have not been well defined, and so we examined in vivo the influences of immunophilins on hormone-dependent gene activation in the Saccharomyces cerevisiae model for glucocorticoid receptor (GR) function. FKBP52 selectively potentiates hormone-dependent reporter gene activation by as much as 20-fold at limiting hormone concentrations, and this potentiation is readily blocked by co-expression of the closely related FKBP51. The mechanism for potentiation is an increase in GR hormone-binding affinity that requires both the Hsp90-binding ability and the prolyl isomerase activity of FKBP52. PMID:12606580

  18. 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

  19. Bacterial L-arabinose isomerases: industrial application for D-tagatose production.

    PubMed

    Boudebbouze, Samira; Maguin, Emmanuelle; Rhimi, Moez

    2011-12-01

    D-tagatose is a natural monosaccharide with a low caloric value and has an anti-hyperglycemiant effect. This hexose has potential applications both in pharmaceutical and agro-food industries. However, the use of D-tagatose remains limited by its production cost. Many production procedures including chemical and biological processes were developed and patented. The most profitable production way is based on the use of L-arabinose isomerase which allows the manufacture of D-tagatose with an attractive rate. Future developments are focused on the generation of L-arabinose isomerases having biochemical properties satisfying the industrial applications. This report provides a brief review of the most recent patents that have been published relating to this area.

  20. Mutations inducing an active-site aperture in Rhizobium sp. sucrose isomerase confer hydrolytic activity.

    PubMed

    Lipski, Alexandra; Watzlawick, Hildegard; Ravaud, Stéphanie; Robert, Xavier; Rhimi, Moez; Haser, Richard; Mattes, Ralf; Aghajari, Nushin

    2013-02-01

    Sucrose isomerase is an enzyme that catalyzes the production of sucrose isomers of high biotechnological and pharmaceutical interest. Owing to the complexity of the chemical synthesis of these isomers, isomaltulose and trehalulose, enzymatic conversion remains the preferred method for obtaining these products. Depending on the microbial source, the ratio of the sucrose-isomer products varies significantly. In studies aimed at understanding and explaining the underlying molecular mechanisms of these reactions, mutations obtained using a random-mutagenesis approach displayed a major hydrolytic activity. Two of these variants, R284C and F164L, of sucrose isomerase from Rhizobium sp. were therefore crystallized and their crystal structures were determined. The three-dimensional structures of these mutants allowed the identification of the molecular determinants that favour hydrolytic activity compared with transferase activity. Substantial conformational changes resulting in an active-site opening were observed, as were changes in the pattern of water molecules bordering the active-site region.

  1. 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.

  2. TXNDC5, a newly discovered disulfide isomerase with a key role in cell physiology and pathology.

    PubMed

    Horna-Terrón, Elena; Pradilla-Dieste, Alberto; Sánchez-de-Diego, Cristina; Osada, Jesús

    2014-12-17

    Thioredoxin domain-containing 5 (TXNDC5) is a member of the protein disulfide isomerase family, acting as a chaperone of endoplasmic reticulum under not fully characterized conditions As a result, TXNDC5 interacts with many cell proteins, contributing to their proper folding and correct formation of disulfide bonds through its thioredoxin domains. Moreover, it can also work as an electron transfer reaction, recovering the functional isoform of other protein disulfide isomerases, replacing reduced glutathione in its role. Finally, it also acts as a cellular adapter, interacting with the N-terminal domain of adiponectin receptor. As can be inferred from all these functions, TXNDC5 plays an important role in cell physiology; therefore, dysregulation of its expression is associated with oxidative stress, cell ageing and a large range of pathologies such as arthritis, cancer, diabetes, neurodegenerative diseases, vitiligo and virus infections. Its implication in all these important diseases has made TXNDC5 a susceptible biomarker or even a potential pharmacological target.

  3. Structure of ribose 5-phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC118.

    PubMed

    Lobley, Carina M C; Aller, Pierre; Douangamath, Alice; Reddivari, Yamini; Bumann, Mario; Bird, Louise E; Nettleship, Joanne E; Brandao-Neto, Jose; Owens, Raymond J; O'Toole, Paul W; Walsh, Martin A

    2012-12-01

    The structure of ribose 5-phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC188 has been determined at 1.72 Å resolution. The structure was solved by molecular replacement, which identified the functional homodimer in the asymmetric unit. Despite only showing 57% sequence identity to its closest homologue, the structure adopted the typical α and β D-ribose 5-phosphate isomerase fold. Comparison to other related structures revealed high homology in the active site, allowing a model of the substrate-bound protein to be proposed. The determination of the structure was expedited by the use of in situ crystallization-plate screening on beamline I04-1 at Diamond Light Source to identify well diffracting protein crystals prior to routine cryocrystallography.

  4. 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

  5. A protein disulfide isomerase gene fusion expression system that increases the extracellular productivity of Bacillus brevis.

    PubMed

    Kajino, T; Ohto, C; Muramatsu, M; Obata, S; Udaka, S; Yamada, Y; Takahashi, H

    2000-02-01

    We have developed a versatile Bacillus brevis expression and secretion system based on the use of fungal protein disulfide isomerase (PDI) as a gene fusion partner. Fusion with PDI increased the extracellular production of heterologous proteins (light chain of immunoglobulin G, 8-fold; geranylgeranyl pyrophosphate synthase, 12-fold). Linkage to PDI prevented the aggregation of the secreted proteins, resulting in high-level accumulation of fusion proteins in soluble and biologically active forms. We also show that the disulfide isomerase activity of PDI in a fusion protein is responsible for the suppression of the aggregation of the protein with intradisulfide, whereas aggregation of the protein without intradisulfide was prevented even when the protein was fused to a mutant PDI whose two active sites were disrupted, suggesting that another PDI function, such as chaperone-like activity, synergistically prevented the aggregation of heterologous proteins in the PDI fusion expression system.

  6. Catalysis of Protein Folding by Protein Disulfide Isomerase and Small-Molecule Mimics

    PubMed Central

    KERSTEEN, ELIZABETH A.; RAINES, RONALD T.

    2010-01-01

    Protein disulfide isomerase (PDI) catalyzes the formation of native disulfide pairings in secretory proteins. The ability of PDI to act as a disulfide isomerase makes it an essential enzyme in eukaryotes. PDI also fulfills other important roles. Recent studies have emphasized the importance of PDI as an oxidant in the endoplasmic reticulum. Intriguing questions remain regarding how PDI is able to catalyze both isomerization and oxidation in vivo. Studies of PDI and its homologs have led to the development of small-molecule folding catalysts that are able to accelerate disulfide isomerization in vitro and in vivo. PDI will continue to provide both an inspiration for the design of such artificial foldases and a benchmark with which to gauge the success of those designs. Here, we review current understanding of the chemistry and biology of PDI, its homologs, and small molecules that mimic its catalytic activity. PMID:13678529

  7. 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.

  8. Evolutionary significance and diversification of the phosphoglucose isomerase genes in vertebrates.

    PubMed

    Tine, Mbaye

    2015-12-18

    Phosphoglucose isomerase (PGI) genes are important multifunctional proteins whose evolution has, until now, not been well elucidated because of the limited number of completely sequenced genomes. Although the multifunctionality of this gene family has been considered as an original and innate characteristic, PGI genes may have acquired novel functions through changes in coding sequences and exon/intron structure, which are known to lead to functional divergence after gene duplication. A whole-genome comparative approach was used to estimate the rates of molecular evolution of this protein family. The results confirm the presence of two isoforms in teleost fishes and only one variant in all other vertebrates. Phylogenetic reconstructions grouped the PGI genes into five main groups: lungfishes/coelacanth/cartilaginous fishes, teleost fishes, amphibians, reptiles/birds and mammals, with the teleost group being subdivided into two subclades comprising PGI1 and PGI2. This PGI partitioning into groups is consistent with the synteny and molecular evolution results based on the estimation of the ratios of nonsynonymous to synonymous changes (Ka/Ks) and divergence rates between both PGI paralogs and orthologs. Teleost PGI2 shares more similarity with the variant found in all other vertebrates, suggesting that it has less evolved than PGI1 relative to the PGI of common vertebrate ancestor. The diversification of PGI genes into PGI1 and PGI2 is consistent with a teleost-specific duplication before the radiation of this lineage, and after its split from the other infraclasses of ray-finned fishes. The low average Ka/Ks ratios within teleost and mammalian lineages suggest that both PGI1 and PGI2 are functionally constrained by purifying selection and may, therefore, have the same functions. By contrast, the high average Ka/Ks ratios and divergence rates within reptiles and birds indicate that PGI may be involved in different functions. The synteny analyses show that the genomic

  9. BIOPHYSICS. Comment on "Extreme electric fields power catalysis in the active site of ketosteroid isomerase".

    PubMed

    Chen, Deliang; Savidge, Tor

    2015-08-28

    Fried et al. (Reports, 19 December 2014, p. 1510) demonstrate electric field-dependent acceleration of biological catalysis using ketosteroid isomerase as a prototypic example. These findings were not extended to aqueous solution because water by itself has field fluctuations that are too large and fast to provide a catalytic effect. Given physiological context, when water electrostatic interactions are considered, electric fields play a less important role in the catalysis.

  10. BIOPHYSICS. Comment on "Extreme electric fields power catalysis in the active site of ketosteroid isomerase".

    PubMed

    Natarajan, Aditya; Yabukarski, Filip; Lamba, Vandana; Schwans, Jason P; Sunden, Fanny; Herschlag, Daniel

    2015-08-28

    Fried et al. (Reports, 19 December 2014, p. 1510) demonstrated a strong correlation between reaction rate and the carbonyl stretching frequency of a product analog bound to ketosteroid isomerase oxyanion hole mutants and concluded that the active-site electric field provides 70% of catalysis. Alternative comparisons suggest a smaller contribution, relative to the corresponding solution reaction, and highlight the importance of atomic-level descriptions.

  11. Role of isopentenyl-diphosphate isomerase in heterologous cyanobacterial (Synechocystis) isoprene production.

    PubMed

    Chaves, Julie E; Romero, Paloma Rueda; Kirst, Henning; Melis, Anastasios

    2016-12-01

    Heterologous production of isoprene (C5H8) hydrocarbons in cyanobacteria, emanating from sunlight, CO2, and water, is now attracting increasing attention. The concept entails application of an isoprene synthase transgene from terrestrial plants, heterologously expressed in cyanobacteria, aiming to reprogram carbon flux in the terpenoid biosynthetic pathway toward formation and spontaneous release of this volatile chemical from the cell and liquid culture. However, flux manipulations and carbon-partitioning reactions between isoprene (the product) and native terpenoid biosynthesis for cellular needs are not yet optimized for isoprene yield. The primary reactant for isoprene biosynthesis is dimethylallyl diphosphate (DMAPP), whereas both DMAPP and its isopentenyl diphosphate (IPP) isomer are needed for cellular terpenoid biosynthesis. The present work addressed the function of an isopentenyl diphosphate (IPP) isomerase in cyanobacteria and its role in carbon partitioning between IPP and DMAPP, both of which serve, in variable ratios, as reactants for the synthesis of different cellular terpenoids. The work was approached upon the heterologous expression in Synechocystis of the "isopentenyl diphosphate isomerase" gene (FNI) from Streptococcus pneumoniae, using isoprene production as a "reporter process" for substrate partitioning between DMAPP and IPP. It is shown that transgenic expression of the FNI gene in Synechocystis resulted in a 250 % increase in the "reporter isoprene" rate and yield, suggesting that the FNI isomerase shifted the endogenous DMAPP-IPP steady-state pool size toward DMAPP, thereby enhancing rates and yield of isoprene production. The work provides insight into the significance and functional role of the IPP isomerase in these photosynthetic microorganisms.

  12. Synthesis of conjugated linoleic acid by the linoleate isomerase complex in food-derived lactobacilli.

    PubMed

    Yang, B; Chen, H; Gu, Z; Tian, F; Ross, R P; Stanton, C; Chen, Y Q; Chen, W; Zhang, H

    2014-08-01

    To assess strains of lactobacilli for their capacity to produce functional fatty acid-conjugated linoleic acid. To assess the linoleate isomerase for CLA production in the most efficient CLA producer. In this study, strains of food-derived lactobacilli were cultured in media with linoleic acid and CLA production was assessed. Most of the selected strains produced CLA at different levels, with Lactobacillus plantarum ZS2058 being the most efficient CLA producer converting over 50% of linoleic acid to c9, t11-CLA and t9, t11-CLA. Some intermediates 10-hydroxy-cis-12-octadecenoic acid, 10-oxo-cis-12-octadecenoic acid and 10-oxo-trans-11-octadecenoic acid were determined via GC-MS. The genes coding the multicomponent linoleate isomerase containing myosin-cross-reactive antigen, short-chain dehydrogenase/oxidoreductase and acetoacetate decarboxylase for CLA production in Lact. plantarum ZS2058 were cloned and expressed in Escherichia coli. With the mixture of recombinant E. coli, c9, t11-CLA and three kinds of intermediates were produced from linoleic acid, which were in line with those in the lactobacilli. The ability for CLA production by lactobacilli exhibited variation. Lactobacillus plantarum and Lact. bulgaricus were the most efficient producers in the selected strains. Lact. plantarum ZS2058 converted linoleic acid to CLAs with 10-hydroxy-cis-12-octadecenoic acid, 10-oxo-cis-12-octadecenoic acid and 10-oxo-trans-11-octadecenoic acid as intermediates. The multiple-step reactions for CLA production catalysed by multicomponent linoleate isomerase in Lact. plantarum ZS2058 were confirmed successfully. Multicomponent linoleate isomerase provides important results for the illustration of the mechanism for CLA production in lactic acid bacteria. Food-derived lactobacilli with CLA production ability offers novel opportunities for functional foods development. © 2014 The Society for Applied Microbiology.

  13. Synthesis of conjugated linoleic acid by the linoleate isomerase complex in food-derived lactobacilli

    PubMed Central

    Yang, B.; Chen, H.; Gu, Z.; Tian, F.; Ross, R. P.; Stanton, C.; Chen, Y. Q.; Chen, W.; Zhang, H.

    2015-01-01

    Aims To assess strains of lactobacilli for their capacity to produce functional fatty acid-conjugated linoleic acid. To assess the linoleate isomerase for CLA production in the most efficient CLA producer. Methods and Results In this study, strains of food-derived lactobacilli were cultured in media with linoleic acid and CLA production was assessed. Most of the selected strains produced CLA at different levels, with Lactobacillus plantarum ZS2058 being the most efficient CLA producer converting over 50% of linoleic acid to c9, t11-CLA and t9, t11-CLA. Some intermediates 10-hydroxy-cis-12-octadecenoic acid, 10-oxo-cis-12-octadecenoic acid and 10-oxo-trans-11-octadecenoic acid were determined via GC-MS. The genes coding the multicomponent linoleate isomerase containing myosin-cross-reactive antigen, short-chain dehydrogenase/oxidoreductase and acetoacetate decarboxylase for CLA production in Lact. plantarum ZS2058 were cloned and expressed in Escherichia coli. With the mixture of recombinant E. coli, c9, t11-CLA and three kinds of intermediates were produced from linoleic acid, which were in line with those in the lactobacilli. Conclusions The ability for CLA production by lactobacilli exhibited variation. Lactobacillus plantarum and Lact. bulgaricus were the most efficient producers in the selected strains. Lact. plantarum ZS2058 converted linoleic acid to CLAs with 10-hydroxy-cis-12-octadecenoic acid, 10-oxo-cis-12-octadecenoic acid and 10-oxo-trans-11-octadecenoic acid as intermediates. The multiple-step reactions for CLA production catalysed by multicomponent linoleate isomerase in Lact. plantarum ZS2058 were confirmed successfully. Significance and Impact of the study Multicomponent linoleate isomerase provides important results for the illustration of the mechanism for CLA production in lactic acid bacteria. Food-derived lactobacilli with CLA production ability offers novel opportunities for functional foods development. PMID:24750362

  14. Substrate specificity of a recombinant D-lyxose isomerase from Providencia stuartii for monosaccharides.

    PubMed

    Kwon, Hyun-Jung; Yeom, Soo-Jin; Park, Chang-Su; Oh, Deok-Kun

    2010-07-01

    The specific activity and catalytic efficiency (k(cat)/K(m)) of the recombinant putative protein from Providencia stuartii was the highest for D-lyxose among the aldose substrates, indicating that it is a D-lyxose isomerase. Gel filtration analysis suggested that the native enzyme is a dimer with a molecular mass of 44 kDa. The maximal activity for D-lyxose isomerization was observed at pH 7.5 and 45 degrees C in the presence of 1 mM Mn(2+). The enzyme exhibited high isomerization activity for aldose substrates with the C2 and C3 hydroxyl groups in the left-hand configuration, such as D-lyxose, D-mannose, L-ribose, D-talose, and L-allose (listed in decreasing order of activity). The enzyme exhibited the highest activity for D-xylulose among all pentoses and hexoses. Thus, D-lyxose was produced at 288 g/l from 500 g/l D-xylulose by D-lyxose isomerase at pH 7.5 and 45 degrees C for 2 h, with a conversion yield of 58% and a volumetric productivity of 144 g l(-1) h(-1). The observed k(cat)/K(m) (920 mM(-1) s(-1)) of P. stuartiid-lyxose isomerase for D-xylulose is higher than any of the k(cat)/K(m) values previously reported for sugar and sugar phosphate isomerases with monosaccharide substrates. These results suggest that the enzyme will be useful as an industrial producer of D-lyxose.

  15. 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.

  16. Distinct sucrose isomerases catalyze trehalulose synthesis in whiteflies, Bemisia argentifolii, and Erwinia rhapontici.

    PubMed

    Salvucci, Michael E

    2003-06-01

    Isomaltulose [alpha-D-glucopyranosyl-(1,6)-D-fructofuranose] and trehalulose [alpha-D-glucopyranosyl-(1,1)-D-fructofuranose] are commercially valuable sucrose-substitutes that are produced in several microorganisms by the palI gene product, a sucrose isomerase. Trehalulose also occurs in the silverleaf whitefly, Bemisia argentifoli, as the major carbohydrate in the insect's honeydew. To determine if the enzyme that synthesizes trehalulose in whiteflies was similar to the well-characterized sucrose isomerase from microbial sources, the properties of the enzymes from whiteflies and the bacterium, Erwinia rhapontici, were compared. Partial purification of both enzymes showed that the enzyme from whiteflies was a 116 kD membrane-associated polypeptide, in contrast to the enzyme from E. rhapontici, which was soluble and 66 kD. The enzyme from E. rhapontici converted sucrose to isomaltulose and trehalulose in a 5:1 ratio, whereas the enzyme from whiteflies produced only trehalulose. Unlike the E. rhapontici enzyme, the whitefly enzyme did not convert isomaltulose to trehalulose, but both enzymes catalyzed the transfer of fructose to trehalulose using sucrose as the glucosyl donor. The results indicate that trehalulose synthase from whiteflies is structurally and functionally distinct from the sucrose isomerases described in bacteria. The whitefly enzyme is the first reported case of an enzyme that converts sucrose to exclusively trehalulose.

  17. Crystal structures of human IPP isomerase: new insights into the catalytic mechanism.

    PubMed

    Zhang, Cheng; Liu, Lin; Xu, Hang; Wei, Zhiyi; Wang, Yanli; Lin, Yajing; Gong, Weimin

    2007-03-09

    Type I isopentenyl diphosphate (IPP): dimethylally diphosphate (DMAPP) isomerase is an essential enzyme in human isoprenoid biosynthetic pathway. It catalyzes isomerization of the carbon-carbon double bonds in IPP and DMAPP, which are the basic building blocks for the subsequent biosynthesis. We have determined two crystal structures of human IPP isomerase I (hIPPI) under different crystallization conditions. High similarity between structures of human and Escherichia coli IPP isomerases proves the conserved catalytic mechanism. Unexpectedly, one of the hIPPI structures contains a natural substrate analog ethanol amine pyrophosphate (EAPP). Based on this structure, a water molecule is proposed to be the direct proton donor for IPP and different conformations of IPP and DMAPP bound in the enzyme are also proposed. In addition, structures of human IPPI show a flexible N-terminal alpha-helix covering the active pocket and blocking the entrance, which is absent in E. coli IPPI. Besides, the active site conformation is not the same in the two hIPPI structures. Such difference leads to a hypothesis that substrate binding induces conformational change in the active site. The inhibition mechanism of high Mn(2+) concentrations is also discussed.

  18. Identification of Amino Acids that Account for Long-Range Interactions in Two Triosephosphate Isomerases from Pathogenic Trypanosomes

    SciTech Connect

    García-Torres, Itzhel; Cabrera, Nallely; Torres-Larios, Alfredo; Rodríguez-Bolaños, Mónica; Díaz-Mazariegos, Selma; Gómez-Puyou, Armando; Perez-Montfort, Ruy

    2012-04-02

    For a better comprehension of the structure-function relationship in proteins it is necessary to identify the amino acids that are relevant for measurable protein functions. Because of the numerous contacts that amino acids establish within proteins and the cooperative nature of their interactions, it is difficult to achieve this goal. Thus, the study of protein-ligand interactions is usually focused on local environmental structural differences. Here, using a pair of triosephosphate isomerase enzymes with extremely high homology from two different organisms, we demonstrate that the control of a seventy-fold difference in reactivity of the interface cysteine is located in several amino acids from two structurally unrelated regions that do not contact the cysteine sensitive to the sulfhydryl reagent methylmethane sulfonate, nor the residues in its immediate vicinity. The change in reactivity is due to an increase in the apparent pKa of the interface cysteine produced by the mutated residues. Our work, which involved grafting systematically portions of one protein into the other protein, revealed unsuspected and multisite long-range interactions that modulate the properties of the interface cysteines and has general implications for future studies on protein structure-function relationships.

  19. The potato sucrose transporter StSUT1 interacts with a DRM-associated protein disulfide isomerase.

    PubMed

    Krügel, Undine; He, Hong-Xia; Gier, Konstanze; Reins, Jana; Chincinska, Izabela; Grimm, Bernhard; Schulze, Waltraud X; Kühn, Christina

    2012-01-01

    Organization of proteins into complexes is crucial for many cellular functions. Recently, the SUT1 protein was shown to form homodimeric complexes, to be associated with lipid raft-like microdomains in yeast as well as in plants and to undergo endocytosis in response to brefeldin A. We therefore aimed to identify SUT1-interacting proteins that might be involved in dimerization, endocytosis, or targeting of SUT1 to raft-like microdomains. Therefore, we identified potato membrane proteins, which are associated with the detergent-resistant membrane (DRM) fraction. Among the proteins identified, we clearly confirmed StSUT1 as part of DRM in potato source leaves. We used the yeast two-hybrid split ubiquitin system (SUS) to systematically screen for interaction between the sucrose transporter StSUT1 and other membrane-associated or soluble proteins in vivo. The SUS screen was followed by immunoprecipitation using affinity-purified StSUT1-specific peptide antibodies and mass spectrometric analysis of co-precipitated proteins. A large overlap was observed between the StSUT1-interacting proteins identified in the co-immunoprecipitation and the detergent-resistant membrane fraction. One of the SUT1-interacting proteins, a protein disulfide isomerase (PDI), interacts also with other sucrose transporter proteins. A potential role of the PDI as escort protein is discussed.

  20. L-Serine-Mediated Neuroprotection Includes the Upregulation of the ER Stress Chaperone Protein Disulfide Isomerase (PDI).

    PubMed

    Dunlop, R A; Powell, J T; Metcalf, J S; Guillemin, G J; Cox, P A

    2017-10-03

    The unfolded protein response (UPR) is a highly evolutionarily conserved response to endoplasmic reticulum (ER) stress, which functions to return cells to homeostasis or send them into apoptosis, depending on the degree of cellular damage. β-N-methylamino-L-alanine (L-BMAA) has been shown to induce ER stress in a variety of models and has been linked to several types of neurodegenerative disease including Guamanian amyotrophic lateral sclerosis/Parkinsonism dementia complex (ALS/PDC). L-Serine, an amino acid critical for cellular metabolism and neurological signaling, has been shown to be protective against L-BMAA-induced neurotoxicity in both animal and cell culture models. While the mechanisms of L-BMAA neurotoxicity have been well characterized, less is known about L-serine neuroprotection. We recently reported that L-serine and L-BMAA generate similar differential expression profiles in a human ER stress/UPR array, despite L-serine being neuroprotective and L-BMAA being linked to neurodegenerative disease. Here, we further investigate the mechanism(s) of L-serine-induced UPR dysregulation by examining key genes and proteins in the ER stress/UPR pathways. We report that L-serine selectively increased protein disulfide isomerase (PDI) protein translation, an ER chaperone involved in refolding misfolded proteins, suggesting it may be modulating the UPR to favor recovery from ER stress. This constitutes a new mechanism for L-serine-mediated neuroprotection and has implications for its use as a therapy for neurodegenerative illnesses.

  1. Role of protein disulfide isomerase and other thiol-reactive proteins in HIV-1 envelope protein-mediated fusion

    SciTech Connect

    Ou Wu . E-mail: wou@niaid.nih.gov; Silver, Jonathan . E-mail: jsilver@nih.gov

    2006-07-05

    Cell-surface protein disulfide isomerase (PDI) has been proposed to promote disulfide bond rearrangements in HIV-1 envelope protein (Env) that accompany Env-mediated fusion. We evaluated the role of PDI in ways that have not been previously tested by downregulating PDI with siRNA and by overexpressing wild-type or variant forms of PDI in transiently and stably transfected cells. These manipulations, as well as treatment with anti-PDI antibodies, had only small effects on infection or cell fusion mediated by NL4-3 or AD8 strains of HIV-1. However, the cell-surface thiol-reactive reagent 5, 5'-dithiobis(2-nitrobenzoic acid) (DTNB) had a much stronger inhibitory effect in our system, suggesting that cell-surface thiol-containing molecules other than PDI, acting alone or in concert, have a greater effect than PDI on HIV-1 Env-mediated fusion. We evaluated one such candidate, thioredoxin, a PDI family member reported to reduce a labile disulfide bond in CD4. We found that the ability of thioredoxin to reduce the disulfide bond in CD4 is enhanced in the presence of HIV-1 Env gp120 and that thioredoxin also reduces disulfide bonds in gp120 directly in the absence of CD4. We discuss the implications of these observations for identification of molecules involved in disulfide rearrangements in Env during fusion.

  2. S-nitrosylation of the thioredoxin-like domains of protein disulfide isomerase and its role in neurodegenerative conditions.

    NASA Astrophysics Data System (ADS)

    Conway, Myra; Harris, Matthew

    2015-04-01

    Correct protein folding and inhibition of protein aggregation is facilitated by a cellular ‘quality control system’ that engages a network of protein interactions including molecular chaperones and the ubiquitin proteasome system. Key chaperones involved in these regulatory mechanisms are the protein disulphide isomerases (PDI) and their homologues, predominantly expressed in the endoplasmic reticulum of most tissues. Redox changes that disrupt ER homeostasis can lead to modification of these enzymes or chaperones with the loss of their proposed neuroprotective role resulting in an increase in protein misfolding. Misfolded protein aggregates have been observed in several disease states and are considered to play a pivotal role in the pathogenesis of neurodegenerative conditions such as Alzheimer’s disease, Parkinson’s disease, and Amyotrophic Lateral sclerosis. This review will focus on the importance of the thioredoxin-like -CGHC- active site of PDI and how our understanding of this structural motif will play a key role in unravelling the pathogenic mechanisms that underpin these neurodegenerative conditions.

  3. Irreversible inhibition of delta 5-3-oxosteroid isomerase by 2-substituted progesterones.

    PubMed Central

    Penning, T M

    1985-01-01

    2 alpha-Cyanoprogesterone (I) and 2-hydroxymethyleneprogesterone (II) were synthesized and screened as irreversible active-site-directed inhibitors of the delta 5-3-oxosteroid isomerase (EC 5.3.3.1) from Pseudomonas testosteroni. Both compounds were found to inhibit the purified bacterial enzyme in a time-dependent manner. In either case the inactivated enzyme could be dialysed without return of activity, indicating that a stable covalent bond had formed between the inhibitor and the enzyme. Inactivation mediated by compounds (I) and (II) followed pseudo-first-order kinetics, and at higher inhibitor concentrations saturation was observed. The competitive inhibitor 17 beta-oestradiol offered protection against the inactivation mediated by both compounds, and initial-rate studies indicated that compounds (I) and (II) can also act as competitive inhibitors yielding Ki values identical with those generated during inactivation experiments. 2 alpha-Cyanoprogesterone (I) and 2-hydroxymethyleneprogesterone (II) thus appear to be active-site-directed. To compare the reactivity of these 2-substituted progesterones with other irreversible inhibitors of the isomerase, 3 beta-spiro-oxiranyl-5 alpha-pregnan-20 beta-ol (III) was synthesized as the C21 analogue of 3 beta-spiro-oxiranyl-5 alpha-androstan-17 beta-ol, which is a potent inactivator of the isomerase [Pollack, Kayser & Bevins (1979) Biochem. Biophys. Res. Commun. 91, 783-790]. Comparison of the bimolecular rate constants for inactivation (k+3/Ki) mediated by compounds (I)-(III) indicated the following order of reactivity: (III) greater than (II) greater than (I). 2-Mercaptoethanol offers complete protection against the inactivation of the isomerase mediated by 2 alpha-cyanoprogesterone (I). Under the conditions of inactivation compound (I) appears to be completely stable, and no evidence could be obtained for enolate ion formation in the presence or absence of enzyme. It is suggested that cyanoprogesterone inactivates

  4. The secreted L-arabinose isomerase displays anti-hyperglycemic effects in mice.

    PubMed

    Rhimi, Moez; Bermudez-Humaran, Luis G; Huang, Yuan; Boudebbouze, Samira; Gaci, Nadia; Garnier, Alexandrine; Gratadoux, Jean-Jacques; Mkaouar, Héla; Langella, Philippe; Maguin, Emmanuelle

    2015-12-21

    The L-arabinose isomerase is an intracellular enzyme which converts L-arabinose into L-ribulose in living systems and D-galactose into D-tagatose in industrial processes and at industrial scales. D-tagatose is a natural ketohexose with potential uses in pharmaceutical and food industries. The D-galactose isomerization reaction is thermodynamically equilibrated, and leads to secondary subproducts at high pH. Therefore, an attractive L-arabinose isomerase should be thermoactive and acidotolerant with high catalytic efficiency. While many reports focused on the set out of a low cost process for the industrial production of D-tagatose, these procedures remain costly. When compared to intracellular enzymes, the production of extracellular ones constitutes an interesting strategy to increase the suitability of the biocatalysts. The L-arabinose isomerase (L-AI) from Lactobacillus sakei was expressed in Lactococcus lactis in fusion with the signal peptide of usp45 (SP(Usp45)). The L-AI protein and activity were detected only in the supernatant of the induced cultures of the recombinant L. lactis demonstrating the secretion in the medium of the intracellular L. sakei L-AI in an active form. Moreover, we showed an improvement in the enzyme secretion using either (1) L. lactis strains deficient for their two major proteases, ClpP and HtrA, or (2) an enhancer of protein secretion in L. lactis fused to the recombinant L-AI with the SP(Usp45). Th L-AI enzyme secreted by the recombinant L. lactis strains or produced intracellularly in E. coli, showed the same functional properties than the native enzyme. Furthermore, when mice are fed with the L. lactis strain secreting the L-AI and galactose, tagatose was produced in vivo and reduced the glycemia index. We report for the first time the secretion of the intracellular L-arabinose isomerase in the supernatant of food grade L. lactis cultures with hardly display other secreted proteins. The secreted L-AI originated from the food

  5. 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

  6. Metal ion roles and the movement of hydrogen during reaction catalyzed by D-xylose isomerase: a joint x-ray and neutron diffraction study.

    PubMed

    Kovalevsky, Andrey Y; Hanson, Leif; Fisher, S Zoe; Mustyakimov, Marat; Mason, Sax A; Forsyth, V Trevor; Blakeley, Matthew P; Keen, David A; Wagner, Trixie; Carrell, H L; Katz, Amy K; Glusker, Jenny P; Langan, Paul

    2010-06-09

    Conversion of aldo to keto sugars by the metalloenzyme D-xylose isomerase (XI) is a multistep reaction that involves hydrogen transfer. We have determined the structure of this enzyme by neutron diffraction in order to locate H atoms (or their isotope D). Two studies are presented, one of XI containing cadmium and cyclic D-glucose (before sugar ring opening has occurred), and the other containing nickel and linear D-glucose (after ring opening has occurred but before isomerization). Previously we reported the neutron structures of ligand-free enzyme and enzyme with bound product. The data show that His54 is doubly protonated on the ring N in all four structures. Lys289 is neutral before ring opening and gains a proton after this; the catalytic metal-bound water is deprotonated to hydroxyl during isomerization and O5 is deprotonated. These results lead to new suggestions as to how changes might take place over the course of the reaction.

  7. 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.

  8. 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.

  9. Characterization of a F280N variant of L-arabinose isomerase from Geobacillus thermodenitrificans identified as a D-galactose isomerase.

    PubMed

    Kim, Baek-Joong; Hong, Seung-Hye; Shin, Kyung-Chul; Jo, Ye-Seul; Oh, Deok-Kun

    2014-11-01

    The double-site variant (C450S-N475K) L-arabinose isomerase (L-AI) from Geobacillus thermodenitrificans catalyzes the isomerization of D-galactose to D-tagatose, a functional sweetener. Using a substrate-docking homology model, the residues near to D-galactose O6 were identified as Met186, Phe280, and Ile371. Several variants obtained by site-directed mutagenesis of these three residues were analyzed, and a triple-site (F280N) variant enzyme exhibited the highest activity for D-galactose isomerization. The k cat/K m of the triple-site variant enzyme for D-galactose was 2.1-fold higher than for L-arabinose, whereas the k cat/K m of the double-site variant enzyme for L-arabinose was 43.9-fold higher than for D-galactose. These results suggest that the triple-site variant enzyme is a D-galactose isomerase. The conversion rate of D-galactose to D-tagatose by the triple-site variant enzyme was approximately 3-fold higher than that of the double-site variant enzyme for 30 min. However, the conversion yields of L-arabinose to L-ribulose by the triple-site and double-site variant enzymes were 10.6 and 16.0 % after 20 min, respectively. The triple-site variant enzyme exhibited increased specific activity, turnover number, catalytic efficiency, and conversion rate for D-galactose isomerization compared to the double-site variant enzyme. Therefore, the amino acid at position 280 determines the substrate specificity for D-galactose and L-arabinose, and the triple-site variant enzyme has the potential to produce D-tagatose on an industrial scale.

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

    USDA-ARS?s Scientific Manuscript database

    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), ...

  11. The Protein-disulfide Isomerase ERp57 Regulates the Steady-state Levels of the Prion Protein*

    PubMed Central

    Torres, Mauricio; Medinas, Danilo B.; Matamala, José Manuel; Woehlbier, Ute; Cornejo, Víctor Hugo; Solda, Tatiana; Andreu, Catherine; Rozas, Pablo; Matus, Soledad; Muñoz, Natalia; Vergara, Carmen; Cartier, Luis; Soto, Claudio; Molinari, Maurizio; Hetz, Claudio

    2015-01-01

    Although the accumulation of a misfolded and protease-resistant form of the prion protein (PrP) is a key event in prion pathogenesis, the cellular factors involved in its folding and quality control are poorly understood. PrP is a glycosylated and disulfide-bonded protein synthesized at the endoplasmic reticulum (ER). The ER foldase ERp57 (also known as Grp58) is highly expressed in the brain of sporadic and infectious forms of prion-related disorders. ERp57 is a disulfide isomerase involved in the folding of a subset of glycoproteins in the ER as part of the calnexin/calreticulin cycle. Here, we show that levels of ERp57 increase mainly in neurons of Creutzfeldt-Jacob patients. Using gain- and loss-of-function approaches in cell culture, we demonstrate that ERp57 expression controls the maturation and total levels of wild-type PrP and mutant forms associated with human disease. In addition, we found that PrP physically interacts with ERp57, and also with the closest family member PDIA1, but not ERp72. Furthermore, we generated a conditional knock-out mouse for ERp57 in the nervous system and detected a reduction in the steady-state levels of the mono- and nonglycosylated forms of PrP in the brain. In contrast, ERp57 transgenic mice showed increased levels of endogenous PrP. Unexpectedly, ERp57 expression did not affect the susceptibility of cells to ER stress in vitro and in vivo. This study identifies ERp57 as a new modulator of PrP levels and may help with understanding the consequences of ERp57 up-regulation observed in human disease. PMID:26170458

  12. The chitin biosynthesis pathway in Entamoeba and the role of glucosamine-6-P isomerase by RNA interference.

    PubMed

    Samanta, Sintu Kumar; Ghosh, Sudip K

    2012-11-01

    Entamoeba histolytica, the causative agent of amoebiasis, infects through its cyst form. A thick chitin wall protects the cyst from the harsh environment outside of the body. It is known that chitin is synthesized only during encystation, but the chitin synthesis pathway (CSP) of Entamoeba is not well characterized. In this report, we have identified the genes involved in chitin biosynthesis from the Entamoeba genome database and verified their expression profile at the transcriptional level in encysting Entamoeba invadens. Semi-quantitative RT-PCR (sqRT-PCR) analysis showed that all the chitin pathway genes are entirely absent or transcribed at low levels in trophozoites. The mRNA expression of most of the CSP genes reached their maximum level between 9 and 12h after the in vitro initiation of encystation. Double-stranded RNA-mediated silencing of glucosamine-6-P isomerase (Gln6Pi) reduced chitin synthesis to 62-64%, which indicates that Gln6Pi might be a key enzyme for regulating chitin synthesis in Entamoeba. The study of different enzymes involved in glycogen metabolism revealed that stored glycogen is converted to glucose during encystation. It is clear from the sqRT-PCR analysis that the rate of glycolysis decreases as encystation proceeds. Encystation up-regulates the expression of glycogen phosphorylase, which is responsible for glycogen degradation. The significant decrease in chitin synthesis in encysting cells treated with a specific inhibitor of glycogen phosphorylase indicates that the glucose obtained from the degradation of stored glycogen in trophozoites might be one of the major sources of glucose for chitin synthesis. Copyright © 2012 Elsevier B.V. All rights reserved.

  13. Structural characterization and comparison of the large subunits of IPM isomerase and homoaconitase from Methanococcus jannaschii.

    PubMed

    Lee, Eun Hye; Lee, Kitaik; Hwang, Kwang Yeon

    2014-04-01

    The aconitase family of proteins includes three classes of hydro-lyase enzymes: aconitases, homoaconitases and isopropylmalate (IPM) isomerases. They have a common Fe-S cluster-binding site and catalyze the isomerization of specific substrates by sequential dehydration and hydration. The archaeon Methanococcus jannaschii contains two aconitase family proteins, IPM isomerase and homoaconitase, which have 50% sequence identity. These two enzymes are heterodimeric proteins composed of large and small subunits encoded by separate genes. Although structures have been reported for the small subunits of the two enzymes, the first structures of oxidized and reduced forms of the large subunit of IPM isomerase (ox-MJ0499 and red-MJ0499, respectively) from M. jannaschii are reported here at 1.8 and 2.7 Å resolution, respectively, together with the structure of the large subunit of homoaconitase (MJ1003) at 2.5 Å resolution. The structures of both proteins have unbound Fe-S clusters and contain a fourth cysteine in the active site. The active site of MJ1003 is homologous to that of aconitase, whereas MJ0499 has significant structural distortion at the active site compared with aconitase. In addition, significant large conformational changes were observed in the active site of red-MJ0499 when compared with ox-MJ0499. The active sites of the two proteins adopt two different states before changing to the Fe-S cluster-bound `activated' state observed in aconitase. MJ1003 has an `open' active site, which forms an active pocket for the cluster, while ox-MJ0499 has a `closed' active site, with four cysteines in disulfide bonds. These data will be helpful in understanding the biochemical mechanism of clustering of the Fe-S protein family.

  14. Studies on linoleic acid 8R-dioxygenase and hydroperoxide isomerase of the fungus Gaeumannomyces graminis.

    PubMed

    Su, C; Brodowsky, I D; Oliw, E H

    1995-01-01

    Linoleic acid is sequentially converted to 7S,8S-dihydroxy-9Z,12Z-octadecadienoic acid by the 8R-dioxygenase and hydroperoxide isomerase of the fungus Gaeumannomyces graminis, which is a common pathogen of wheat. The objective of this study was to separate and characterize the two enzyme activities. The isomerase activity was found mainly in the microsomal fraction of the mycelia and the 8R-dioxygenase in the cytosol. The 8R-dioxygenase could be partially purified by ammonium sulfate precipitation, gel filtration, ion exchange chromatography or isoelectric focusing. The 8R-dioxygenase was unstable during purification, but it could be stabilized by glutathione, glutathione peroxidase and ethylenediaminetetraacetic acid. Several protease inhibitors reduced the enzyme activity. Gel filtration with Sephacryl S-300 showed that most 8R-dioxygenase activity was eluted with the front with little retention. Isoelectric focusing in the presence of ethylene glycol (20%) indicated an isoelectric point of pl 6.1-6.3. The enzyme was retained on strong anion exchange columns at pH 7.4 and could be eluted with 0.3-0.5 M NaCl. Incubation of the enzyme with 0.1 mM linoleic acid led to partial inactivation, which may indicate product inhibition. Paracetamol and the lipoxygenase inhibitor ICI 230,487 at 30 microM inhibited the 8R-dioxygenase by 44 and 58%, respectively. 8R-hydroperoxy-9Z,12Z-octadecadienoic acid was isolated from incubations of linoleic acid with the partially purified enzyme or with the cytosol in the presence of p-hydroxymercuribenzoate. The hydroperoxide was rapidly converted by the hydroperoxide isomerase in the microsomal fractions to 7S,8S-dihydroxy-9Z,12Z-octadecadienoic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

  15. Inhibition of RPE65 Retinol Isomerase Activity by Inhibitors of Lipid Metabolism*

    PubMed Central

    Eroglu, Abdulkerim; Gentleman, Susan; Poliakov, Eugenia; Redmond, T. Michael

    2016-01-01

    RPE65 is the isomerase catalyzing conversion of all-trans-retinyl ester (atRE) into 11-cis-retinol in the retinal visual cycle. Crystal structures of RPE65 and site-directed mutagenesis reveal aspects of its catalytic mechanism, especially retinyl moiety isomerization, but other aspects remain to be determined. To investigate potential interactions between RPE65 and lipid metabolism enzymes, HEK293-F cells were transfected with expression vectors for visual cycle proteins and co-transfected with either fatty acyl:CoA ligases (ACSLs) 1, 3, or 6 or the SLC27A family fatty acyl-CoA synthase FATP2/SLCA27A2 to test their effect on isomerase activity. These experiments showed that RPE65 activity was reduced by co-expression of ACSLs or FATP2. Surprisingly, however, in attempting to relieve the ACSL-mediated inhibition, we discovered that triacsin C, an inhibitor of ACSLs, also potently inhibited RPE65 isomerase activity in cellulo. We found triacsin C to be a competitive inhibitor of RPE65 (IC50 = 500 nm). We confirmed that triacsin C competes directly with atRE by incubating membranes prepared from chicken RPE65-transfected cells with liposomes containing 0–1 μm atRE. Other inhibitors of ACSLs had modest inhibitory effects compared with triascin C. In conclusion, we have identified an inhibitor of ACSLs as a potent inhibitor of RPE65 that competes with the atRE substrate of RPE65 for binding. Triacsin C, with an alkenyl chain resembling but not identical to either acyl or retinyl chains, may compete with binding of the acyl moiety of atRE via the alkenyl moiety. Its inhibitory effect, however, may reside in its nitrosohydrazone/triazene moiety. PMID:26719343

  16. 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.

  17. 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.

  18. Overexpression, purification, crystallization and preliminary X-ray crystal analysis of Bacillus pallidus d-arabinose isomerase

    PubMed Central

    Takeda, Kosei; Yoshida, Hiromi; Takada, Goro; Izumori, Ken; Kamitori, Shigehiro

    2008-01-01

    d-Arabinose isomerase catalyzes the isomerization of d-arabinose to d-ribulose. Bacillus pallidus d-arabinose isomerase has broad substrate specificity and can catalyze the isomerization of d-arabinose, l-fucose, l-xylose, l-galactose and d-­altrose. Recombinant B. pallidus d-arabinose isomerase was overexpressed, purified and crystallized. A crystal of the enzyme was obtained by the sitting-drop method at room temperature and belonged to the orthorhombic space group P21212, with unit-cell parameters a = 144.9, b = 127.9, c = 109.5 Å. Diffraction data were collected to 2.3 Å resolution. PMID:18931442

  19. Crystal structure and putative mechanism of 3-methylitaconate-delta-isomerase from Eubacterium barkeri.

    PubMed

    Velarde, Milko; Macieira, Sofia; Hilberg, Markus; Bröker, Gerd; Tu, Shang-Min; Golding, Bernard T; Pierik, Antonio J; Buckel, Wolfgang; Messerschmidt, Albrecht

    2009-08-21

    3-Methylitaconate-Delta-isomerase (Mii) participates in the nicotinate fermentation pathway of the anaerobic soil bacterium Eubacterium barkeri (order Clostridiales) by catalyzing the reversible conversion of (R)-3-methylitaconate (2-methylene-3-methylsuccinate) to 2,3-dimethylmaleate. The enzyme is also able to catalyze the isomerization of itaconate (methylenesuccinate) to citraconate (methylmaleate) with ca 10-fold higher K(m) but > 1000-fold lower k(cat). The gene mii from E. barkeri was cloned and expressed in Escherichia coli. The protein produced with a C-terminal Strep-tag exhibited the same specific activity as the wild-type enzyme. The crystal structure of Mii from E. barkeri has been solved at a resolution of 2.70 A. The asymmetric unit of the P2(1)2(1)2(1) unit cell with parameters a = 53.1 A, b = 142.3 A, and c = 228.4 A contains four molecules of Mii. The enzyme belongs to a group of isomerases with a common structural feature, the so-called diaminopimelate epimerase fold. The monomer of 380 amino acid residues has two topologically similar domains exhibiting an alpha/beta-fold. The active site is situated in a cleft between these domains. The four Mii molecules are arranged as a tetramer with 222 symmetry for the N-terminal domains. The C-terminal domains have different relative positions with respect to the N-terminal domains resulting in a closed conformation for molecule A and two distinct open conformations for molecules B and D. The C-terminal domain of molecule C is disordered. The Mii active site contains the putative catalytic residues Lys62 and Cys96, for which mechanistic roles are proposed based on a docking experiment of the Mii substrate complex. The active sites of Mii and the closely related PrpF, most likely a methylaconitate Delta-isomerase, have been compared. The overall architecture including the active-site Lys62, Cys96, His300, and Ser17 (Mii numbering) is similar. This positioning of (R)-3-methylitaconate allows Cys96 (as

  20. Characterization of an L-arabinose isomerase from Bacillus thermoglucosidasius for D-tagatose production.

    PubMed

    Seo, Myung-Ji

    2013-01-01

    L-Arabinose isomerase from Bacillus thermoglucosidasius KCTC 1828 (BTAI) was expressed in Escherichia coli. The optimal temperature and pH for the activity of the purified BTAI were 40 °C and pH 7.0. The Mn(2+) ion was an activator of BTAI activity. The kinetic parameters of BTAI for D-galactose were a K(m) of 175 mM and a k(cat)/K(m) of 2.8 mM(-1)min(-1). The conversion ratio by BTAI to D-tagatose reached 45.6% at 40 °C.

  1. Crystal Structure and Substrate Specificity of D-Galactose-6-Phosphate Isomerase Complexed with Substrates

    PubMed Central

    Lee, Jung-Kul; Pan, Cheol-Ho

    2013-01-01

    D-Galactose-6-phosphate isomerase from Lactobacillus rhamnosus (LacAB; EC 5.3.1.26), which is encoded by the tagatose-6-phosphate pathway gene cluster (lacABCD), catalyzes the isomerization of D-galactose-6-phosphate to D-tagatose-6-phosphate during lactose catabolism and is used to produce rare sugars as low-calorie natural sweeteners. The crystal structures of LacAB and its complex with D-tagatose-6-phosphate revealed that LacAB is a homotetramer of LacA and LacB subunits, with a structure similar to that of ribose-5-phosphate isomerase (Rpi). Structurally, LacAB belongs to the RpiB/LacAB superfamily, having a Rossmann-like αβα sandwich fold as has been identified in pentose phosphate isomerase and hexose phosphate isomerase. In contrast to other family members, the LacB subunit also has a unique α7 helix in its C-terminus. One active site is distinctly located at the interface between LacA and LacB, whereas two active sites are present in RpiB. In the structure of the product complex, the phosphate group of D-tagatose-6-phosphate is bound to three arginine residues, including Arg-39, producing a different substrate orientation than that in RpiB, where the substrate binds at Asp-43. Due to the proximity of the Arg-134 residue and backbone Cα of the α6 helix in LacA to the last Asp-172 residue of LacB with a hydrogen bond, a six-carbon sugar-phosphate can bind in the larger pocket of LacAB, compared with RpiB. His-96 in the active site is important for ring opening and substrate orientation, and Cys-65 is essential for the isomerization activity of the enzyme. Two rare sugar substrates, D-psicose and D-ribulose, show optimal binding in the LacAB-substrate complex. These findings were supported by the results of LacA activity assays. PMID:24015281

  2. Inhibition of hexose transport by glucose in a glucose-6-phosphate isomerase mutant of Saccharomyces cerevisiae.

    PubMed

    Alonso, A; Pascual, C; Romay, C; Herrera, L; Kotyk, A

    1989-01-01

    The rate of hexose transport was approximately 60% lower for both the high- and the low-affinity components of hexose uptake when a glucose-6-phosphate isomerase mutant of Saccharomyces cerevisiae was preincubated with glucose, as compared with preincubation with water. Similarly the Jmax value of the high-affinity system of the mutant was 25-35% of the corresponding Jmax value for normal cells incubated with glucose. Accumulation of glucose 6-phosphate or of some other metabolite, such as fructose 6-phosphate or trehalose, may be responsible for this striking inhibition.

  3. A coleopteran triosephosphate isomerase: X-ray structure and phylogenetic impact of insect sequences.

    PubMed

    Knobeloch, D; Schmidt, A; Scheerer, P; Krauss, N; Wessner, H; Scholz, Ch; Küttner, G; von Rintelen, T; Wessel, A; Höhne, W

    2010-02-01

    A coleopteran triosephosphate isomerase (TIM) from Tenebrio molitor (yellow mealworm beetle) was recombinantly expressed in Escherichia coli and characterized with respect to thermal stability, kinetic parameters and oligomeric state. The enzyme was successfully crystallized and the structure determined by X-ray analysis to 2.0 A resolution. This is the first example of an invertebrate TIM. We compare structural features with known structures of TIMs from microorganisms, plants and vertebrates, and discuss the utility of the Tenebrio TIM sequence, together with several newly sequenced insect TIMs, for molecular phylogenetic analysis.

  4. Chemical modification of chalcone isomerase by mercurials and tetrathionate. Evidence for a single cysteine residue in the active site

    SciTech Connect

    Bednar, R.A.; Fried, W.B.; Lock, Y.W.; Pramanik, B. )

    1989-08-25

    Chalcone isomerase from soybean is inactivated by stoichiometric amounts of p-mercuribenzoate or HgCl{sub 2}. Spectral titration of the enzyme with p-mercuribenzoate indicates that a single thiol group is modified. Treatment of modified enzyme with KCN or thiols results in a complete restoration of enzyme activity demonstrating that the inactivation is not due to irreversible protein denaturation. A product of the enzymatic reaction, naringenin, provides complete kinetic protection against inactivation by both mercurials. The binding constant (33 microM) for naringenin determined from the concentration dependence of the protection agrees with the inhibition constant (34 microM) for naringenin as a competitive inhibitor of the catalytic reaction. This agreement demonstrates that the observed kinetic protection results from the specific binding of naringenin to the active site. Incubation of native chalcone isomerase with sodium tetrathionate (0.1 M) results in a slow time-dependent loss of enzymatic activity. The inactivation of chalcone isomerase by tetrathionate and N-ethylmaleimide becomes very rapid in the presence of 6 M urea, indicating that the native tertiary structure is responsible for the low reactivity of the enzymatic thiol. The stoichiometric modification of reduced and denatured chalcone isomerase by ({sup 3}H) N-ethylmaleimide indicates that the enzyme contains only a single cysteine residue and does not contain any disulfides. The evidence presented suggests that the only half-cystine residue in chalcone isomerase is located in the active site and thereby provides the first clue to the location of the active site in chalcone isomerase.

  5. An unusual isopentenyl diphosphate isomerase found in the mevalonate pathway gene cluster from Streptomyces sp. strain CL190

    PubMed Central

    Kaneda, Kazuhide; Kuzuyama, Tomohisa; Takagi, Motoki; Hayakawa, Yoichi; Seto, Haruo

    2001-01-01

    A gene cluster encoding five enzymes of the mevalonate pathway had been cloned from Streptomyces sp. strain CL190. This gene cluster contained an additional ORF, orfD, encoding an unknown protein that was detected in some archaebacteria and some Gram-positive bacteria including Staphylococcus aureus. The recombinant product of orfD was purified as a soluble protein and characterized. The molecular mass of the enzyme was estimated to be 37 kDa by SDS-polyacrylamide gel electrophoresis and 155 kDa by gel filtration chromatography, suggesting that the enzyme is most likely to be a tetramer. The purified enzyme contained flavin mononucleotide (FMN) with the amount per tetramer being 1.4 to 1.6 mol/mol. The enzyme catalyzed the isomerization of isopentenyl diphosphate (IPP) to produce dimethylallyl diphosphate (DMAPP) in the presence of both FMN and NADPH. The Escherichia coli plasmid expressing orfD could complement the disrupted IPP isomerase gene in E. coli. These results indicate that orfD encodes an unusual IPP isomerase showing no sequence similarity to those of IPP isomerases identified to date. Based on the difference in enzymatic properties, we classify the IPP isomerases into two types: Type 2 for FMN- and NAD(P)H-dependent enzymes, and type 1 for the others. In view of the critical role of this isomerase in S. aureus and of the different enzymatic properties of mammalian (type 1) and S. aureus (type 2) isomerases, this unusual enzyme is considered to be a suitable molecular target for the screening of antibacterial drugs specific to S. aureus. PMID:11158573

  6. X-ray analysis of D-xylose isomerase at 1.9 A: native enzyme in complex with substrate and with a mechanism-designed inactivator.

    PubMed Central

    Carrell, H L; Glusker, J P; Burger, V; Manfre, F; Tritsch, D; Biellmann, J F

    1989-01-01

    The structures of crystalline D-xylose isomerase (D-xylose ketol-isomerase; EC 5.3.1.5) from Streptomyces rubiginosus and of its complexes with substrate and with an active-site-directed inhibitor have been determined by x-ray diffraction techniques and refined to 1.9-A resolution. This study identifies the active site, as well as two metal-binding sites. The metal ions are important in maintaining the structure of the active-site region and one of them binds C3-O and C5-O of the substrate forming a six-membered ring. This study has revealed a very close contact between histidine and C1 of a substrate, suggesting that this is the active-site base that abstracts a proton from substrate. The mechanism-based inhibitor is a substrate analog and is turned over by the enzyme to give a product that alkylates this same histidine, reinforcing our interpretation. The changes in structure of the native enzyme, the enzyme with bound substrate, and the alkylated enzyme indicate that the mechanism involves an "open-chain" conformation of substrate and that the intermediate in the isomerization reaction is probably a cis-ene diol because the active-site histidine is correctly placed to abstract a proton from C1 or C2 of the substrate. A water molecule binds to C1O and C2O of the substrate and so may act as a proton donor or acceptor in the enolization of a ring-opened substrate. PMID:2734296

  7. Microbial cyclophilins: specialized functions in virulence and beyond.

    PubMed

    Dimou, Maria; Venieraki, Anastasia; Katinakis, Panagiotis

    2017-08-08

    Cyclophilins belong to the superfamily of peptidyl-prolyl cis/trans isomerases (PPIases, EC: 5.2.1.8), the enzymes that catalyze the cis/trans isomerization of peptidyl-prolyl peptide bonds in unfolded and partially folded polypeptide chains and native state proteins. Cyclophilins have been extensively studied, since they are involved in multiple cellular processes related to human pathologies, such as neurodegenerative disorders, infectious diseases, and cancer. However, the presence of cyclophilins in all domains of life indicates a broader biological importance. In this mini-review, we summarize current advances in the study of microbial cyclophilins. Apart from their anticipated role in protein folding and chaperoning, cyclophilins are involved in several other biological processes, such as cellular signal transduction, adaptation to stress, control of pathogens virulence, and modulation of host immune response. Since many existing family members do not have well-defined functions and novel ones are being characterized, the requirement for further studies on their biological role and molecular mechanism of action is apparent.

  8. 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.

  9. [Significance of antibodies to the citrullinated glucose-6-phosphate isomerase peptides in rheumatoid arthritis].

    PubMed

    Wu, D; Sun, L; Li, C H; Yang, L; Zhao, J X; Liu, X Y

    2016-12-18

    To detect the anti-citrullinated glucose-6-phosphate isomerase (GPI) 70-88 peptide antibody (anti-C-GPI(70-88) antibody), anti-citrullinated GPI 435-453 peptide antibody (anti-C-GPI(435-453) antibody), anti-GPI 70-88 peptide antibody (anti-GPI(70-88) antibody) and anti-GPI 435-453 peptide antibody(anti-GPI(435-453) antibody) in the serum of rheumatoid arthritis (RA) patients, and examine the diagnostic values of the anti-C-GPI peptide antibodies in RA. The anti-C-GPI(70-88) antibody, anti-C-GPI(435-453) antibody, anti-GPI(70-88) antibody and anti-GPI(435-453) antibody were detected by enzyme-linked immunosorbent assay (ELISA) in 191 RA patients, 129 other rheumatic diseases and 74 healthy controls. The clinical and laboratory data of the patients with RA were collected, and the values of anti-C-GPI peptide antibodies in the diagnosis of RA and the relationships of anti-C-GPI peptide antibodies with the clinical and laboratory parameters analyzed. (1) The mean titers of the anti-C-GPI(70-88) antibody and the anti-C-GPI(435-453) antibody in the RA patients (respectively, 68.71 ± 4.20 and 51.78 ± 3.13) were significantly higher than those with other rheumatic diseases and healthy individuals (P <0.05). However, the mean titers of the anti-GPI(70-88) antibody and anti-GPI(435-453) antibody in the RA patients were similar to those with other rheumatic diseases and healthy individuals. (2) The diagnostic sensitivity and specificity of the anti-C-GPI(70-88) antibody for RA were 41.88% and 84.50% respectively; and the diagnostic sensitivity and specificity of the anti-C-GPI(435-453) antibody for RA were 46.05% and 86.05% respectively. The sensitivity of combined detection of the two anti-C-GPI peptide antibodies was 50.79%, and the specificity was 81.40%. (3) The positive rates of the anti-C-GPI(70-88) antibody and the anti-C-GPI(435-453) antibody were 35% and 45% respectively in those patients with negative anti-cyclic citrullinated peptide antibody, anti

  10. Sucrose isomerase and its mutants from Erwinia rhapontici can synthesise α-arbutin.

    PubMed

    Zhou, Xing; Zheng, Yuantao; Wei, Xingming; Yang, Kedi; Yang, Xiangkai; Wang, Yuting; Xu, Liming; Du, Liqin; Huang, Ribo

    2011-10-01

    Sucrose isomerase (SI) from Erwinia rhapontici is an intramolecular isomerase that is normally used to synthesise isomaltulose from sucrose by a mechanism of intramolecular transglycosylation. In this study, it was found that SI could synthesise α-arbutin using hydroquinone and sucrose as substrates, via an intermolecular transglycosylation reaction. Five phenylalanine residues (F185, F186, F205, F297, and F321) in the catalytic pocket of SI were chosen for sitedirected mutagenesis. Mutants F185I, F321I, and F321W, whose hydrolytic activities were enhanced after the mutation, could synthesise α-arbutin through intermolecular transglycosylation with a more than two-fold increase in the molar transfer ratio compared with wild type SI. The F297A mutant showed a strong ability to synthesise a novel α-arbutin derivative and a four-fold increase in its specific activity for intermolecular transglycosylation over the wild type. Our findings may lead to a new way to synthesise novel glucoside products such as α-arbutin derivatives by simply manipulating the Phe residues in the catalytic pocket. From the structure superposition, our strategy of manipulating these Phe residues may be applicable to other similar transglycosylating enzymes.

  11. 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.

  12. 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.

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

    PubMed

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

    2015-12-01

    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.

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

    PubMed

    Marsolier, J; Perichon, M; DeBarry, J D; Villoutreix, B O; Chluba, J; Lopez, T; Garrido, C; Zhou, X Z; Lu, K P; Fritsch, L; Ait-Si-Ali, S; Mhadhbi, M; Medjkane, S; Weitzman, J B

    2015-04-16

    Infectious agents develop intricate mechanisms to interact with host cell pathways and hijack their genetic and epigenetic machinery to change host cell phenotypic states. Among the Apicomplexa phylum of obligate intracellular parasites, which cause veterinary and human diseases, Theileria is the only genus that transforms its mammalian host cells. Theileria infection of bovine leukocytes induces proliferative and invasive phenotypes associated with activated signalling pathways, notably JNK and AP-1 (ref. 2). The transformed phenotypes are reversed by treatment with the theilericidal drug buparvaquone. We used comparative genomics to identify a homologue of the peptidyl-prolyl isomerase PIN1 in T. annulata (TaPIN1) that 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 and in silico 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 isomerization is thus a conserved mechanism that is important in cancer and is used by Theileria parasites to manipulate host oncogenic signalling.

  15. 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

  16. 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.

  17. 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

    2014-01-08

    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.

  18. Human Cellular Retinaldehyde-Binding Protein Has Secondary Thermal 9-cis-Retinal Isomerase Activity

    PubMed Central

    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

    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 1H-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 towards 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

  19. TXNDC5, a Newly Discovered Disulfide Isomerase with a Key Role in Cell Physiology and Pathology

    PubMed Central

    Horna-Terrón, Elena; Pradilla-Dieste, Alberto; Sánchez-de-Diego, Cristina; Osada, Jesús

    2014-01-01

    Thioredoxin domain-containing 5 (TXNDC5) is a member of the protein disulfide isomerase family, acting as a chaperone of endoplasmic reticulum under not fully characterized conditions As a result, TXNDC5 interacts with many cell proteins, contributing to their proper folding and correct formation of disulfide bonds through its thioredoxin domains. Moreover, it can also work as an electron transfer reaction, recovering the functional isoform of other protein disulfide isomerases, replacing reduced glutathione in its role. Finally, it also acts as a cellular adapter, interacting with the N-terminal domain of adiponectin receptor. As can be inferred from all these functions, TXNDC5 plays an important role in cell physiology; therefore, dysregulation of its expression is associated with oxidative stress, cell ageing and a large range of pathologies such as arthritis, cancer, diabetes, neurodegenerative diseases, vitiligo and virus infections. Its implication in all these important diseases has made TXNDC5 a susceptible biomarker or even a potential pharmacological target. PMID:25526565

  20. 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

  1. 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.

  2. Structural analysis of arabinose-5-phosphate isomerase from Bacteroides fragilis and functional implications.

    PubMed

    Chiu, Hsiu Ju; Grant, Joanna C; Farr, Carol L; Jaroszewski, Lukasz; Knuth, Mark W; Miller, Mitchell D; Elsliger, Marc André; Deacon, Ashley M; Godzik, Adam; Lesley, Scott A; Wilson, Ian A

    2014-10-01

    The crystal structure of arabinose-5-phosphate isomerase (API) from Bacteroides fragilis (bfAPI) was determined at 1.7 Å resolution and was found to be a tetramer of a single-domain sugar isomerase (SIS) with an endogenous ligand, CMP-Kdo (cytidine 5'-monophosphate-3-deoxy-D-manno-oct-2-ulosonate), bound at the active site. API catalyzes the reversible isomerization of D-ribulose 5-phosphate to D-arabinose 5-phosphate in the first step of the Kdo biosynthetic pathway. Interestingly, the bound CMP-Kdo is neither the substrate nor the product of the reaction catalyzed by API, but corresponds to the end product in the Kdo biosynthetic pathway and presumably acts as a feedback inhibitor for bfAPI. The active site of each monomer is located in a surface cleft at the tetramer interface between three monomers and consists of His79 and His186 from two different adjacent monomers and a Ser/Thr-rich region, all of which are highly conserved across APIs. Structure and sequence analyses indicate that His79 and His186 may play important catalytic roles in the isomerization reaction. CMP-Kdo mimetics could therefore serve as potent and specific inhibitors of API and provide broad protection against many different bacterial infections.

  3. Structural analysis of arabinose-5-phosphate isomerase from Bacteroides fragilis and functional implications

    PubMed Central

    Chiu, Hsiu-Ju; Grant, Joanna C.; Farr, Carol L.; Jaroszewski, Lukasz; Knuth, Mark W.; Miller, Mitchell D.; Elsliger, Marc-André; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Wilson, Ian A.

    2014-01-01

    The crystal structure of arabinose-5-phosphate isomerase (API) from Bacteroides fragilis (bfAPI) was determined at 1.7 Å resolution and was found to be a tetramer of a single-domain sugar isomerase (SIS) with an endogenous ligand, CMP-Kdo (cytidine 5′-monophosphate-3-deoxy-d-manno-oct-2-ulosonate), bound at the active site. API catalyzes the reversible isomerization of d-ribulose 5-phosphate to d-arabinose 5-phosphate in the first step of the Kdo biosynthetic pathway. Interestingly, the bound CMP-Kdo is neither the substrate nor the product of the reaction catalyzed by API, but corresponds to the end product in the Kdo biosynthetic pathway and presumably acts as a feedback inhibitor for bfAPI. The active site of each monomer is located in a surface cleft at the tetramer interface between three monomers and consists of His79 and His186 from two different adjacent monomers and a Ser/Thr-rich region, all of which are highly conserved across APIs. Structure and sequence analyses indicate that His79 and His186 may play important catalytic roles in the isomerization reaction. CMP-Kdo mimetics could therefore serve as potent and specific inhibitors of API and provide broad protection against many different bacterial infections. PMID:25286848

  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.

  5. 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.

  6. Bioconversion of D-galactose into D-tagatose by expression of L-arabinose isomerase.

    PubMed

    Roh, H J; Kim, P; Park, Y C; Choi, J H

    2000-02-01

    D-Tagatose is a potential bulking agent in food as a non-calorific sweetener. To produce D-tagatose from cheaper resources, plasmids harbouring the L-arabinose isomerase gene (araA) from Escherichia coli, Bacillus subtilis and Salmonella typhimurium were constructed because L-arabinose isomerase was suggested previously as an enzyme that mediates the bioconversion of galactose into tagatose as well as that of arabinose to ribulose. The constructed plasmids were named pTC101, pTC105 and pTC106, containing araA from E. coli, B. subtilis and S. typhimurium respectively. In the cultures of recombinant E. coli with pTC101, pTC105 and pTC106, tagatose was produced from galactose in 9.9, 7.1 and 6.9% yields respectively. The enzyme extract of E. coli with the plasmid pTC101 also converted galactose into tagatose with a 96.4% yield.

  7. Characterization of a monoclonal antibody that specifically inhibits triosephosphate isomerase activity of Taenia solium.

    PubMed

    Víctor, Sanabria-Ayala; Yolanda, Medina-Flores; Araceli, Zavala-Carballo; Lucía, Jiménez; Abraham, Landa

    2013-08-01

    In the present study, we obtained and characterized partially a monoclonal antibody (4H11D10B11 mAb) against triosephosphate isomerase from Taenia solium (TTPI). This antibody recognized the enzyme by both ELISA and western blot and was able to inhibit its enzymatic activity in 74%. Moreover, the antigen-binding fragments (Fabs), products of digestion of the monoclonal antibody with papain, retained almost the same inhibitory effect. We determined the binding site by ELISA; synthetic peptides containing sequences from different non-conserved regions of the TTPI were confronted to the 4H11D10B11 mAb. The epitope recognized by the monoclonal antibody was located on peptide TTPI-56 (ATPAQAQEVHKVVRDWIRKHVDAGIADKARI), and an analysis of mimotopes, obtained with the 4H11D10B11 mAb, suggests that the epitope spans the sequence WIRKHVDAGIAD, residues 193-204 of the enzyme. This epitope is located within helix 6, next to loop 6, an essential active loop during catalysis. The antibody did not recognize triosephosphate isomerase from man and pig, definitive and intermediary hosts of T. solium, respectively. Furthermore, it did not bind to the catalytic site, since kinetic analysis demonstrated that inhibition had a non-competitive profile.

  8. Optimization of lactulose synthesis from whey lactose by immobilized β-galactosidase and glucose isomerase.

    PubMed

    Song, Yoon-Seok; Lee, Hee-Uk; Park, Chulhwan; Kim, Seung-Wook

    2013-03-22

    In the present study, commercially available whey was used as a lactose source, and immobilized β-galactosidase and glucose isomerase were used to synthesize lactulose from whey lactose in the absence of fructose. Optimal reaction conditions, such as lactose concentration, temperature, ionic strength of the buffer, and ratio of immobilized enzymes, were determined to improve lactulose synthesis using immobilized enzymes. Lactulose synthesis using immobilized enzymes improved markedly after optimizing the reaction conditions. When the lactulose synthesis was carried out at 53.5°C using 20% (w/v) whey lactose, 12U/ml of immobilized β-galactosidase and 60U/ml of immobilized glucose isomerase in 100mM sodium phosphate buffer at pH 7.5, the lactulose concentration and specific productivity were 7.68g/l and 0.32mg/Uh, respectively. Additionally, when the immobilized enzymes were reused for lactulose synthesis, their catalytic activity was 57.1% after 7 repeated uses. Copyright © 2013 Elsevier Ltd. All rights reserved.

  9. The effect of specific proline residues on the kinetic stability of the triosephosphate isomerases of two trypanosomes.

    PubMed

    Guzmán-Luna, Valeria; Quezada, Andrea G; Díaz-Salazar, A Jessica; Cabrera, Nallely; Pérez-Montfort, Ruy; Costas, Miguel

    2017-04-01

    The effect of specific residues on the kinetic stability of two closely related triosephosphate isomerases (from Trypanosoma cruzi, TcTIM and Trypanosoma brucei, TbTIM) has been studied. Based on a comparison of their β-turn occurrence, we engineered two chimerical enzymes where their super secondary β-loop-α motifs 2 ((βα)2 ) were swapped. Differential scanning calorimetry (DSC) experiments showed that the (βα)2 motif of TcTIM inserted into TbTIM (2Tc) increases the kinetic stability. On the other hand, the presence of the (βα)2 motif of TbTIM inserted into TcTIM (2Tb) gave a chimerical protein difficult to purify in soluble form and with a significantly reduced kinetic stability. The comparison of the contact maps of the (βα)2 of TbTIM and TcTIM showed differences in the contact pattern of residues 43 and 49. In TcTIM these residues are prolines, located at the N-terminal of loop-2 and the C-terminal of α-helix-2. Twelve mutants were engineered involving residues 43 and 49 to study the effect over the unfolding activation energy barrier (EA ). A systematic analysis of DSC data showed a large decrease on the EA of TcTIM (ΔEA ranging from 468 to 678 kJ/mol) when the single and double proline mutations are present. The relevance of Pro43 to the kinetic stability is also revealed by mutation S43P, which increased the free energy of the transition state of TbTIM by 17.7 kJ/mol. Overall, the results indicate that protein kinetic stability can be severely affected by punctual mutations, disturbing the complex network of interactions that, in concerted action, determine protein stability. Proteins 2017; 85:571-579. © 2016 Wiley Periodicals, Inc.

  10. Phycourobilin in Trichromatic Phycocyanin from Oceanic Cyanobacteria Is Formed Post-translationally by a Phycoerythrobilin Lyase-Isomerase*S⃞

    PubMed Central

    Blot, Nicolas; Wu, Xian-Jun; Thomas, Jean-Claude; Zhang, Juan; Garczarek, Laurence; Böhm, Stephan; Tu, Jun-Ming; Zhou, Ming; Plöscher, Matthias; Eichacker, Lutz; Partensky, Frédéric; Scheer, Hugo; Zhao, Kai-Hong

    2009-01-01

    Most cyanobacteria harvest light with large antenna complexes called phycobilisomes. The diversity of their constituting phycobiliproteins contributes to optimize the photosynthetic capacity of these microorganisms. Phycobiliprotein biosynthesis, which involves several post-translational modifications including covalent attachment of the linear tetrapyrrole chromophores (phycobilins) to apoproteins, begins to be well understood. However, the biosynthetic pathway to the blue-green-absorbing phycourobilin (λmax ∼ 495 nm) remained unknown, although it is the major phycobilin of cyanobacteria living in oceanic areas where blue light penetrates deeply into the water column. We describe a unique trichromatic phycocyanin, R-PC V, extracted from phycobilisomes of Synechococcus sp. strain WH8102. It is evolutionarily remarkable as the only chromoprotein known so far that absorbs the whole wavelength range between 450 and 650 nm. R-PC V carries a phycourobilin chromophore on its α-subunit, and this can be considered an extreme case of adaptation to blue-green light. We also discovered the enzyme, RpcG, responsible for its biosynthesis. This monomeric enzyme catalyzes binding of the green-absorbing phycoerythrobilin at cysteine 84 with concomitant isomerization to phycourobilin. This reaction is analogous to formation of the orange-absorbing phycoviolobilin from the red-absorbing phycocyanobilin that is catalyzed by the lyase-isomerase PecE/F in some freshwater cyanobacteria. The fusion protein, RpcG, and the heterodimeric PecE/F are mutually interchangeable in a heterologous expression system in Escherichia coli. The novel R-PC V likely optimizes rod-core energy transfer in phycobilisomes and thereby adaptation of a major phytoplankton group to the blue-green light prevailing in oceanic waters. PMID:19182270

  11. Acrosin-binding protein (ACRBP) and triosephosphate isomerase (TPI) are good markers to predict boar sperm freezing capacity.

    PubMed

    Vilagran, Ingrid; Castillo, Judit; Bonet, Sergi; Sancho, Sílvia; Yeste, Marc; Estanyol, Josep M; Oliva, Rafael

    2013-09-15

    Sperm cryopreservation is the most efficient method for storing boar sperm samples for a long time. However, one of the inconveniences of this method is the large variation between and within boars in the cryopreservation success of their sperm. The aim of the present work was thus to find reliable and useful predictive biomarkers of the good and poor capacity to withstand the freeze-thawing process in boar ejaculates. To find these biomarkers, the amount of proteins present in the total proteome in sperm cells were compared between good freezability ejaculates (GFE) and poor freezability ejaculates (PFE) using the two-dimensional difference gel electrophoresis technique. Samples were classified as GFE and PFE using progressive motility and viability of the sperm at 30 and 240 minutes after thawing, and the proteomes from each group, before starting cryopreservation protocols, were compared. Because two proteins, acrosin binding protein (ACRBP) and triosephosphate isomerase (TPI), presented the highest significant differences between GFE and PFE groups in two-dimensional difference gel electrophoresis assessment, Western blot analyses for ACRBP and TPI were also performed for validation. ACRBP normalized content was significantly lower in PFE than in GFE (P < 0.05), whereas the TPI amounts were significantly lower in GFE (P < 0.05) than in PFE. The association of ACRBP and TPI with postthaw sperm viability and motility was confirmed using Pearson's linear correlation. In conclusion, ACRBP and TPI can be used as markers of boar sperm freezability before starting the cryopreservation procedure, thereby avoiding unnecessary costs involved in this practice.

  12. Elevated Levels of Protein Disulfide Isomerase and Binding Immunoglobulin Protein Implicated in Spinal Cord Injury Paraplegia Patients with Pressure Ulcers.

    PubMed

    Chen, Weiyan; Liu, Jing; Zhao, Liang; Wang, Cuijiang; Li, Zengjun; Liu, Ting

    2016-07-01

    To explore the associations between two endoplasmic reticulum (ER) stress proteins, protein disulfide isomerase (PDI), binding immunoglobulin protein (BIP), and the development and progression of pressure ulcers (PUs) in spinal cord injury (SCI) paraplegia patients. ELISA kits were used to measure the levels of serum PDI and BIP in 67 SCI paraplegia patients with PUs and 61 SCI paraplegia patients without PUs. The associations between PDI and BIP, PU formation, PU staging, and pressure ulcer scale for healing (PUSH) score were analyzed. The patients in the PU group had higher levels of PDI and BIP than those in the non-PU group (both p < 0.05). Furthermore, the levels of PDI were positively correlated with those of BIP (r = 0.707, p < 0.0001). There were significant differences in the PDI and BIP levels among the different stages of PU (all p < 0.05). As the PU stages progressed, the levels of PDI and BIP first increased, then decreased, and finally peaked at stage III of the PUs. The PUSH scores significantly declined 7 days after debridement for the PU stage II (p < 0.01) but showed no significant difference between stages III and IV at 7 days after debridement (p > 0.05). The PUSH scores also decreased at 28 days after debridement for stages II, III, and IV (all p < 0.01). Higher PUSH scores indicated a longer time of debridement accompanied by a longer wound surface healing time (p < 0.05). ER stress proteins may be involved in the process of PU formation and healing; moreover, the levels of PDI and BIP were also associated with the severity of the PUs. Finally, we found that the PUSH scores can be used as a reference to evaluate PU severity and healing.

  13. Gold color in onions (Allium cepa): a natural mutation of the chalcone isomerase gene resulting in a premature stop codon.

    PubMed

    Kim, S; Jones, R; Yoo, K S; Pike, L M

    2004-11-01

    Unusual gold-colored onions were selected from a F3 family originating from a cross between US-type yellow and Brazilian yellow onions. HPLC analysis showed that the gold onions contained a significantly reduced amount of quercetin, the most abundant flavonoid in onions. This result indicated that an early step in the flavonoid biosynthesis pathway might be abnormal in these onions. The expression of flavonoid synthesis genes isolated from onions was examined in gold onions and compared to that in onions of other colors by RT-PCR. The results showed that all genes were transcribed in gold onions as in red onions. In order to identify any critical mutations in flavonoid synthesis genes encoding enzymes involved in early steps of the pathway, the genomic sequence of chalcone isomerase (CHI) was obtained. A premature stop codon and a subsequent single base-pair addition causing a frameshift were identified in the coding region of the CHI gene in the gold onions. Co-segregation of the mutant allele of the CHI gene and the gold phenotype was investigated in the original F2 segregating population. Genotyping of three color groups (red, yellow and gold) of F2 onions revealed perfect co-segregation of the mutant CHI allele with the gold phenotype. All tested gold F2 onions were homozygous for the mutant CHI allele. This perfect co-segregation implies that the presence of a premature stop codon in the gold CHI gene results in an inactive CHI. Inactivation of CHI results in a block in the flavonoid biosynthesis pathway and the accumulation of chalcone derivatives, including a yellow pigment which might be responsible for the gold color in onions.

  14. Functional analysis of protein disulfide isomerases in blood feeding, viability and oocyte development in Haemaphysalis longicornis ticks.

    PubMed

    Liao, Min; Boldbaatar, Damdinsuren; Gong, Haiyan; Huang, Penglong; Umemiya, Rika; Harnnoi, Thasaneeya; Zhou, Jinlin; Tanaka, Tetsuya; Suzuki, Hiroshi; Xuan, Xuenan; Fujisaki, Kozo

    2008-03-01

    Three protein disulfide isomerases from Haemaphysalis longicornis ticks (designated as HlPDI-1, HlPDI-2, and HlPDI-3) were previously identified. In order to further analyze their biological functions, the dsRNA of each HlPDI gene and one dsRNA combination of HlPDI-1/HlPDI-3 were separately injected into female ticks. Reduction of gene and protein expression of HlPDIs by RNA interference (RNAi) was demonstrated by real-time PCR, RT-PCR and Western blot analysis. In single dsRNA-injected groups, HlPDI-1 RNAi impacted tick blood feeding and oviposition, HlPDI-2 RNAi impacted tick viability and HlPDI-3 RNAi had no significant impact by itself. However, the injection of a combination of HlPDI-1/HlPDI-3 dsRNA had synergistic effects on tick viability. Furthermore, the midgut and cuticle were severely damaged in HlPDI-2 dsRNA-injected ticks and HlPDI-1/HlPDI-3 dsRNA-injected ticks, respectively, and disruption of HlPDI genes led to a significant reduction of disulfide bond-containing vitellogenin (Vg) expression in ticks. These results indicate that PDIs from H. longicornis are involved in blood feeding, viability and oocyte development, probably by mediating the formation of disulfide bond-containing proteins of the ticks and the formation of basement membrane and cuticle components such as extracellular matrix (ECM). This is the first report on the functional analysis of PDI family molecules as well as the interactions of PDI and other molecules in blood-feeding arthropods.

  15. Molecular analysis of a series of alleles in humans with reduced activity at the triosephosphate isomerase locus.

    PubMed Central

    Watanabe, M.; Zingg, B. C.; Mohrenweiser, H. W.

    1996-01-01

    Individuals with 50% of expected triosephosphate isomerase (TPI) enzyme activity have been previously identified in families during the screening of approximately 2,000 newborn children for quantitative variation in activity of 12 erythrocyte enzymes. The frequency of the trait was 9/1,713 individuals in the Caucasian population and 7/168 individuals among the African-American population studied. Genetic transmission of the trait was confirmed in all families. The frequency of the presumptive deficiency allele(s) at the TPI locus was greater than expected, given the reported incidence of clinical TPI deficiency. We report the molecular characterization of the variant alleles from seven African-American and three Caucasian individuals in this group of unrelated individuals. Three amino acid substitutions--a Gly-->Ala substitution at residue 72, a Val-->Met at residue 154, and a previously described Glu-->Asp substitution at residue 104--were identified in the Caucasian individuals. The substitutions occur at residues that are not directly involved in the active site but are highly conserved through evolutionary time, suggesting important roles for these residues in maintenance of subunit structure and conformation. The variant allele in the seven African-American individuals had nucleotide changes at positions -8 and -5 (5' of) from the transcription-initiation site. In three of these individuals, an additional T-->G substitution was detected in a TATA box-like sequence located 24 nucleotides 5' of the transcription-initiation site and on the same chromosome as the -5/-8 substitutions. Thus, molecular alterations at the TPI locus were detected in 10 unrelated individuals in whom segregation of a phenotype of reduced TPI activity previously had been identified. Images Figure 3 PMID:8571957

  16. The phosphoglucose isomerase from the hyperthermophilic archaeon Pyrococcus furiosus is a unique glycolytic enzyme that belongs to the cupin superfamily.

    PubMed

    Verhees, C H; Huynen, M A; Ward, D E; Schiltz, E; de Vos, W M; van der Oost, J

    2001-11-02

    Pyrococcus furiosus uses a variant of the Embden-Meyerhof pathway during growth on sugars. All but one of the genes that encode the glycolytic enzymes of P. furiosus have previously been identified, either by homology searching of its genome or by reversed genetics. We here report the isolation of the missing link of the pyrococcal glycolysis, the phosphoglucose isomerase (PGI), which was purified to homogeneity from P. furiosus and biochemically characterized. The P. furiosus PGI, a dimer of identical 23.5-kDa subunits, catalyzes the reversible isomerization of glucose 6-phosphate to fructose 6-phosphate, with K(m) values of 1.99 and 0.63 mm, respectively. An optimum pH of 7.0 has been determined in both directions, and at its optimum temperature of 90 degrees C the enzyme has a half-life of 2.4 h. The N-terminal sequence was used for the identification of the pgiA gene in the P. furiosus genome. The pgiA transcription start site has been determined, and a monocistronic messenger was detected in P. furiosus during growth on maltose and pyruvate. The pgiA gene was functionally expressed in Escherichia coli BL21(DE3). The deduced amino acid sequence of this first archaeal PGI revealed that it is not related to its bacterial and eukaryal counterparts. In contrast, this archaeal PGI shares similarity with the cupin superfamily that consists of a variety of proteins that are generally involved in sugar metabolism in both prokaryotes and eukaryotes. As for the P. furiosus PGI, distinct phylogenetic origins have previously been reported for other enzymes from the pyrococcal glycolytic pathway. Apparently, convergent evolution by recruitment of several unique enzymes has resulted in the unique Pyrococcus glycolysis.

  17. Chemical- and thermal-induced unfolding of Leishmania donovani ribose-5-phosphate isomerase B: a single-tryptophan protein.

    PubMed

    Kaur, Preet Kamal; Supin, Jakka S; Rashmi, S; Singh, Sushma

    2014-08-01

    Ribose-5-phosphate isomerase B (RpiB), a crucial enzyme of pentose phosphate pathway, was proposed to be a potential drug target for visceral leishmaniasis. In this study, we have analyzed the biophysical properties of Leishmania donovani RpiB (LdRpiB) enzyme to gain insight into its unfolding pathway under various chemical and thermal denaturation conditions by using fluorescence and CD spectroscopy. LdRpiB inactivation precedes the structural transition at lower concentrations of both urea and guanidine hydrochloride (GdHCl). 8-Anilinonapthalene 1-sulfonic (ANS) binding experiments revealed the presence of molten globule intermediate at 1.5 M GdHCl and a nonnative intermediate state at 6-M urea concentration. Acrylamide quenching experiments further validated the above findings, as solvent accessibility of tryptophan residues increased with increase in GdHCl and urea concentration. The recombinant LdRpiB was completely unfolded at 6 M GdHCl, whereas the enzyme molecule was resistant to complete unfolding even at 8-M urea concentration. The GdHCl- and urea-mediated unfolding involves a three-state transition process. Thermal-induced denaturation revealed complete loss of enzyme activity at 65 °C with only 20 % secondary structure loss. The formation of the well-ordered β-sheet structures of amyloid fibrils was observed after 55 °C which increased linearly till 85 °C as detected by thioflavin T dye. This study depicts the stability of the enzyme in the presence of chemical and thermal denaturants and stability-activity relationship of the enzyme. The presence of the intermediate states may have major implications in the way the enzyme binds to its natural ligand under various conditions. Also, the present study provides insights into the properties of intermediate entities of this important enzyme.

  18. Molecular analysis of a series of alleles in humans with reduced activity at the triosephosphate isomerase locus

    SciTech Connect

    Watanabe, M.; Zingg, B.C.; Mohrenweiser, H.W.

    1996-02-01

    Individuals with 50% of expected triosephosphate isomerase (TPI) enzyme activity have been previously identified in families during the screening of {approximately}2,000 newborn children for quantitative variation in activity of 12 erythrocyte enzymes. The frequency of the trait was 9/1,713 individuals in the Caucasian population and 7/168 individuals among the African-American population studied. Genetic transmission of the trait was confirmed in all families. The frequency of the presumptive deficiency allele(s) at the TPI locus was greater than expected, given the reported incidence of clinical TPI deficiency. We report the molecular characterization of the variant alleles from seven African-American and three Caucasian individuals in this group of unrelated individuals. Three amino acid substitutions - a Gly {yields} Ala substitution at residue 72, a Val {yields} Met at residue 154, and a previously described Glu {yields} Asp substitution at residue 104 - were identified in the Caucasian individuals. The substitutions occur at residues that are not directly involved in the active site but are highly conserved through evolutionary time, suggesting important roles for these residues in maintenance of subunit structure and conformation. The variant allele in the seven African-American individuals had nucleotide changes at positions -8 and -5 (5{prime} of) from the transcription-initiation site. In three of these individuals, an additional T {yields} G substitution was detected in a TATA box-like sequence located 24 nucleotides 5{prime} of the transcription-initiation site and on the same chromosome as the -5/-8 substitutions. Thus, molecular alterations at the TPI locus were detected in 10 unrelated individuals in whom segregation of a phenotype of reduced TPI activity previously had been identified. 41 refs., 3 figs., 2 tabs.

  19. Cis-trans isomerism in a square-planar platinum(II) complex bearing bulky fluorinated phosphane ligands.

    PubMed

    Bernès, Sylvain; Meléndez, Francisco J; Torrens, Hugo

    2016-04-01

    Transition-metal complexes bearing fluorinated phosphane and thiolate ligands has been an area of study in recent years and the chemical context of the current work is related to the metal-assisted functionalization of fluorinated derivatives. The cis and trans isomers of the square-planar complex bis[(pentafluorophenyl)diphenylphosphane-κP]bis(2,3,5,6-tetrafluorobenzenethiolato-κS)platinum(II), [Pt(C6HF4S)2{P(C6H5)2(C6F5)}2], have been crystallized from a single chromatographic fraction and characterized by X-ray diffraction analysis. The stabilization of the cis isomer results from weak intramolecular π-stacking interactions and possibly from the formation of a C-F...Pt contact, characterized by an F...Pt separation of 2.957 (6) Å. The natural bond orbital analysis (NBO) for this isomer confirms that the corresponding F → Pt charge transfer accounts for 6.92 kcal mol(-1) in the isomer stabilization. Such interactions are not present in the centrosymmetric trans isomer.

  20. Molecular Switch for Sub-Diffraction Laser Lithography by Photoenol Intermediate-State Cis-Trans Isomerization.

    PubMed

    Mueller, Patrick; Zieger, Markus M; Richter, Benjamin; Quick, Alexander S; Fischer, Joachim; Mueller, Jonathan B; Zhou, Lu; Nienhaus, Gerd Ulrich; Bastmeyer, Martin; Barner-Kowollik, Christopher; Wegener, Martin

    2017-06-27

    Recent developments in stimulated-emission depletion (STED) microscopy have led to a step change in the achievable resolution and allowed breaking the diffraction limit by large factors. The core principle is based on a reversible molecular switch, allowing for light-triggered activation and deactivation in combination with a laser focus that incorporates a point or line of zero intensity. In the past years, the concept has been transferred from microscopy to maskless laser lithography, namely direct laser writing (DLW), in order to overcome the diffraction limit for optical lithography. Herein, we propose and experimentally introduce a system that realizes such a molecular switch for lithography. Specifically, the population of intermediate-state photoenol isomers of α-methyl benzaldehydes generated by two-photon absorption at 700 nm fundamental wavelength can be reversibly depleted by simultaneous irradiation at 440 nm, suppressing the subsequent Diels-Alder cycloaddition reaction which constitutes the chemical core of the writing process. We demonstrate the potential of the proposed mechanism for STED-inspired DLW by covalently functionalizing the surface of glass substrates via the photoenol-driven STED-inspired process exploiting reversible photoenol activation with a polymerization initiator. Subsequently, macromolecules are grown from the functionalized areas and the spatially coded glass slides are characterized by atomic-force microscopy. Our approach allows lines with a full-width-at-half-maximum of down to 60 nm and line gratings with a lateral resolution of 100 nm to be written, both surpassing the diffraction limit.

  1. Interplay between metal binding and cis/trans isomerization in legume lectins: structural and thermodynamic study of P. angolensis lectin.

    PubMed

    Garcia-Pino, Abel; Buts, Lieven; Wyns, Lode; Loris, Remy

    2006-08-04

    The interplay between metal binding, carbohydrate binding activity, stability and structure of the lectin from Pterocarpus angolensis was investigated. Removal of the metals leads to a more flexible form of the protein with significantly less conformational stability. Crystal structures of this metal-free form show significant structural rearrangements, although some structural features that allow the binding of sugars are retained. We propose that substitution of an asparagine residue at the start of the C-terminal beta-strand of the legume lectin monomer hinders the trans-isomerization of the cis-peptide bond upon demetallization and constitutes an intramolecular switch governing the isomer state of the non-proline bond and ultimately the lectin phenotype.

  2. Light-controlled ion channels formed by amphiphilic small molecules regulate ion conduction via cis-trans photoisomerization.

    PubMed

    Liu, Tao; Bao, Chunyan; Wang, Haiyan; Lin, Yao; Jia, Huijuan; Zhu, Linyong

    2013-11-11

    Light-regulated ion channel-transport across lipid bilayers was realized using structurally simple azobenzene-based amphiphilic small molecules. UV or visible irradiation triggers molecular photoisomerization, which induces structural and membrane affinity changes in self-assembled channels, thus resulting in light-regulated ion transmembrane transport.

  3. Functional aspects of extracellular cyclophilins.

    PubMed

    Hoffmann, Henrik; Schiene-Fischer, Cordelia

    2014-07-01

    The cyclophilin family of peptidyl prolyl cis/trans isomerases includes several isoforms found to be secreted in response to different stimuli, thus existing both in the interior and the exterior of cells. The extracellular fractions of the cyclophilins CypA and CypB are involved in the control of cell-cell communication. By binding to the cell membrane receptor CD147 and cell surface heparans they elicit a variety of intracellular signaling cascades involved in inflammatory processes. Increased levels of cyclophilins in inflammatory tissues and body fluids are considered as an inflammatory response to injury. Thus, the extracellular portion of cyclophilins probably plays an important role in human diseases associated with acute or chronic inflammation like rheumatoid arthritis, sepsis, asthma and cardiovascular diseases. Specific inhibition of the cyclophilins in the extracellular space may open an effective therapeutic approach for treating inflammatory diseases.

  4. Kinetic measurements of phosphoglucose isomerase and phosphomannose isomerase by direct analysis of phosphorylated aldose-ketose isomers using tandem mass spectrometry

    NASA Astrophysics Data System (ADS)

    Gao, Hong; Chen, Ye; Leary, Julie A.

    2005-02-01

    A mass spectrometry based method for the direct determination of kinetic constants for phosphoglucose isomerase (PGI) and phosphomannose isomerase (PMI) is described. PGI catalyzes the interconversion between glucose-6-phosphate (Glc6P) and fructose-6-phosphate (Fru6P) and PMI performs the same function between mannose-6-phosphate (Man6P) and Fru6P. These two enzymes are essential in the pathways of glycolytic or oxidative metabolism of carbohydrates and have been considered as potential therapeutic targets. Traditionally, they are assayed either by spectrophotometric detection of Glc6P with one or more coupling enzymes or by a colorimetric detection of Fru6P. However, no suitable assay for Man6P has been developed yet to study the reaction of PMI in the direction from Fru6P to Man6P. In the work presented herein, a general assay for the isomeric substrate-product pair between Glc6P and Fru6P or between Man6P and Fru6P was developed, with the aim of directly studying the kinetics of PGI and PMI in both directions. The 6-phosphorylated aldose and ketose isomers were distinguished based on their corresponding tandem mass spectra (MS2) obtained on a quadrupole ion trap mass spectrometer, and a multicomponent quantification method was utilized to determine the composition of binary mixtures. Using this method, the conversion between Fru6P and Glc6P and that between Fru6P and Man6P are directly monitored. The equilibrium constants for the reversible reactions catalyzed by PGI and PMI are measured to be 0.3 and 1.1, respectively, and the kinetic parameters for both substrates of PGI and PMI are also determined. The values of KM and Vmax for Fru6P as substrate of PMI are reported to be 0.15 mM and 7.78 [mu]mol/(min mg), respectively. All other kinetic parameters measured correlate well with those obtained using traditional methods, demonstrating the accuracy and reliability of this assay.

  5. Isoform-specific Inhibition of Cyclophilins

    PubMed Central

    Daum, Sebastian; Schumann, Michael; Mathea, Sebastian; Aumüller, Tobias; Balsley, Molly A.; Constant, Stephanie L.; de Lacroix, Boris Féaux; Kruska, Fabian; Braun, Manfred; Schiene-Fischer, Cordelia

    2009-01-01

    Cyclophilins belong to the enzyme class of peptidyl prolyl cis/trans isomerases which catalyze the cis/trans isomerization of prolyl bonds in peptides and proteins in different folding states. Cyclophilins have been shown to be involved in a multitude of cellular functions like cell growth, proliferation, and motility. Among the 20 human cyclophilin isoenzymes, the two most abundant members of the cyclophilin family CypA and CypB exhibit specific cellular functions in several inflammatory diseases, cancer development and HCV replication. A small-molecule inhibitor on the basis of aryl 1-indanylketones has now been shown to discriminate between CypA and CypB in vitro. CypA binding of this inhibitor has been characterized by fluorescence anisotropy- and isothermal titration calorimetry-based cyclosporin competition assays. Inhibition of CypA- but not CypB-mediated chemotaxis of mouse CD4+ T cells by the inhibitor provided biological proof of discrimination in vivo. PMID:19480458

  6. 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

    ... plants are exempt from the requirement of a tolerance when used as plant-incorporated protectant inert... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Phosphomannose isomerase in all plants... ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS PROCEDURES AND REQUIREMENTS FOR...

  7. 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

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

    PubMed

    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

    2016-06-15

    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 (Atm(KD/-)) 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 Atm(KD/-), 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.

  9. Open reading frame 176 in the photosynthesis gene cluster of Rhodobacter capsulatus encodes idi, a gene for isopentenyl diphosphate isomerase.

    PubMed Central

    Hahn, F M; Baker, J A; Poulter, C D

    1996-01-01

    Isopentenyl diphosphate (IPP) isomerase catalyzes an essential activation step in the isoprenoid biosynthetic pathway. A database search based on probes from the highly conserved regions in three eukaryotic IPP isomerases revealed substantial similarity with ORF176 in the photosynthesis gene cluster in Rhodobacter capsulatus. The open reading frame was cloned into an Escherichia coli expression vector. The encoded 20-kDa protein, which was purified in two steps by ion exchange and hydrophobic interaction chromatography, catalyzed the interconversion of IPP and dimethylallyl diphosphate. Thus, the photosynthesis gene cluster encodes all of the enzymes required to incorporate IPP into the ultimate carotenoid and bacteriochlorophyll metabolites in R. capsulatus. More recent searches uncovered additional putative open reading frames for IPP isomerase in seed-bearing plants (Oryza sativa, Arabadopsis thaliana, and Clarkia breweri), a worm (Caenorhabiditis elegans), and another eubacterium (Escherichia coli). The R. capsulatus enzyme is the smallest of the IPP isomerases to be identified thus far and may consist mostly of a fundamental catalytic core for the enzyme. PMID:8550491

  10. Single active-site histidine in D-xylose isomerase from Streptomyces violaceoruber. Identification by chemical derivatization and peptide mapping.

    PubMed

    Vangrysperre, W; Ampe, C; Kersters-Hilderson, H; Tempst, P

    1989-10-01

    Group-specific chemical modifications of D-xylose isomerase from Streptomyces violaceruber indicated that complete loss of activity is fully correlated with the acylation of a single histidine. Active-site protection, by the ligand combination of xylitol plus Mg2+, completely blocked diethyl pyrocarbonate derivatization of this particular residue [Vangrysperre, Callens, Kersters-Hilderson & De Bruyne (1988) Biochem. J. 250, 153-160]. Differential peptide mapping between D-xylose isomerase, which has previously been treated with diethyl pyrocarbonate in the presence or absence of xylitol plus Mg2+, allowed specific isolation and sequencing of a peptide containing this active-site histidine. For this purpose we used two essentially new techniques: first, a highly reproducible peptide cleavage protocol for protease-resistant, carbethoxylated proteins with guanidinium hydrochloride as denaturing agent and subtilisin for proteolysis; and second, reverse-phase liquid chromatography with dual-wavelength detection at 214 and 238 nm, and calculation of absorbance ratios. It allowed us to locate the single active-site histidine at position 54 in the primary structure of Streptomyces violaceoruber D-xylose isomerase. The sequence around this residue is conserved in D-xylose isomerases from a diversity of micro-organisms, suggesting that this is a structurally and/or functionally essential part of the molecule.

  11. Identification of a d-Arabinose-5-Phosphate Isomerase in the Gram-Positive Clostridium tetani.

    PubMed

    Cech, David L; Markin, Katherine; Woodard, Ronald W

    2017-09-01

    d-Arabinose-5-phosphate (A5P) isomerases (APIs) catalyze the interconversion of d-ribulose-5-phosphate and d-arabinose-5-phosphate. Various Gram-negative bacteria, such as the uropathogenic Escherichia coli strain CFT073, contain multiple API paralogs (KdsD, GutQ, KpsF, and c3406) that have been assigned various cellular functions. The d-arabinose-5-phosphate formed by these enzymes seems to play important roles in the biosynthesis of lipopolysaccharide (LPS) and group 2 K-antigen capsules, as well as in the regulation of the cellular d-glucitol uptake and uropathogenic infectivity/virulence. The genome of a Gram-positive pathogenic bacterium, Clostridium tetani, contains a gene encoding a putative API, C. tetani API (CtAPI), even though C. tetani lacks both LPS and capsid biosynthetic genes. To better understand the physiological role of d-arabinose-5-phosphate in this Gram-positive organism, recombinant CtAPI was purified and characterized. CtAPI displays biochemical characteristics similar to those of APIs from Gram-negative organisms and complements the API deficiency of an E. coli API knockout strain. Thus, CtAPI represents the first d-arabinose-5-phosphate isomerase to be identified and characterized from a Gram-positive bacterium.IMPORTANCE The genome of Clostridium tetani, a pathogenic Gram-positive bacterium and the causative agent of tetanus, contains a gene (the CtAPI gene) that shares high sequence similarity with those of genes encoding d-arabinose-5-phosphate isomerases. APIs play an important role within Gram-negative bacteria in d-arabinose-5-phosphate production for lipopolysaccharide biosynthesis, capsule formation, and regulation of cellular d-glucitol uptake. The significance of our research is in identifying and characterizing CtAPI, the first Gram-positive API. Our findings show that CtAPI is specific to the interconversion of arabinose-5-phosphate and ribulose-5-phosphate while having no activity with the other sugars and sugar phosphates

  12. Bioethanol production from steam-pretreated corn stover through an isomerase mediated process.

    PubMed

    De Bari, Isabella; Cuna, Daniela; Di Matteo, Vincenzo; Liuzzi, Federico

    2014-03-25

    Agricultural by-products such as corn stover are considered strategic raw materials for the production of second-generation bioethanol from renewable and non-food sources. This paper describes the conversion of steam-pretreated corn stover to ethanol utilising a multi-step process including enzymatic hydrolysis, isomerisation, and fermentation of mixed hydrolysates with native Saccharomyces cerevisiae. An immobilised isomerase enzyme was used for the xylose isomerisation along with high concentrations of S. cerevisiae. The objective was to assess the extent of simultaneity of the various conversion steps, through a detailed analysis of process time courses, and to test this process scheme for the conversion of lignocellulosic hydrolysates containing several inhibitors of the isomerase enzyme (e.g. metal ions, xylitol and glycerol). The process was tested on two types of hydrolysate after acid-catalysed steam pretreatment: (a) the water soluble fraction (WSF) in which xylose was the largest carbon source and (b) the entire slurry, containing both cellulose and hemicellulose carbohydrates, in which glucose predominated. The results indicated that the ethanol concentration rose when the inoculum concentration was increased in the range 10-75 g/L. However, when xylose was the largest carbon source, the metabolic yields were higher than 0.51g(ethanol)/g(consumed) sugars probably due to the use of yeast internal cellular resources. This phenomenon was not observed in the fermentation of mixed hydrolysates obtained from the entire pretreated product and in which glucose was the largest carbon source. The ethanol yield from biomass suspensions with dry matter (DM) concentrations of 11-12% (w/v) was 70% based on total sugars (glucose, xylose, galactose). The results suggest that xylulose uptake was more effective in mixed hydrolysates containing glucose levels similar to, or higher than, xylose. Analysis of the factors that limit isomerase activity in lignocellulosic

  13. Down-regulation of phosphoglucose isomerase/autocrine motility factor expression sensitizes human fibrosarcoma cells to oxidative stress leading to cellular senescence.

    PubMed

    Funasaka, Tatsuyoshi; Hu, Huankai; Hogan, Victor; Raz, Avraham

    2007-12-14

    Phosphoglucose isomerase/autocrine motility factor (PGI/AMF) is a housekeeping gene product present in all cells, is an essential enzyme of catabolic glycolysis and anabolic gluconeogenesis, and regulates tumor cell growth and metastasis. Because glycolytic enzyme up-regulation of expression contributes to glycolytic flux, leading to increased of cell growth and a resistance to cellular stress of normal fibroblasts whereas down-regulation of PGI/AMF leads to mesenchymal-to-epithelial transition in tumor cells, we examined the involvement of PGI/AMF in overcoming cellular senescence in cancer cells. PGI/AMF cellular expression in HT1080 human fibrosarcoma was down-regulated by small interfering RNA methodology, which resulted in an increased sensitivity to oxidative stress and oxidative stress-induced cellular senescence. Signaling analysis revealed that the senescence pathway involving p21 cyclin-dependent kinase inhibitor was up-regulated in PGI/AMF knockdown cells and that superoxide dismutase is the upstream regulator protein of p21-mediated cellular senescence. A specific inhibitor of PGI/AMF induced cellular senescence and p21 expression in tumor cells exposed to an oxidative stress environment. Taken together, the results presented here suggest that PGI/AMF is involved in oxidative stress-induced cellular senescence and should bring novel insights into the control of cellular growth leading to a new methodology for cancer treatment.

  14. Domain a' of protein disulfide isomerase plays key role in inhibiting alpha-synuclein fibril formation.

    PubMed

    Cheng, Han; Wang, Lei; Wang, Chih-chen

    2010-07-01

    alpha-Synuclein (alpha Syn) is the main component of Lewy bodies formed in midbrain dopaminergic neurons which is a pathological characteristic of Parkinson's disease. It has been recently showed to induce endoplasmic reticulum (ER) stress and impair ER functions. However, the mechanism of how ER responds to alpha Syn toxicity is poorly understood. In the present study, we found that protein disulfide isomerase (PDI), a stress protein abundant in ER, effectively inhibits alpha Syn fibril formation in vitro. In PDI molecule with a structure of abb'xa'c, domain a' was found to be essential and sufficient for PDI to inhibit alpha Syn fibril formation. PDI was further found to be more avid for binding with intermediate species formed during alpha Syn fibril formation, and the binding was more intensive in the later lag phase. Our results provide new insight into the role of PDI in protecting ER from the deleterious effects of misfolded protein accumulation in many neurodegenerative diseases.

  15. Assisted refolding of recombinant prochymosin with the aid of protein disulphide isomerase.

    PubMed Central

    Tang, B; Zhang, S; Yang, K

    1994-01-01

    Protein disulphide isomerase (PDI) was shown to be able to accelerate the refolding of unfolded recombinant prochymosin and to enhance the overall yield of active protein. Unlike previous reports in this study PDI was found to be active at pH values as high as 11. The coincidence of the similar apparent optimum pH values of uncatalysed and PDI-catalysed reactions suggests that conditions favourable to spontaneous refolding of proteins may help PDI to catalyse thiol/disulphide interchange. Under the conditions described here no exogenously added dithiothreitol was required for PDI-catalysed renaturation, implying that the disulphide form of PDI was reduced to its active form by the free thiol groups in prochymosin molecules. PMID:8037666

  16. Novel approach for identifying key residues in enzymatic reactions: proton abstraction in ketosteroid isomerase.

    PubMed

    Ito, Mika; Brinck, Tore

    2014-11-20

    We propose a computationally efficient approach for evaluating the individual contributions of many different residues to the catalytic efficiency of an enzymatic reaction. This approach is based on the fragment molecular orbital (FMO) method, and it defines the energy of a deletion form, i.e., the energy of the system when a particular residue is deleted. Using this approach, we found that, among 10 investigated residues, three, Tyr14, Asp99, and Tyr55, in this order, significantly reduce the activation energy of the proton abstraction from a substrate, cyclopent-2-enone, catalyzed by ketosteroid isomerase (KSI). The relative activation energies estimated in this study are in good agreement with available previous experimental and theoretical data obtained for the similar proton abstraction with a native substrate and substitution mutants of KSI. It was thus indicated that the new approach is efficient for rationally evaluating the catalytic effects of multiple residues on an enzymatic reaction.

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

    PubMed

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

    2014-12-19

    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.

  18. Enzyme-enzyme interaction in the chloroplast: glyceraldehyde-3-phosphate dehydrogenase, triose phosphate isomerase and aldolase.

    PubMed

    Anderson, L E; Goldhaber-Gordon, I M; Li, D; Tang, X Y; Xiang, M; Prakash, N

    1995-01-01

    Apparent physical interaction between pea chloroplast (Pisum sativum L.) glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) and aldolase (EC 4.1.2.13) is seen in phase-partitioning, fluorescent-anisotropy and isoelectric-focusing experiments. Similarly, results obtained in phase-partitioning and isoelectric-focusing experiments indicate physical interaction between aldolase and triose-phosphate isomerase (EC 5.3.1.1). Kinetic experiments suggest that both aldolase-bound glyceraldehyde-3-phosphate can act as substrate for glyceraldehyde-3-phosphate dehydrogenase. These results are consistent with the notion that there is interaction between these three enzymes both during photosynthetic CO2 fixation and during glycolysis in the chloroplast.

  19. Isolation and sequence analysis of the gene encoding triose phosphate isomerase from Zygosaccharomyces bailii.

    PubMed

    Merico, A; Rodrigues, F; Côrte-Real, M; Porro, D; Ranzi, B M; Compagno, C

    2001-06-30

    The ZbTPI1 gene encoding triose phosphate isomerase (TIM) was cloned from a Zygosaccharomyces bailii genomic library by complementation of the Saccharomyces cerevisiae tpi1 mutant strain. The nucleotide sequence of a 1.5 kb fragment showed an open reading frame (ORF) of 746 bp, encoding a protein of 248 amino acid residues. The deduced amino acid sequence shares a high degree of homology with TIMs from other yeast species, including some highly conserved regions. The analysis of the promoter sequence of the ZbTPI1 revealed the presence of putative motifs known to have regulatory functions in S. cerevisiae. The GenBank Accession No. of ZbTPI1 is AF325852.

  20. Triosephosphate Isomerase Gene Characterization and Potential Zoonotic Transmission of Giardia duodenalis

    PubMed Central

    Sulaiman, Irshad M.; Fayer, Ronald; Bern, Caryn; Gilman, Robert H.; Trout, James M.; Schantz, Peter M.; Das, Pradeep; Lal, Altaf A.

    2003-01-01

    To address the source of infection in humans and public health importance of Giardia duodenalis parasites from animals, nucleotide sequences of the triosephosphate isomerase (TPI) gene were generated for 37 human isolates, 15 dog isolates, 8 muskrat isolates, 7 isolates each from cattle and beavers, and 1 isolate each from a rat and a rabbit. Distinct genotypes were found in humans, cattle, beavers, dogs, muskrats, and rats. TPI and small subunit ribosomal RNA (SSU rRNA) gene sequences of G. microti from muskrats were also generated and analyzed. Phylogenetic analysis on the TPI sequences confirmed the formation of distinct groups. Nevertheless, a major group (assemblage B) contained most of the human and muskrat isolates, all beaver isolates, and the rabbit isolate. These data confirm that G. duodenalis from certain animals can potentially infect humans and should be useful in the detection, differentiation, and taxonomy of Giardia spp. PMID:14718089

  1. Production of Ethanol from d-Xylose by Using d-Xylose Isomerase and Yeasts

    PubMed Central

    Gong, Cheng-Shung; Chen, Li-Fu; Flickinger, Michael C.; Chiang, Lin-Chang; Tsao, George T.

    1981-01-01

    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. PMID:16345717

  2. Lack of Variation at Phosphoglucose Isomerase (Pgi) in Bumblebees: Implications for Conservation Genetics Studies

    PubMed Central

    Ellis, Jonathan S.; Turner, Lucy M.; Knight, Mairi E.

    2013-01-01

    Assessing genetic variation underlying ecologically important traits is increasingly of interest and importance in population and conservation genetics. For some groups generally useful markers exist for examining the relative role of selection and drift in shaping genetic diversity e.g. the major histocompatibility complex in vertebrates and self-incompatibility loci in plants. For invertebrates there is no such generally useful locus. However, phosphoglucose isomerase (Pgi) has been proposed as a useful functional marker in the conservation genetics of invertebrates. Where thermal microclimate varies, balanced polymorphisms may be maintained due to trade-offs between thermally stable and kinetically advantageous allelic forms. We here report very low levels of Pgi variation in bumblebees rendering this locus to be of little use as an adaptive marker in a conservation genetics context in this group. Potential explanations for this lack of variation are considered. PMID:23750269

  3. 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

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

    PubMed Central

    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.

    2016-01-01

    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

  5. Drosophila peptidyl-prolyl isomerase Pin1 modulates circadian rhythms via regulating levels of PERIOD.

    PubMed

    Kang, So Who; Lee, Euna; Cho, Eunjoo; Seo, Ji Hye; Ko, Hyuk Wan; Kim, Eun Young

    2015-07-31

    In animal circadian clock machinery, the phosphorylation program of PERIOD (PER) leads to the spatio-temporal regulation of diverse PER functions, which are crucial for the maintenance of ~24-hr circadian rhythmicity. The peptidyl-prolyl isomerase PIN1 modulates the diverse functions of its substrates by inducing conformational changes upon recognizing specific phosphorylated residues. Here, we show that overexpression of Drosophila pin1, dodo (dod), lengthens the locomotor behavioral period. Using Drosophila S2 cells, we demonstrate that Dod associates preferentially with phosphorylated species of PER, which delays the phosphorylation-dependent degradation of PER. Consistent with this, PER protein levels are higher in flies overexpressing dod. Taken together, we suggest that Dod plays a role in the maintenance of circadian period by regulating PER metabolism.

  6. Mannose production from fructose by free and immobilized D-lyxose isomerases from Providencia stuartii.

    PubMed

    Park, Chang-Su; Kwon, Hyun-Jung; Yeom, Soo-Jin; Oh, Deok-Kun

    2010-09-01

    A recombinant D-lyxose isomerase from Providencia stuartii was immobilized on Duolite A568 beads which gave the highest conversion of D-fructose to D-mannose among the various immobilization beads evaluated. Maximum activities of both the free and immobilized enzymes for fructose isomerization were at pH 7.5 and 45 degrees C in the presence of 1 mM Mn(2+). Enzyme half-lives were 14 and 30 h at 35 degrees C and 3.4 and 5.1 h at 45 degrees C, respectively. The immobilized enzyme in 300 g fructose/l (replaced hourly), produced 75 g mannose/l at 35 degrees C = 25% (w/w) yield with a productivity of 75 g mannose l(-1) h(-1) after 23 cycles.

  7. Galactaro δ-Lactone Isomerase: Lactone Isomerization by a Member of the Amidohydrolase Superfamily

    PubMed Central

    2015-01-01

    Agrobacterium tumefaciens strain C58 can utilize d-galacturonate as a sole source of carbon via a pathway in which the first step is oxidation of d-galacturonate to d-galactaro-1,5-lactone. We have identified a novel enzyme, d-galactarolactone isomerase (GLI), that catalyzes the isomerizaton of d-galactaro-1,5-lactone to d-galactaro-1,4-lactone. GLI, a member of the functionally diverse amidohydrolase superfamily, is a homologue of LigI that catalyzes the hydrolysis of 2-pyrone-4,6-dicarboxylate in lignin degradation. The ability of GLI to catalyze lactone isomerization instead of hydrolysis can be explained by the absence of the general basic catalysis used by 2-pyrone-4,6-dicarboxylate lactonase. PMID:24450804

  8. Galactaro δ-lactone isomerase: lactone isomerization by a member of the amidohydrolase superfamily.

    PubMed

    Bouvier, Jason T; Groninger-Poe, Fiona P; Vetting, Matthew; Almo, Steven C; Gerlt, John A

    2014-02-04

    Agrobacterium tumefaciens strain C58 can utilize d-galacturonate as a sole source of carbon via a pathway in which the first step is oxidation of d-galacturonate to D-galactaro-1,5-lactone. We have identified a novel enzyme, D-galactarolactone isomerase (GLI), that catalyzes the isomerizaton of D-galactaro-1,5-lactone to D-galactaro-1,4-lactone. GLI, a member of the functionally diverse amidohydrolase superfamily, is a homologue of LigI that catalyzes the hydrolysis of 2-pyrone-4,6-dicarboxylate in lignin degradation. The ability of GLI to catalyze lactone isomerization instead of hydrolysis can be explained by the absence of the general basic catalysis used by 2-pyrone-4,6-dicarboxylate lactonase.

  9. Inheritance and subcellular localization of triose-phosphate isomerase in dwarf mountain pine (Pinus mugo).

    PubMed

    Odrzykoski, I J

    2001-01-01

    Several trees with expected heterozygous phenotype for triose-phosphate isomerase (TPI) were discovered in a population of dwarf mountain pine (Pinus mugo Turra) from southern Poland. As the inheritance of this enzyme in pines has not been reported, segregation of allelic variants was tested in eight trees with putative heterozygous phenotypes for two loci, TpiA and TPIB: Linkage between these and some other isozyme loci were studied and evidence for linkage has been found between TpiA and PgdA (r = 0.10) and between TpiB and DiaD (r = 0.36), but in single trees only. The subcellular localization of TPI isozymes was determined by comparing isoenzymes from the total extract with those found in fraction enriched in plastids, prepared by differential gradient centrifugation of cellular organelles. The more slowly migrating TPI-B isozyme is located in plastids.

  10. Enzymatic conversion of flavonoids using bacterial chalcone isomerase and enoate reductase.

    PubMed

    Gall, Mechthild; Thomsen, Maren; Peters, Christin; Pavlidis, Ioannis V; Jonczyk, Patrick; Grünert, Philipp P; Beutel, Sascha; Scheper, Thomas; Gross, Egon; Backes, Michael; Geissler, Torsten; Ley, Jakob P; Hilmer, Jens-Michael; Krammer, Gerhard; Palm, Gottfried J; Hinrichs, Winfried; Bornscheuer, Uwe T

    2014-01-27

    Flavonoids are a large group of plant secondary metabolites with a variety of biological properties and are therefore of interest to many scientists, as they can lead to industrially interesting intermediates. The anaerobic gut bacterium Eubacterium ramulus can catabolize flavonoids, but until now, the pathway has not been experimentally confirmed. In the present work, a chalcone isomerase (CHI) and an enoate reductase (ERED) could be identified through whole genome sequencing and gene motif search. These two enzymes were successfully cloned and expressed in Escherichia coli in their active form, even under aerobic conditions. The catabolic pathway of E. ramulus was confirmed by biotransformations of flavanones into dihydrochalcones. The engineered E. coli strain that expresses both enzymes was used for the conversion of several flavanones, underlining the applicability of this biocatalytic cascade reaction. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Redox-coupled structural changes of the catalytic a' domain of protein disulfide isomerase.

    PubMed

    Inagaki, Koya; Satoh, Tadashi; Yagi-Utsumi, Maho; Le Gulluche, Anne-Charlotte; Anzai, Takahiro; Uekusa, Yoshinori; Kamiya, Yukiko; Kato, Koichi

    2015-09-14

    Protein disulfide isomerase functions as a folding catalyst in the endoplasmic reticulum. Its b' and a' domains provide substrate-binding sites and undergo a redox-dependent domain rearrangement coupled to an open-closed structural change. Here we determined the first solution structure of the a' domain in its oxidized form and thereby demonstrate that oxidation of the a' domain induces significant conformational changes not only in the vicinity of the active site but also in the distal b'-interfacial segment. Based on these findings, we propose that this conformational transition triggers the domain segregation coupled with the exposure of the hydrophobic surface. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  12. 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.

  13. Lack of variation at phosphoglucose isomerase (pgi) in bumblebees: implications for conservation genetics studies.

    PubMed

    Ellis, Jonathan S; Turner, Lucy M; Knight, Mairi E

    2013-01-01

    Assessing genetic variation underlying ecologically important traits is increasingly of interest and importance in population and conservation genetics. For some groups generally useful markers exist for examining the relative role of selection and drift in shaping genetic diversity e.g. the major histocompatibility complex in vertebrates and self-incompatibility loci in plants. For invertebrates there is no such generally useful locus. However, phosphoglucose isomerase (Pgi) has been proposed as a useful functional marker in the conservation genetics of invertebrates. Where thermal microclimate varies, balanced polymorphisms may be maintained due to trade-offs between thermally stable and kinetically advantageous allelic forms. We here report very low levels of Pgi variation in bumblebees rendering this locus to be of little use as an adaptive marker in a conservation genetics context in this group. Potential explanations for this lack of variation are considered.

  14. The Arabidopsis Cyclophilin Gene Family1

    PubMed Central

    Romano, Patrick G.N.; Horton, Peter; Gray, Julie E.

    2004-01-01

    Database searching has allowed the identification of a number of previously unreported single and multidomain isoform members of the Arabidopsis cyclophilin gene family. In addition to the cyclophilin-like peptidyl-prolyl cis-trans isomerase domain, the latter contain a variety of other domains with characterized functions. Transcriptional analysis showed they are expressed throughout the plant, and different isoforms are present in all parts of the cell including the cytosol, nucleus, mitochondria, secretory pathway, and chloroplast. The abundance and diversity of cyclophilin isoforms suggests that, like their animal counterparts, plant cyclophilins are likely to be important proteins involved in a wide variety of cellular processes. As well as fulfilling the basic role of protein folding, they may also play important roles in mRNA processing, protein degradation, and signal transduction and thus may be crucial during both development and stress responsiveness. PMID:15051864

  15. Cyclophilin function in Cancer; lessons from virus replication.

    PubMed

    Lavin, Paul T M; Mc Gee, Margaret M

    2015-01-01

    Cyclophilins belong to a group of proteins that possess peptidyl prolyl isomerase activity and catalyse the cis-trans conversion of proline peptide bonds. Cyclophilin members play important roles in protein folding and as molecular chaperones, in addition to a well-established role as host factors required for completion of the virus life cycle. Members of the cyclophilin family are overexpressed in a range of human malignancies including hepatocellular cancer, pancreatic cancer, nonsmall cell lung cancer, gastric cancer, colorectal cancer and glioblastoma multiforme, however, their precise role in tumourigenesis remains unclear. In recent years, mounting evidence supports a role for prolyl isomerisation during mammalian cell division; a process with striking similarity to plasma membrane remodelling during virus replication. Here, we summarise our current understanding of the role of cyclophilins in cancer. We review the function of cyclophilins during mammalian cell division and during HIV-1 infection, and highlight common processes involving members of the ESCRT and Rab GTPase families.

  16. Current implications of cyclophilins in human cancers.

    PubMed

    Lee, Jinhwa; Kim, Sung Soo

    2010-07-19

    Cyclophilins (Cyps), the intracellular receptor for immunosuppressant cyclosporine A (CsA), play important cellular roles through activities of peptidyl-prolyl cis-trans isomerase (PPIase) and chaperones. Cyps are structurally conserved and found in both prokaryotic and eukaryotic organisms, including humans which contain 16 Cyp isoforms. Although human Cyps were identified about 25 years ago, their physiological and pathological roles have only been the focus of attention recently because of their possible involvement in diseases and ailments such as HIV infection, hepatitis B and C viral infection, atherosclerosis, ER stress-related diseases and neurodegenerative diseases, etc. There are reports for upregulated Cyps in many human cancers and there are also strong correlations found between Cyps overexpression and malignant transformation. This review discusses the important and diverse roles of Cyps overexpression in human cancers. Understanding biological functions of Cyps will eventually lead to improved strategies for cancer treatment and prevention.

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

    PubMed Central

    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

  18. Strigolactones, a novel carotenoid-derived plant hormone.

    PubMed

    Al-Babili, Salim; Bouwmeester, Harro J

    2015-01-01

    Strigolactones (SLs) are carotenoid-derived plant hormones and signaling molecules. When released into the soil, SLs indicate the presence of a host to symbiotic fungi and root parasitic plants. In planta, they regulate several developmental processes that adapt plant architecture to nutrient availability. Highly branched/tillered mutants in Arabidopsis, pea, and rice have enabled the identification of four SL biosynthetic enzymes: a cis/trans-carotene isomerase, two carotenoid cleavage dioxygenases, and a cytochrome P450 (MAX1). In vitro and in vivo enzyme assays and analysis of mutants have shown that the pathway involves a combination of new reactions leading to carlactone, which is converted by a rice MAX1 homolog into an SL parent molecule with a tricyclic lactone moiety. In this review, we focus on SL biosynthesis, describe the hormonal and environmental factors that determine this process, and discuss SL transport and downstream signaling as well as the role of SLs in regulating plant development.

  19. Elucidating cdc25’s Oncogenic Mechanism in Breast Cancer Using Pin1, a Negative Mitotic Regulator

    DTIC Science & Technology

    2001-07-01

    Davies, P., and Lu, K. P. (1999a). The prolyl isomerase Pinl restores the function of Alzheimer-associated phosphorylated tau protein. Nature 399, 784...characterization of a 14 kDa human protein as a novel parvulin-like peptidyl prolyl cis/trans isomerase. Febs Letters 446, 278- 82. Verdecia, M. A., Bowman...isomerization regulates dephosphorylation of Cdc25C and tau proteins. Mol Cell 6, 873-83. 18 Contains unpublished and proprietary information. - Not

  20. 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

  1. 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

  2. Substrate specificity of platypus venom L-to-D-peptide isomerase.

    PubMed

    Bansal, Paramjit S; Torres, Allan M; Crossett, Ben; Wong, Karen K Y; Koh, Jennifer M S; Geraghty, Dominic P; Vandenberg, Jamie I; Kuchel, Philip W

    2008-04-04

    The L-to-D-peptide isomerase from the venom of the platypus (Ornithorhyncus anatinus) is the first such enzyme to be reported for a mammal. In delineating its catalytic mechanism and broader roles in the animal, its substrate specificity was explored. We used N-terminal segments of defensin-like peptides DLP-2 and DLP-4 and natriuretic peptide OvCNP from the venom as substrates. The DLP analogues IMFsrs and ImFsrs (srs is a solubilizing chain; lowercase letters denote D-amino acid) were effective substrates for the isomerase; it appears to recognize the N-terminal tripeptide sequence Ile-Xaa-Phe-. A suite of 26 mutants of these hexapeptides was synthesized by replacing the second residue (Met) with another amino acid, viz. Ala, alpha-aminobutyric acid, Ile, Leu, Lys, norleucine, Phe, Tyr, and Val. It was shown that mutant peptides incorporating norleucine and Phe are substrates and exhibit L- or D-amino acid isomerization, but mutant peptides that contain residues with shorter, beta-branched or long side chains with polar terminal groups, viz. Ala, alpha-aminobutyric acid, Ile, Val, Leu, Lys, and Tyr, respectively, are not substrates. It was demonstrated that at least three N-terminal amino acid residues are absolutely essential for L-to-D-isomerization; furthermore, the third amino acid must be a Phe residue. None of the hexapeptides based on LLH, the first three residues of OvCNP, were substrates. A consistent 2-base mechanism is proposed for the isomerization; abstraction of a proton by 1 base is concomitant with delivery of a proton by the conjugate acid of a second base.

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

    PubMed

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

    2015-09-01

    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.

  4. Improved xylose fermentation of Kluyveromyces marxianus at elevated temperature through construction of a xylose isomerase pathway.

    PubMed

    Wang, Rongliang; Li, Lulu; Zhang, Biao; Gao, Xiaolian; Wang, Dongmei; Hong, Jiong

    2013-08-01

    To improve the xylose fermentation ability of Kluyveromyces marxianus, a xylose assimilation pathway through xylose isomerase was constructed. The genes encoding xylose reductase (KmXyl1) and xylitol dehydrogenase (KmXyl2) were disrupted in K. marxianus YHJ010 and the resultant strain was named YRL002. A codon-optimized xylose isomerase gene from Orpinomyces was transformed into K. marxianus YRL002 and expressed under GAPDH promoter. The transformant was adapted in the SD medium containing 1 % casamino acid with 2 % xylose as sole carbon source. After 32 times of trans-inoculation, a strain named YRL005, which can grow at a specific growth rate of 0.137/h with xylose as carbon source, was obtained. K. marxianus YRL005 could ferment 30.15 g/l of xylose and produce 11.52 g/l ethanol with a yield of 0.38 g/g, production rate of 0.069 g/l/h at 42 °C, and also could ferment 16.60 g/l xylose to produce 5.21 g/l ethanol with a yield of 0.31 g/g, and production rate of 0.054 g/l h at 45 °C. Co-fermentation with 2 % glucose could not improve the amount and yield of ethanol fermented from xylose obviously, but it could improve the production rate. Furthermore, K. marxianus YRL005 can ferment with the corn cob hydrolysate, which contained 20.04 g/l xylose to produce 8.25 g/l ethanol. It is a good platform to construct thermo-tolerant xylose fermentation yeast.

  5. FrnE, a Cadmium-Inducible Protein in Deinococcus radiodurans, Is Characterized as a Disulfide Isomerase Chaperone In Vitro and for Its Role in Oxidative Stress Tolerance In Vivo

    PubMed Central

    Khairnar, Nivedita P.; Joe, Min-Ho; Misra, H. S.; Lim, Sang-Yong

    2013-01-01

    Deinococcus radiodurans R1 exposed to a lethal dose of cadmium shows differential expression of a large number of genes, including frnE (drfrnE) and some of those involved in DNA repair and oxidative stress tolerance. The drfrnE::nptII mutant of D. radiodurans showed growth similar to that of the wild type, but its tolerance to 10 mM cadmium and 10 mM diamide decreased by ∼15- and ∼3-fold, respectively. These cells also showed nearly 6 times less resistance to gamma radiation at 12 kGy and ∼2-fold-higher sensitivity to 40 mM hydrogen peroxide than the wild type. In trans expression of drFrnE increased cytotoxicity of dithiothreitol (DTT) in the dsbA mutant of Escherichia coli. Recombinant drFrnE showed disulfide isomerase activity and could maintain insulin in its reduced form in the presence of DTT. While an equimolar ratio of wild-type protein could protect malate dehydrogenase completely from thermal denaturation at 42°C, the C22S mutant of drFrnE provided reduced protection to malate dehydrogenase from thermal inactivation. These results suggested that drFrnE is a protein disulfide isomerase in vitro and has a role in oxidative stress tolerance of D. radiodurans possibly by protecting the damaged cellular proteins from inactivation. PMID:23603741

  6. Electrostatic contributions to binding of transition state analogues can be very different from the corresponding contributions to catalysis: phenolates binding to the oxyanion hole of ketosteroid isomerase.

    PubMed

    Warshel, Arieh; Sharma, Pankaz K; Chu, Zhen T; Aqvist, Johan

    2007-02-13

    The relationship between binding of transition state analogues (TSAs) and catalysis is an open problem. A recent study of the binding of phenolate TSAs to ketosteroid isomerase (KSI) found a small change in the binding energy with a change in charge delocalization of the TSAs. This has been taken as proof that electrostatic effects do not contribute in a major way to catalysis. Here we reanalyze the relationship between the binding of the TSAs and the chemical catalysis by KSI as well as the binding of the transition state (TS), by computer simulation approaches. Since the simulations reproduce the relevant experimental results, they can be used to quantify the different contributions to the observed effects. It is found that the binding of the TSA and the chemical catalysis represent different thermodynamic cycles with very different electrostatic contributions. While the binding of the TSA involves a small electrostatic contribution, the chemical catalysis involves a charge transfer process and a major electrostatic contribution due to the preorganization of the active site. Furthermore, it is found that the electrostatic preorganization contributions to the binding of the enolate intermediate of KSI and the TS are much larger than the corresponding effect for the binding of the TSAs. This reflects the dependence of the preorganization on the orientation of the nonpolar form of the TSAs relative to the oxyanion hole. It seems to us that this work provides an excellent example of the need for computational studies in analyzing key experimental findings about enzyme catalysis.

  7. Analysis of the relationship between Chalcone Isomerase gene expression level and rutin production in Ficus deltoidea var. deltoidea and F. deltoidea var. angustifolia

    NASA Astrophysics Data System (ADS)

    Najid, Najihah Mohd; Zain, Che Radziah Che Mohd; Zainal, Zamri

    2016-11-01

    Ficus deltoidea (moraceae) is a herbal plant with medicinal values. Previous studies reported that the F. deltoidea contains a high level of bioactive compounds such as flavonoids. A cDNA encodes for chalcone isomerase was identified from F. deltoidea, designated as FdCHI, which involved in the isomerization of naringenin chalcone to naringenin. Naringenin is a key branch point for the synthesis of rutin, which is believed involved