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Sample records for 5-enolpyruvylshikimate 3-phosphate synthase

  1. Crystal structure of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase from the ESKAPE pathogen Acinetobacter baumannii.

    PubMed

    Sutton, Kristin A; Breen, Jennifer; Russo, Thomas A; Schultz, L Wayne; Umland, Timothy C

    2016-03-01

    The enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the sixth step of the seven-step shikimate pathway. Chorismate, the product of the pathway, is a precursor for the biosynthesis of aromatic amino acids, siderophores and metabolites such as folate, ubiquinone and vitamin K. The shikimate pathway is present in bacteria, fungi, algae, plants and apicomplexan parasites, but is absent in humans. The EPSP synthase enzyme produces 5-enolpyruvylshikimate 3-phosphate and phosphate from phosphoenolpyruvate and shikimate 3-phosphate via a transferase reaction, and is the target of the herbicide glyphosate. The Acinetobacter baumannii gene encoding EPSP synthase, aroA, has previously been demonstrated to be essential during host infection for the growth and survival of this clinically important drug-resistant ESKAPE pathogen. Prephenate dehydrogenase is also encoded by the bifunctional A. baumannii aroA gene, but its activity is dependent upon EPSP synthase since it operates downstream of the shikimate pathway. As part of an effort to evaluate new antimicrobial targets, recombinant A. baumannii EPSP (AbEPSP) synthase, comprising residues Ala301-Gln756 of the aroA gene product, was overexpressed in Escherichia coli, purified and crystallized. The crystal structure, determined to 2.37 Å resolution, is described in the context of a potential antimicrobial target and in comparison to EPSP synthases that are resistant or sensitive to the herbicide glyphosate. PMID:26919521

  2. Characterization of Streptococcus pneumoniae 5-enolpyruvylshikimate 3-phosphate synthase and its activation by univalent cations.

    PubMed

    Du, W; Wallis, N G; Mazzulla, M J; Chalker, A F; Zhang, L; Liu, W S; Kallender, H; Payne, D J

    2000-01-01

    The aroA gene (Escherichia coli nomenclature) encoding 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase from the gram-positive pathogen Streptococcus pneumoniae has been identified, cloned and overexpressed in E. coli, and the enzyme purified to homogeneity. It was shown to catalyze a reversible conversion of shikimate 3-phosphate (S3P) and phosphoenolpyruvate (PEP) to EPSP and inorganic phosphate. Activation by univalent cations was observed in the forward reaction, with NH+4, Rb+ and K+ exerting the greatest effects. Km(PEP) was lowered by increasing [NH+4] and [K+], whereas Km(S3P) rose with increasing [K+], but fell with increasing [NH+4]. Increasing [NH+4] and [K+] resulted in an overall increase in kcat. Glyphosate (GLP) was found to be a competitive inhibitor with PEP, but the potency of inhibition was profoundly affected by [NH+4] and [K+]. For example, increasing [NH+4] and [K+] reduced Ki(GLP versus PEP) up to 600-fold. In the reverse reaction, the enzyme catalysis was less sensitive to univalent cations. Our analysis included univalent cation concentrations comparable with those found in bacterial cells. Therefore, the observed effects of these metal ions are more likely to reflect the physiological behavior of EPSP synthase and also add to our understanding of how to inhibit this enzyme in the host organism. As there is a much evidence to suggest that EPSP synthase is essential for bacterial survival, its discovery in the serious gram-positive pathogen S. pneumoniae and its inhibition by GLP indicate its potential as a broad-spectrum antibacterial target. PMID:10601870

  3. A novel 5-enolpyruvylshikimate-3-phosphate synthase from Rahnella aquatilis with significantly reduced glyphosate sensitivity.

    PubMed

    Peng, Ri-He; Tian, Yong-Sheng; Xiong, Ai-Sheng; Zhao, Wei; Fu, Xiao-Yan; Han, Hong-Juan; Chen, Chen; Jin, Xiao-Fen; Yao, Quan-Hong

    2012-01-01

    The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19) is a key enzyme in the shikimate pathway for the production of aromatic amino acids and chorismate-derived secondary metabolites in plants, fungi, and microorganisms. It is also the target of the broad-spectrum herbicide glyphosate. Natural glyphosate resistance is generally thought to occur within microorganisms in a strong selective pressure condition. Rahnella aquatilis strain GR20, an antagonist against pathogenic agrobacterial strains of grape crown gall, was isolated from the rhizosphere of grape in glyphosate-contaminated vineyards. A novel gene encoding EPSPS was identified from the isolated bacterium by complementation of an Escherichia coli auxotrophic aroA mutant. The EPSPS, named AroA(R. aquatilis), was expressed and purified from E. coli, and key kinetic values were determined. The full-length enzyme exhibited higher tolerance to glyphosate than the E. coli EPSPS (AroA(E. coli)), while retaining high affinity for the substrate phosphoenolpyruvate. Transgenic plants of AroA(R. aquatilis) were also observed to be more resistant to glyphosate at a concentration of 5 mM than that of AroA(E. coli). To probe the sites contributing to increased tolerance to glyphosate, mutant R. aquatilis EPSPS enzymes were produced with the c-strand of subdomain 3 and the f-strand of subdomain 5 (Thr38Lys, Arg40Val, Arg222Gln, Ser224Val, Ile225Val, and Gln226Lys) substituted by the corresponding region of the E. coli EPSPS. The mutant enzyme exhibited greater sensitivity to glyphosate than the wild type R. aquatilis EPSPS with little change of affinity for its first substrate, shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP). The effect of the residues on subdomain 5 on glyphosate resistance was more obvious. PMID:22870190

  4. In silico peptide prediction for antibody generation to recognize 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in genetically modified organisms.

    PubMed

    Marani, Mariela M; Costa, Joana; Mafra, Isabel; Oliveira, Maria Beatriz P P; Camperi, Silvia A; Leite, José Roberto de Souza Almeida

    2015-03-01

    For the prospective immunorecognition of 5-enolpyruvylshikimate-3-phosphate synthase (CP4-EPSPS) as a biomarker protein expressed by transgenic soybean, an extensive in silico evaluation of the referred protein was performed. The main objective of this study was the selection of a set of peptides that could function as potential immunogens for the production of novel antibodies against CP4-EPSPS protein. For this purpose, the protein was in silico cleaved with trypsin/chymotrypsin and the resultant peptides were extensively analyzed for further selection of the best candidates for antibody production. The analysis enabled the successful proposal of four peptides with potential immunogenicity for their future use as screening biomarkers of genetically modified organisms. To our knowledge, this is the first attempt to select and define potential linear epitopes for the immunization of animals and, subsequently, to generate adequate antibodies for CP4-EPSPS recognition. The present work will be followed by the synthesis of the candidate peptides to be incubated in animals for antibody generation and potential applicability for the development of an immunosensor for CP4-EPSPS detection. PMID:25620523

  5. A Novel 5-Enolpyruvylshikimate-3-Phosphate Synthase Shows High Glyphosate Tolerance in Escherichia coli and Tobacco Plants

    PubMed Central

    Zhang, Shengxue; Yang, Xuewen; Chen, Rongrong; Zhang, Yuwen; Lu, Wei; Liu, Yan; Wang, Jianhua; Lin, Min; Wang, Guoying

    2012-01-01

    A key enzyme in the shikimate pathway, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is the primary target of the broad-spectrum herbicide glyphosate. Identification of new aroA genes coding for EPSPS with a high level of glyphosate tolerance is essential for the development of glyphosate-tolerant crops. In the present study, the glyphosate tolerance of five bacterial aroA genes was evaluated in the E. coli aroA-defective strain ER2799 and in transgenic tobacco plants. All five aroA genes could complement the aroA-defective strain ER2799, and AM79 aroA showed the highest glyphosate tolerance. Although glyphosate treatment inhibited the growth of both WT and transgenic tobacco plants, transgenic plants expressing AM79 aroA tolerated higher concentration of glyphosate and had a higher fresh weight and survival rate than plants expressing other aroA genes. When treated with high concentration of glyphosate, lower shikimate content was detected in the leaves of transgenic plants expressing AM79 aroA than transgenic plants expressing other aroA genes. These results suggest that AM79 aroA could be a good candidate for the development of transgenic glyphosate-tolerant crops. PMID:22715408

  6. Translocation of the precursor of 5-enolpyruvylshikimate-3-phosphate synthase into chloroplasts of higher plants in vitro

    PubMed Central

    Della-Cioppa, Guy; Bauer, S. Christopher; Klein, Barbara K.; Shah, Dilip M.; Fraley, Robert T.; Kishore, Ganesh M.

    1986-01-01

    5-enolPyruvylshikimate-3-phosphate synthase (EPSP synthase; 3-phosphoshikimate 1-carboxyvinyl-transferase; EC 2.5.1.19) is a chloroplast-localized enzyme of the shikimate pathway in plants. This enzyme is the target for the nonselective herbicide glyphosate (N-phosphonomethylglycine). We have previously isolated a full-length cDNA clone of EPSP synthase from Petunia hybrida. DNA sequence analysis suggested that the enzyme is synthesized as a cytosolic precursor (pre-EPSP synthase) with an amino-terminal transit peptide. Based on the known amino terminus of the mature enzyme, and the 5′ open reading frame of the cDNA, the transit peptide of pre-EPSP synthase would be maximally 72 amino acids long. To confirm this prediction and to assay directly for translocation of pre-EPSP synthase into chloroplasts in vitro, we cloned the full-length cDNA into an SP6 transcription system to produce large amounts of mRNA for in vitro translation. The translation products, when analyzed by NaDodSO4/PAGE autoradiography, indicate a relative molecular mass for pre-EPSP synthase of ≈55 kDa. Uptake studies with intact chloroplasts, in vitro, indicate that pre-EPSP synthase was rapidly taken up into chloroplasts and proteolytically cleaved to the mature ≈48-kDa enzyme. The transit peptide was shown to be essential for import of the precursor enzyme into the chloroplast. To our knowledge, post-translational import into chloroplasts of a precursor enzyme involved in amino acid biosynthesis has not been reported previously. Furthermore, enzymatic analysis of translation products indicates that pre-EPSP synthase is catalytically active and has a similar sensitivity to the herbicide glyphosate as the mature enzyme. To our knowledge, pre-EPSP synthase represents the only example of a catalytically competent chloroplast-precursor enzyme. Images PMID:16593759

  7. Evolution of a Double Amino Acid Substitution in the 5-Enolpyruvylshikimate-3-Phosphate Synthase in Eleusine indica Conferring High-Level Glyphosate Resistance1

    PubMed Central

    Yu, Qin; Jalaludin, Adam; Han, Heping; Chen, Ming; Sammons, R. Douglas; Powles, Stephen B.

    2015-01-01

    Glyphosate is the most important and widely used herbicide in world agriculture. Intensive glyphosate selection has resulted in the widespread evolution of glyphosate-resistant weed populations, threatening the sustainability of this valuable once-in-a-century agrochemical. Field-evolved glyphosate resistance due to known resistance mechanisms is generally low to modest. Here, working with a highly glyphosate-resistant Eleusine indica population, we identified a double amino acid substitution (T102I + P106S [TIPS]) in the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene in glyphosate-resistant individuals. This TIPS mutation recreates the biotechnology-engineered commercial first generation glyphosate-tolerant EPSPS in corn (Zea mays) and now in other crops. In E. indica, the naturally evolved TIPS mutants are highly (more than 180-fold) resistant to glyphosate compared with the wild type and more resistant (more than 32-fold) than the previously known P106S mutants. The E. indica TIPS EPSPS showed very high-level (2,647-fold) in vitro resistance to glyphosate relative to the wild type and is more resistant (600-fold) than the P106S variant. The evolution of the TIPS mutation in crop fields under glyphosate selection is likely a sequential event, with the P106S mutation being selected first and fixed, followed by the T102I mutation to create the highly resistant TIPS EPSPS. The sequential evolution of the TIPS mutation endowing high-level glyphosate resistance is an important mechanism by which plants adapt to intense herbicide selection and a dramatic example of evolution in action. PMID:25717039

  8. Glyphosate inhibition of 5-enolpyruvylshikimate 3-phosphate synthease from suspension-cultured cells of Nicotiana silvestris

    SciTech Connect

    Rubin, J.L.; Gaines, C.G.; Jensen, R.A.

    1984-07-01

    Treatment of isogenic suspension-cultured cells of Nicotiana silvestris Speg, et Comes with glyphosate (N-(phosphonomethyl)glycine) led to elevated levels of intracellular shikimate (364-fold increase by 1.0 millimolar glyphosate). In the presence of glyphosate, it is likely that most molecules of shikimate originate from the action of 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase-Mn since this isozyme, in contrast to the DAHP synthase-Co isozyme, is insensitive to inhibition by glyphosate. 5-Enolpyruvylshikimate 3-phosphate (EPSP) synthase (EC 2.5.1.19) from N. silvestris was sensitive to micromolar concentrations of glyphosate and possessed a single inhibitor binding site. Rigorous kinetic studies of EPSP synthase required resolution from the multiple phosphatase activities present in crude extracts, a result achieved by ion-exchange column chromatography. Although EPSP synthase exhibited a broad pH profile (50% of maximal activity between pH 6.2 and 8.5), sensitivity to glyphosate increased dramatically with increasing pH within this range. In accordance with these data and the pK/sub a/ values of glyphosate, it is likely that the ionic form of glyphosate inhibiting EPSP synthase is COO/sup -/CH/sub 2/NH/sub 2//sup +/CH/sub 2/PO/sub 3//sup 2 -/, and that a completely ionized phosphono group is essential for inhibition. At pH 7.0, inhibition was competitive with respect to phosphoenolpyruvate (K/sub i/ = 1.25 micromolar) and uncompetitive with respect to shikimate-3-P (K/sub i/ = 18.3 micromolar). All data were consistent with a mechanism of inhibition in which glyphosate competes with PEP for binding to an (enzyme:shikimate-3-P) complex and ultimately forms the dead-end complex of (enzyme:shikimate-3-P:glyphosate). 36 references, 8 figures, 1 table.

  9. Distinguishing between weedy Amaranthus species based on intron one sequences from the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS)gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Hybridization between Amaranthus species and the potential for herbicide resistance to be transferred by hybridization are of growing concern in the weed science community. It is important to confirm suspect hybrid populations early to develop an effective control strategy. With this in mind, a PC...

  10. A Novel Naturally Occurring Class I 5-Enolpyruvylshikimate-3-Phosphate Synthase from Janibacter sp. Confers High Glyphosate Tolerance to Rice

    PubMed Central

    Yi, Shu-yuan; Cui, Ying; Zhao, Yan; Liu, Zi-duo; Lin, Yong-jun; Zhou, Fei

    2016-01-01

    As glyphosate is a broad spectrum herbicide extensively used in agriculture worldwide, identification of new aroA genes with high level of glyphosate tolerance is essential for the development and breeding of transgenic glyphosate-tolerant crops. In this study, an aroA gene was cloned from a Janibacter sp. strain isolated from marine sediment (designated as aroAJ. sp). The purified aroAJ. sp enzyme has a Km value of 30 μM for PEP and 83 μM for S3P, and a significantly higher Ki value for glyphosate (373 μM) than aroAE. coli. AroAJ. sp is characterized as a novel and naturally occurring class I aroA enzyme with glyphosate tolerance. Furthermore, we show that aroAJ. sp can be used as an effective selectable marker in both japonica and indica rice cultivar. Transgenic rice lines were tested by herbicide bioassay and it was confirmed that they could tolerate up to 3360 g/ha glyphosate, a dosage four-fold that of the recommended agricultural application level. To our knowledge, it is the first report of a naturally occurring novel class I aroA gene which can be efficiently utilized to study and develop transgenic glyphosate-tolerant crops, and can facilitate a more economical and simplified weed control system. PMID:26754957

  11. Glyphosate-Resistant Goosegrass. Identification of a Mutation in the Target Enzyme 5-enolpyruvylshikimate-3-phosphate Synthase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The spontaneous occurrence of resistance to the herbicide glyphosate in weed species has been an extremely infrequent event, despite over 20 years of extensive use. Recently, a glyphosate-resistant biotype of goosegrass (Eleusine indica) was identified in Malaysia exhibiting an LD50 value approxima...

  12. Characterization and site-directed mutagenesis of a novel class II 5-enopyruvylshikimate-3-phosphate (EPSP) synthase from the deep-sea bacterium Alcanivorax sp. L27.

    PubMed

    Zhang, Yi; Yi, Licong; Lin, Yongjun; Zhang, Lili; Shao, Zongze; Liu, Ziduo

    2014-09-01

    The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is a key enzyme in the aromatic amino acid biosynthetic pathway in microorganisms and plants, which catalyzes the formation of 5-enolpyruvylshikimate-3-phosphate (EPSP) from shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP). In this study, a novel AroA-encoding gene was identified from the deep sea bacterium Alcanivorax sp. L27 through screening the genomic library and termed as AroAA.sp. A phylogenetic analysis revealed that AroAA.sp (1317 bp and 438 amino acids) is a class II AroA. This enzyme exhibited considerable activity between pH 5.5 and pH 8.0 and notable activity at low temperatures. The KM for PEP and IC50 [glyphosate] values (the concentration of glyphosate that inhibited enzyme activity by 50%) of AroAA.sp were 78 μM and 1.5 mM, respectively. Furthermore, site-directed mutagenesis revealed that the G100A mutant had a 30-fold increase in the IC50 [glyphosate] value; while the L105P mutant showed only 20% catalytic activity compared to wild-type AroAA.sp. The specific activity of the wild-type AroAA.sp, the G100A mutant and the L105P mutant were 7.78 U/mg, 7.26 U/mg and 1.76 U/mg, respectively. This is the first report showing that the G100A mutant of AroA displays considerably improved glyphosate resistance and demonstrates that Leu105 is essential for the enzyme's activity. PMID:25039062

  13. Molecular and phylogenetic characterization of the homoeologous EPSP Synthase genes of allohexaploid wheat, Triticum aestivum (L.)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: 5-Enolpyruvylshikimate-3-phosphate synthase (EPSPS) is the sixth and penultimate enzyme in the shikimate biosynthesis pathway. The EPSPS genes of allohexaploid wheat (Triticum aestivum, AABBDD) have not been well characterized. Herein, the three homoeologous copies of the wheat EPSPS gen...

  14. 40 CFR 174.523 - CP4 Enolpyruvylshikimate-3-phosphate (CP4 EPSPS) synthase in all plants; exemption from the...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... PROCEDURES AND REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.523... requirement of a tolerance. Residues of the CP4 Enolpyruvylshikimate-3-phosphate (CP4 EPSPS) synthase enzyme in all plants are exempt from the requirement of a tolerance when used as...

  15. 40 CFR 174.523 - CP4 Enolpyruvylshikimate-3-phosphate (CP4 EPSPS) synthase in all plants; exemption from the...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... PROCEDURES AND REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.523... requirement of a tolerance. Residues of the CP4 Enolpyruvylshikimate-3-phosphate (CP4 EPSPS) synthase enzyme in all plants are exempt from the requirement of a tolerance when used as...

  16. 40 CFR 174.523 - CP4 Enolpyruvylshikimate-3-phosphate (CP4 EPSPS) synthase in all plants; exemption from the...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... PROCEDURES AND REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.523... requirement of a tolerance. Residues of the CP4 Enolpyruvylshikimate-3-phosphate (CP4 EPSPS) synthase enzyme in all plants are exempt from the requirement of a tolerance when used as...

  17. 40 CFR 174.523 - CP4 Enolpyruvylshikimate-3-phosphate (CP4 EPSPS) synthase in all plants; exemption from the...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... PROCEDURES AND REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.523... requirement of a tolerance. Residues of the CP4 Enolpyruvylshikimate-3-phosphate (CP4 EPSPS) synthase enzyme in all plants are exempt from the requirement of a tolerance when used as...

  18. 40 CFR 174.523 - CP4 Enolpyruvylshikimate-3-phosphate (CP4 EPSPS) synthase in all plants; exemption from the...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... PROCEDURES AND REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.523... requirement of a tolerance. Residues of the CP4 Enolpyruvylshikimate-3-phosphate (CP4 EPSPS) synthase enzyme in all plants are exempt from the requirement of a tolerance when used as...

  19. The Inositol-3-Phosphate Synthase Biosynthetic Enzyme Has Distinct Catalytic and Metabolic Roles

    PubMed Central

    Frej, Anna D.; Clark, Jonathan; Le Roy, Caroline I.; Lilla, Sergio; Thomason, Peter A.; Otto, Grant P.; Churchill, Grant; Insall, Robert H.; Claus, Sandrine P.; Hawkins, Phillip; Stephens, Len

    2016-01-01

    Inositol levels, maintained by the biosynthetic enzyme inositol-3-phosphate synthase (Ino1), are altered in a range of disorders, including bipolar disorder and Alzheimer's disease. To date, most inositol studies have focused on the molecular and cellular effects of inositol depletion without considering Ino1 levels. Here we employ a simple eukaryote, Dictyostelium discoideum, to demonstrate distinct effects of loss of Ino1 and inositol depletion. We show that loss of Ino1 results in an inositol auxotrophy that can be rescued only partially by exogenous inositol. Removal of inositol supplementation from the ino1− mutant resulted in a rapid 56% reduction in inositol levels, triggering the induction of autophagy, reduced cytokinesis, and substrate adhesion. Inositol depletion also caused a dramatic generalized decrease in phosphoinositide levels that was rescued by inositol supplementation. However, loss of Ino1 triggered broad metabolic changes consistent with the induction of a catabolic state that was not rescued by inositol supplementation. These data suggest a metabolic role for Ino1 that is independent of inositol biosynthesis. To characterize this role, an Ino1 binding partner containing SEL1L1 domains (Q54IX5) and having homology to mammalian macromolecular complex adaptor proteins was identified. Our findings therefore identify a new role for Ino1, independent of inositol biosynthesis, with broad effects on cell metabolism. PMID:26951199

  20. The Inositol-3-Phosphate Synthase Biosynthetic Enzyme Has Distinct Catalytic and Metabolic Roles.

    PubMed

    Frej, Anna D; Clark, Jonathan; Le Roy, Caroline I; Lilla, Sergio; Thomason, Peter A; Otto, Grant P; Churchill, Grant; Insall, Robert H; Claus, Sandrine P; Hawkins, Phillip; Stephens, Len; Williams, Robin S B

    2016-05-15

    Inositol levels, maintained by the biosynthetic enzyme inositol-3-phosphate synthase (Ino1), are altered in a range of disorders, including bipolar disorder and Alzheimer's disease. To date, most inositol studies have focused on the molecular and cellular effects of inositol depletion without considering Ino1 levels. Here we employ a simple eukaryote, Dictyostelium discoideum, to demonstrate distinct effects of loss of Ino1 and inositol depletion. We show that loss of Ino1 results in an inositol auxotrophy that can be rescued only partially by exogenous inositol. Removal of inositol supplementation from the ino1(-) mutant resulted in a rapid 56% reduction in inositol levels, triggering the induction of autophagy, reduced cytokinesis, and substrate adhesion. Inositol depletion also caused a dramatic generalized decrease in phosphoinositide levels that was rescued by inositol supplementation. However, loss of Ino1 triggered broad metabolic changes consistent with the induction of a catabolic state that was not rescued by inositol supplementation. These data suggest a metabolic role for Ino1 that is independent of inositol biosynthesis. To characterize this role, an Ino1 binding partner containing SEL1L1 domains (Q54IX5) and having homology to mammalian macromolecular complex adaptor proteins was identified. Our findings therefore identify a new role for Ino1, independent of inositol biosynthesis, with broad effects on cell metabolism. PMID:26951199

  1. Comparison of DFT and ab initio QM/MM methods for modelling reaction in chorismate synthase

    NASA Astrophysics Data System (ADS)

    Lawan, Narin; Ranaghan, Kara E.; Manby, Frederick R.; Mulholland, Adrian J.

    2014-07-01

    Quantum mechanics/molecular mechanics (QM/MM) methods are a popular tool in the investigation of enzyme reactions. Here, we compare B3LYP density functional theory (DFT) and ab initio QM/MM methods for modelling the conversion of 5-enolpyruvylshikimate-3-phosphate to chorismate in chorismate synthase. Good agreement with experimental data is only obtained at the SCS-MP2/CHARMM27 level for a reaction mechanism in which phosphate elimination precedes proton transfer. B3LYP predicts reaction energetics that are qualitatively wrong, stressing the need for ab initio QM/MM methods, and caution in interpretation of DFT results for this enzyme.

  2. An Unusual Cation-Binding Site and Distinct Domain-Domain Interactions Distinguish Class II Enolpyruvylshikimate-3-phosphate Synthases.

    PubMed

    Light, Samuel H; Krishna, Sankar N; Minasov, George; Anderson, Wayne F

    2016-03-01

    Enolpyruvylshikimate-3-phosphate synthase (EPSPS) catalyzes a critical step in the biosynthesis of a number of aromatic metabolites. An essential prokaryotic enzyme and the molecular target of the herbicide glyphosate, EPSPSs are the subject of both pharmaceutical and commercial interest. Two EPSPS classes that exhibit low sequence homology, differing substrate/glyphosate affinities, and distinct cation activation properties have previously been described. Here, we report structural studies of the monovalent cation-binding class II Coxiella burnetii EPSPS (cbEPSPS). Three cbEPSPS crystal structures reveal that the enzyme undergoes substantial conformational changes that alter the electrostatic potential of the active site. A complex with shikimate-3-phosphate, inorganic phosphate (Pi), and K(+) reveals that ligand induced domain closure produces an unusual cation-binding site bordered on three sides by the N-terminal domain, C-terminal domain, and the product Pi. A crystal structure of the class I Vibrio cholerae EPSPS (vcEPSPS) clarifies the basis of differential class I and class II cation responsiveness, showing that in class I EPSPSs a lysine side chain occupies the would-be cation-binding site. Finally, we identify distinct patterns of sequence conservation at the domain-domain interface and propose that the two EPSPS classes have evolved to differently optimize domain opening-closing dynamics. PMID:26813771

  3. Steady-state kinetic evaluation of the reverse reaction for Escherichia coli 5-enolpyruvoylshikimate-3-phosphate synthase.

    PubMed

    Gruys, K J; Marzabadi, M R; Pansegrau, P D; Sikorski, J A

    1993-08-01

    Recently it has been found that the kinetic mechanism for Escherichia coli 5-enolpyruvoylshikimate-3-phosphate synthase (EPSPS) in the forward direction is random with synergistic binding of substrates and inhibitors (K. J. Gruys, M. C. Walker, and J. A. Sikorski, 1992, Biochemistry 31, 5534). This work, however, did not address the reverse reaction with 5-enolpyruvoylshikimate-3-phosphate (EPSP) and phosphate (Pi) as substrates where a similar question of random versus ordered addition of substrates remained. Previous transient-state kinetic results led to a proposal for an equilibrium-ordered mechanism, where binding of EPSP occurs first followed by Pi (K. S. Anderson, and K. A. Johnson, 1990, Chem. Rev. 90, 1131). Steady-state kinetic results of the reverse reaction presented here suggest that, like the forward reaction, addition of substrates occurs randomly. Initial velocity studies with EPSP and Pi show a normal intersecting pattern in the reciprocal plots, consistent with a random or steady-state-ordered mechanism, but not with equilibrium-ordered addition of substrates. Inhibition of the EPSPS reverse reaction by 5-amino-S3P or the S3P-glyphosate hybrid molecule gave the expected competitive patterns versus EPSP, but mixed noncompetitive patterns versus Pi. These results also disfavor an equilibrium-ordered model, but again are consistent with a random or steady-state-ordered mechanism. A more quantitative mechanistic analysis of the inhibition data to determine the true rather than apparent Ki values provides evidence for a random over a steady-state-ordered addition of substrates. These results in combination with previous findings lead to the conclusion that the mechanism is random addition of EPSP and Pi since it is the only possible model for substrate addition that is consistent with the cumulative data from all kinetic (transient- as well as steady-state) and direct binding studies. PMID:8346911

  4. New EPSP synthase inhibitors: synthesis and evaluation of an aromatic tetrahedral intermediate mimic containing a 3-malonate ether as a 3-phosphate surrogate.

    PubMed

    Miller, M J; Cleary, D G; Ream, J E; Snyder, K R; Sikorski, J A

    1995-12-01

    A new analog of the EPSP synthase enzyme reaction intermediate 1, containing a 3-malonate ether moiety in place of the usual 3-phosphate group, was synthesized from 3,5-dihydroxybenzoic acid. This simple, synthetically accessible aromatic compound (5) is an effective competitive inhibitor versus S3P with an apparent Ki of 1.3 +/- 0.22 microM. This result demonstrates that a simple benzene ring can be a suitable achiral substitute for the more complex shikimate ring in the design of EPSP synthase inhibitors. Furthermore, the greater potency of 5 versus the phenol 6, glycolate 7 and the gallic acid analog 8 demonstrates the requirement for multiple anionic charges at the dihydroxybenzoate 5-position in order to attain effective inhibition of this enzyme. However, this 3-malonate ether substituted compound was at least 10-fold less effective as a bisubstrate inhibitor than the corresponding 3-phosphate. This suggests that tetrahedral intermediate mimics possessing a 3-malonate ether moiety are less effective than their corresponding 3-phosphates in accessing the optimal enzyme conformation stabilizing 1. PMID:8770393

  5. Safety evaluation of the double mutant 5-enol pyruvylshikimate-3-phosphate synthase (2mEPSPS) from maize that confers tolerance to glyphosate herbicide in transgenic plants.

    PubMed

    Herouet-Guicheney, Corinne; Rouquié, David; Freyssinet, Martine; Currier, Thomas; Martone, Aris; Zhou, Junguo; Bates, Elizabeth E M; Ferullo, Jean-Marc; Hendrickx, Koen; Rouan, Dominique

    2009-07-01

    Glyphosate tolerance can be conferred by decreasing the herbicide's ability to inhibit the enzyme 5-enol pyruvylshikimate-3-phosphate synthase, which is essential for the biosynthesis of aromatic amino acids in all plants, fungi, and bacteria. Glyphosate tolerance is based upon the expression of the double mutant 5-enol pyruvylshikimate-3-phosphate synthase (2mEPSPS) protein. The 2mEPSPS protein, with a lower binding affinity for glyphosate, is highly resistant to the inhibition by glyphosate and thus allows sufficient enzyme activity for the plants to grow in the presence of herbicides that contain glyphosate. Based on both a review of published literature and experimental studies, the potential safety concerns related to the transgenic 2mEPSPS protein were assessed. The safety evaluation supports that the expressed protein is innocuous. The 2mEPSPS enzyme does not possess any of the properties associated with known toxins or allergens, including a lack of amino acid sequence similarity to known toxins and allergens, a rapid degradation in simulated gastric and intestinal fluids, and no adverse effects in mice after intravenous or oral administration (at 10 or 2000 mg/kg body weight, respectively). In conclusion, there is a reasonable certainty of no harm resulting from the inclusion of the 2mEPSPS protein in human food or in animal feed. PMID:19303906

  6. Divergent properties and phylogeny of cyanobacterial 5-enol-pyruvyl-shikimate-3-phosphate synthases: evidence for horizontal gene transfer in the Nostocales.

    PubMed

    Forlani, Giuseppe; Bertazzini, Michele; Barillaro, Donatella; Rippka, Rosmarie

    2015-01-01

    As it represents the target of the successful herbicide glyphosate, great attention has been paid to the shikimate pathway enzyme 5-enol-pyruvyl-shikimate-3-phosphate (EPSP) synthase. However, inconsistent results have been reported concerning the sensitivity of the enzyme from cyanobacteria, and consequent inhibitory effects on cyanobacterial growth. The properties of EPSP synthase were investigated in a set of 42 strains representative of the large morphological diversity of these prokaryotes. Publicly available protein sequences were analyzed, and related to enzymatic features. In most cases, the native protein showed an unusual homodimeric composition and a general sensitivity to micromolar doses of glyphosate. By contrast, eight out of 15 Nostocales strains were found to possess a monomeric EPSP synthase, whose activity was inhibited only at concentrations exceeding 1 mM. Sequence analysis showed that these two forms are only distantly related, the latter clustering separately in a clade composed of diverse bacterial phyla. The results are consistent with the occurrence of a horizontal gene transfer event involving an evolutionarily distant organism. Moreover, data suggest that the existence of class I (glyphosate-sensitive) and class II (glyphosate-tolerant) EPSP synthases representing two distinct phylogenetic clades is an oversimplification because of the limited number of analyzed samples. PMID:25229999

  7. A novel 5-enolpyruvoylshikimate-3-phosphate (EPSP) synthase transgene for glyphosate resistance stimulates growth and fecundity in weedy rice (Oryza sativa) without herbicide

    PubMed Central

    Wang, Wei; Xia, Hui; Yang, Xiao; Xu, Ting; Si, Hong Jiang; Cai, Xing Xing; Wang, Feng; Su, Jun; Snow, Allison A; Lu, Bao-Rong

    2014-01-01

    Understanding evolutionary interactions among crops and weeds can facilitate effective weed management. For example, gene flow from crops to their wild or weedy relatives can lead to rapid evolution in recipient populations. In rice (Oryza sativa), transgenic herbicide resistance is expected to spread to conspecific weedy rice (Oryza sativa f. spontanea) via hybridization. Here, we studied fitness effects of transgenic over-expression of a native 5-enolpyruvoylshikimate-3-phosphate synthase (epsps) gene developed to confer glyphosate resistance in rice. Controlling for genetic background, we examined physiological traits and field performance of crop–weed hybrid lineages that segregated for the presence or absence of this novel epsps transgene. Surprisingly, we found that transgenic F2 crop–weed hybrids produced 48–125% more seeds per plant than nontransgenic controls in monoculture- and mixed-planting designs without glyphosate application. Transgenic plants also had greater EPSPS protein levels, tryptophan concentrations, photosynthetic rates, and per cent seed germination compared with nontransgenic controls. Our findings suggest that over-expression of a native rice epsps gene can lead to fitness advantages, even without exposure to glyphosate. We hypothesize that over-expressed epsps may be useful to breeders and, if deployed, could result in fitness benefits in weedy relatives following transgene introgression. PMID:23905647

  8. A novel 5-enolpyruvoylshikimate-3-phosphate (EPSP) synthase transgene for glyphosate resistance stimulates growth and fecundity in weedy rice (Oryza sativa) without herbicide.

    PubMed

    Wang, Wei; Xia, Hui; Yang, Xiao; Xu, Ting; Si, Hong Jiang; Cai, Xing Xing; Wang, Feng; Su, Jun; Snow, Allison A; Lu, Bao-Rong

    2014-04-01

    Understanding evolutionary interactions among crops and weeds can facilitate effective weed management. For example, gene flow from crops to their wild or weedy relatives can lead to rapid evolution in recipient populations. In rice (Oryza sativa), transgenic herbicide resistance is expected to spread to conspecific weedy rice (Oryza sativa f. spontanea) via hybridization. Here, we studied fitness effects of transgenic over-expression of a native 5-enolpyruvoylshikimate-3-phosphate synthase (epsps) gene developed to confer glyphosate resistance in rice. Controlling for genetic background, we examined physiological traits and field performance of crop-weed hybrid lineages that segregated for the presence or absence of this novel epsps transgene. Surprisingly, we found that transgenic F2 crop-weed hybrids produced 48-125% more seeds per plant than nontransgenic controls in monoculture- and mixed-planting designs without glyphosate application. Transgenic plants also had greater EPSPS protein levels, tryptophan concentrations, photosynthetic rates, and per cent seed germination compared with nontransgenic controls. Our findings suggest that over-expression of a native rice epsps gene can lead to fitness advantages, even without exposure to glyphosate. We hypothesize that over-expressed epsps may be useful to breeders and, if deployed, could result in fitness benefits in weedy relatives following transgene introgression. PMID:23905647

  9. Phosphorus-31, sup 15 N, and sup 13 C NMR of glyphosate: Comparison of pH titrations to the herbicidal dead-end complex with 5-enolpyruvoylshikimate-3-phosphate synthase

    SciTech Connect

    Castellino, S.; Leo, G.C.; Sammons, R.D.; Sikorski, J.A. )

    1989-05-02

    The herbicidal dead-end ternary complex (E{sup S3P}{sub Glyph}) of glyphosate (N-(phosphonomethyl)glycine) with 5-enolpyruvoylshikimate-3-phosphate synthase (EPSPS) and the substrate shikimate 3-phosphate (S3P) has been characterized by {sup 31}P, {sup 15}N, and {sup 13}C NMR. The NMR spectra of EPSPS-bound glyphosate show unique chemical shifts ({delta}) for each of the three nuclei. By {sup 31}P NMR, glyphosate in the dead-end complex is a distinct species 3.5 ppm downfield from free glyphosate. The {sup 13}C signal of glyphosate in the dead-end complex is shifted 4 ppm downfield from that of free glyphosate. The {sup 15}N signal for glyphosate (99%) in the dead-end complex is 5 ppm further downfield than that of any free zwitterionic species and 10 ppm downfield from that of the average free species at pH 10.1. The structures of each ionic state of glyphosate are modeled with force field calculations by using MacroModel. A correlation is made for the {sup 31}P {delta} and the C-P-O bond angle, and the {sup 13}C and {sup 15}N {delta} values are postulated to be related to C-C-O and C-N-C bond angles, respectively. The downfield {sup 31}P chemical shift perturbation for S3P in the EPSPS binary complex is consistent with ionization of the 3-phosphate of S3P upon binding. Comparison with the S3P {sup 31}P {delta} vs pH titration curve specifies predominantly the dianion of the 3-phosphate in the E{sup S3P} binary complex, while the E{sup S3P}{sub Glyph} complex indicates net protonation at the 3-phosphate. Chemical shift perturbations of this latter type may be explained by changes in the O-P-O bond angle.

  10. Mechanism of the Enzymatic Synthesis of 4-(Hydroxymethyl)-2- furancarboxaldehyde-phosphate (4-HFC-P) from Glyceraldehyde-3-phosphate Catalyzed by 4-HFC-P Synthase.

    PubMed

    Wang, Yu; Jones, Michael K; Xu, Huimin; Ray, W Keith; White, Robert H

    2015-05-19

    A single enzyme, 4-(hydroxymethyl)-2-furancarboxaldehyde-phosphate synthase (MfnB), from the methanogen Methanocaldococcus jannaschii catalyzed at least 10 separate chemical reactions in converting two molecules of glyceraldehyde-3-P (GA-3-P) to 4-(hydroxymethyl)-2-furancarboxaldehyde-P (4-HFC-P), the first discrete intermediate in the biosynthetic pathway to the furan moiety of the coenzyme methanofuran. Here we describe the biochemical characterization of the recombinantly expressed MfnB to understand its catalytic mechanism. Site-directed mutagenesis showed that the strictly conserved residues (Asp25, Lys27, Lys85, and Asp151) around the active site are all essential for enzyme catalysis. Matrix-assisted laser desorption/ionization analysis of peptide fragments of MfnB incubated with GA-3-P followed by NaBH₄ reduction and trypsin digestion identified a peptide with a mass/charge ratio of 1668.8 m/z present only in the D25N, D151N, and K155R mutants, which is consistent with Lys27 having increased by a mass of 58 m/z, indicating that Lys27 forms a Schiff base with a methylglyoxal-like intermediate. In addition, incubation of MfnB with GA-3-P in the presence of deuterated water or incubation of MfnB with C-2 deuterated GA-3-P showed essentially no deuterium incorporated into the 4-HFC-P. Combined with structural analysis and molecular docking, we predict the potential binding sites for two GA-3P molecules in the active site. On the basis of our observations, a possible catalytic mechanism of MfnB is proposed in this study. A phosphate elimination reaction and a triose phosphate isomerase-like reaction occur at the GA-3-P binding site I and II, respectively, prior to the aldol condensation between the enzyme-bound enol form of methylglyoxal and dihydroxyacetone phosphate (DHAP), after which the catalytic cycle is completed by a cyclization and two dehydration reactions assisted by several general acids/bases at the same active site. PMID:25905665

  11. Characterization of glyphosate resistance in cloned Amaranthus palmeri plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Glyphosate resistant Palmer amaranth from Georgia (GA) possesses multiple copies of the target site, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) of this herbicide. Cloned plants of glyphosate-resistant Palmer amaranth biotypes from Mississippi (MS) were compared with GA populations using le...

  12. Gene Amplification Is A Mechanism For Rapid Weed Evolution To Herbicide Resistance

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The herbicide glyphosate became widely used in the U.S. and other parts of the world following the introduction of glyphosate-resistant crops. These crops were created by introduction of a modified 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene, the herbicide target site. Increased use of ...

  13. Identification of genetic elements associated with EPSPS gene amplification

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Weed populations can have high genetic plasticity and rapid responses to environmental selection pressures. For example, 100-fold amplification of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene evolved to confer resistance to glyphosate, the world's most important herbicide, in the wee...

  14. Mechanism of glyceraldehyde-3-phosphate transfer from aldolase to glyceraldehyde-3-phosphate dehydrogenase.

    PubMed

    Kvassman, J; Pettersson, G; Ryde-Pettersson, U

    1988-03-01

    The catalytic interaction of glyceraldehyde-3-phosphate dehydrogenase with glyceraldehyde 3-phosphate has been examined by transient-state kinetic methods. The results confirm previous reports that the apparent Km for oxidative phosphorylation of glyceraldehyde 3-phosphate decreases at least 50-fold when the substrate is generated in a coupled reaction system through the action of aldolase on fructose 1,6-bisphosphate, but lend no support to the proposal that glyceraldehyde 3-phosphate is directly transferred between the two enzymes without prior release to the reaction medium. A theoretical analysis is presented which shows that the kinetic behaviour of the coupled two-enzyme system is compatible in all respects tested with a free-diffusion mechanism for the transfer of glyceraldehyde 3-phosphate from the producing enzyme to the consuming one. PMID:3350006

  15. Iodination of glyceraldehyde 3-phosphate dehydrogenase

    PubMed Central

    Thomas, Jean O.; Harris, J. Ieuan

    1970-01-01

    1. A high degree of homology in the positions of tyrosine residues in glyceraldehyde 3-phosphate dehydrogenase from lobster and pig muscle, and from yeast, prompted an examination of the reactivity of tyrosine residues in the enzyme. 2. Iodination of the enzyme from lobster muscle with low concentrations of potassium tri-[125I]-iodide led to the identification of tyrosine residues of differing reactivity. Tyrosine-46 appeared to be the most reactive in the native enzyme. 3. When the monocarboxymethylated enzyme was briefly treated with small amounts of iodine, iodination could be confined almost entirely to tyrosine-46 in the lobster enzyme; tyrosine-39 or tyrosine-42, or both, were also beginning to react. 4. These three tyrosine residues were also those that reacted most readily in the carboxymethylated pig and yeast enzymes. 5. The difficulties in attaining specific reaction of the native enzyme are considered. 6. The differences between our results and those of other workers are discussed. ImagesPLATE 1PLATE 2 PMID:5530750

  16. THE HEME BINDING PROPERTIES OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE

    PubMed Central

    Hannibal, Luciana; Collins, Daniel; Brassard, Julie; Chakravarti, Ritu; Vempati, Rajesh; Dorlet, Pierre; Santolini, Jérôme; Dawson, John H.; Stuehr, Dennis J.

    2012-01-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a glycolytic enzyme that also functions in transcriptional regulation, oxidative stress, vesicular trafficking, and apoptosis. Because GAPDH is required for cellular heme insertion into inducible nitric oxide synthase (Chakravarti et al, PNAS 2010, 107(42):18004-9), we extensively characterized the heme binding properties of GAPDH. Substoichiometric amounts of ferric heme bound to GAPDH (1 heme per GAPDH tetramer) to form a low-spin complex with UV-visible maxima at 362, 418 and 537 nm, and when reduced to ferrous gave maxima at 424, 527 and 559 nm. Ferric heme association and dissociation rate constants at 10 °C were kon =17,800 M−1s−1 and koff1 = 7.0 × 10−3 s−1; koff2 = 3.3 × 10−4 s−1 respectively, giving approximate affinities of 19–390 nM. Ferrous heme bound more poorly to GAPDH and dissociated with a koff = 4.2 × 10−3 s−1. Magnetic circular dichroism (MCD), resonance Raman (rR) and EPR spectroscopic data on the ferric, ferrous, and ferrous-CO complexes of GAPDH showed that the heme is bis-ligated with His as the proximal ligand. The distal ligand in ferric complex was not displaced by CN− or N3− but in ferrous complex was displaceable by CO at a rate of 1.75 s−1 (for [CO]>0.2 mM). Studies with heme analogs revealed selectivity toward the coordinating metal and porphyrin ring structure. GAPDH-heme was isolated from bacteria induced to express rabbit GAPDH in the presence of δ-amino levulinic acid. Our finding of heme binding to GAPDH expands the protein’s potential roles. The strength, selectivity, reversibility, and redox sensitivity of heme binding to GAPDH is consistent with it performing heme sensing or heme chaperone-like functions in cells. PMID:22957700

  17. Structure of RNA 3'-phosphate cyclase bound to substrate RNA.

    PubMed

    Desai, Kevin K; Bingman, Craig A; Cheng, Chin L; Phillips, George N; Raines, Ronald T

    2014-10-01

    RNA 3'-phosphate cyclase (RtcA) catalyzes the ATP-dependent cyclization of a 3'-phosphate to form a 2',3'-cyclic phosphate at RNA termini. Cyclization proceeds through RtcA-AMP and RNA(3')pp(5')A covalent intermediates, which are analogous to intermediates formed during catalysis by the tRNA ligase RtcB. Here we present a crystal structure of Pyrococcus horikoshii RtcA in complex with a 3'-phosphate terminated RNA and adenosine in the AMP-binding pocket. Our data reveal that RtcA recognizes substrate RNA by ensuring that the terminal 3'-phosphate makes a large contribution to RNA binding. Furthermore, the RNA 3'-phosphate is poised for in-line attack on the P-N bond that links the phosphorous atom of AMP to N(ε) of His307. Thus, we provide the first insights into RNA 3'-phosphate termini recognition and the mechanism of 3'-phosphate activation by an Rtc enzyme. PMID:25161314

  18. Metabolism of L-glyceraldehyde 3-phosphate in Escherichia coli

    SciTech Connect

    Kalyananda, M.K.G.S.

    1985-01-01

    E. coli is able to incorporate L-glyceraldehyde and L-glyceraldehyde 3-phosphate into phospholipids, L-(3-/sup 3/H)Glyceraldehyde was synthesized and the purity and the chemical identity of the product were checked by paper chromatography. L-(3-/sup 3/H)Glyceraldehyde 3-phosphate was synthesized from L-(3-/sup 3/H)glyceraldehyde in a reaction catalyzed by glycerokinase. E. coli extract contains a new enzyme activity which catalyzes an NADPH dependent reduction of L-glyceraldehyde 3-phosphate into sn-glycerol 3-phosphate. A procedure, specifically suitable for assaying the reductase activity in the crude extract, was developed. A more convenient spectrophotometric assay method was employed for the purified enzyme. At moderate concentrations sulfhydryl group inhibitors had no effect on the enzyme activity of L-GAP reductase. At 100..mu..M concentration Zn/sup +2/ inhibited the enzyme activity by about 30% while Mn/sup +2/ elevated the activity by about the same margin. Mg/sup +2/, Ca/sup +2/ and Fe/sup +2/ were without effect at this concentration. L-Glyceraldehyde 3-phosphate is known to be bactericidal at 1.25 ..mu..M concentration and the D-enantiomer is without effect. Furthermore, methylglyoxal is known to be bactericidal at or above 0.5 mM concentration. Strains of E. coli resistant to 1 mM methylglyoxal were isolated. The cell extract prepared from the mutant possessed increased capacity to transform methylglyoxal into D-lactate via a glutathione dependent reaction. These mutants were less sensitive to 2.5 mM DL-GAP suggesting that conversion of L-glyceraldehyde 3-phosphate into methylglyoxal may at least partly be responsible for the bactericidal activity of L-GAP.

  19. [Sn-glycerol-3-phosphate acyltransferases (GPATs) in plants].

    PubMed

    Liu, Cong; Xiao, Dan-Wang; Shi, Chun-Lin; Hu, Xue-Fang; Wu, Ke-Bin; Guan, Chun-Yun; Xiong, Xing-Hua

    2013-12-01

    Sn-glycerol-3-phosphate acyltransferase (GPAT) catalyzes the acylation at sn-1 position of glycerol-3-phosphate to produce lysophosphatidic acid (LPA) in an acyl-CoA or acyl-ACP-dependent manner, which is the initial and rate-determining step of TAG biosynthetic pathway. Some GPATs have sn-2 transfer activity. Part members of the GPAT gene family have been cloned from different plant species. Based on their subcellular localizations, GPATs can be classified into three types, plastid GPATs, mitochondria GPATs and endoplasmic reticulum GPATs. GPATs exhibit diverse biochemical properties and are involved in synthesis of several lipids such as TAG, suberin, and cutin which play important roles in the growth and development of plants. This review summarized the current understanding of the chromosomal locus and gene structure of GPAT genes and the subcellular localization, sn-2 regiospecificity, substrates specialty, and functions of GPATs in plants. PMID:24645344

  20. Model of early self-replication based on covalent complementarity for a copolymer of glycerate-3-phosphate and glycerol-3-phosphate

    NASA Astrophysics Data System (ADS)

    Weber, Arthur L.

    1989-03-01

    Glyceraldehyde-3-phosphate acts as the substrate in a model of early self-replication of a phosphodiester copolymer of glycerate-3-phosphate and glycerol-3-phosphate. This model of self-replication is based on covalent complementarity in which information transfer is mediated by a single covalent bond, in contrast to multiple weak interactions that establish complementarity in nucleic acid replication. This replication model is connected to contemporary biochemistry through its use of glyceraldehyde-3-phosphate, a central metabolite of glycolysis and photosynthesis.

  1. Model of early self-replication based on covalent complementarity for a copolymer of glycerate-3-phosphate and glycerol-3-phosphate

    NASA Technical Reports Server (NTRS)

    Weber, Arthur L.

    1989-01-01

    Glyceraldehyde-3-phosphate acts as the substrate in a model of early self-replication of a phosphodiester copolymer of glycerate-3-phosphate and glycerol-3-phosphate. This model of self-replication is based on covalent complementarity in which information transfer is mediated by a single covalent bond, in contrast to multiple weak interactions that establish complementarity in nucleic acid replication. This replication model is connected to contemporary biochemistry through its use of glyceraldehyde-3-phosphate, a central metabolite of glycolysis and photosynthesis.

  2. Metabolism of L-glyceraldehyde 3-phosphate in Escherichia coli

    SciTech Connect

    Kalyananda, M.K.G.S.; Engel, R.; Tropp, B.E.

    1987-06-01

    When either /sup 3/H-labeled L-glyceraldehyde or /sup 3/H-labeled L-glyceraldehyde 3-phosphate (GAP) was added to cultures of Escherichia coli, the phosphoglycerides were labeled. More than 81% of the label appeared in the backbone of the phosphoglycerides. Chromatographic analyses of the labeled phosphoglycerides revealed that the label was normally distributed into phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. These results suggest that L-glyceraldehyde is phosphorylated and the resultant L-GAP is converted into sn-glycerol 3-phosphate (G3P) before being incorporated into the bacterial phosphoglycerides. Cell-free bacterial extracts catalyzed an NADPH-dependent reduction of L-GAP to sn-G3P. The partially purified enzyme was specific for L-GAP and recognized neither D-GAP nor dihydroxyacetone phosphate as a substrate. NADH could not replace NADPH as a coenzyme. The L-GAP:NADPH oxidoreductase had an apparent K/sub m/ of 28 and 35 ..mu..M for L-GAP and NADPH, respectively. The enzyme was insensitive to sulfhydryl reagents and had a pH optimum of approximately 6.6. The phosphonic acid analog of GAP, 3-hydroxy-4-oxobutyl-1-phosphonate, was a substrate for the reductase, with an apparent K/sub m/ of 280 ..mu..M.

  3. [Cooperative properties of D-glyceraldehyde-3-phosphate dehydrogenase].

    PubMed

    Nagradova, N K

    1977-03-01

    The structure of the active center of glyceraldehyde-3-phosphate dehydrogenase and the arrangement of subunits in the tetrameric molecule is delineated. The mechanism of cooperative effects in the oligomer is considered, and the involvement of various regions of the active center and of different-subunit contact area in the realization of the cooperative phenomena is discussed. A special attention is paid to the effect of NAD+ bound to one of the subunits of the tetramer on the structure of an adjacent subunit and to the problem of the participation of the coenzyme in the creation of anion-binding sites of the enzyme. The conditions of reversible dissociation of the tetrameric apoenzyme molecule into dimers are depicted, and the role of NAD+ in the organization of the quaternary structure of the dehydrogenase is discussed. The problem of catalytic activity of the dimeric form of the enzyme is argued. PMID:193581

  4. Phosphatidylinositol 4-phosphate and phosphatidylinositol 3-phosphate regulate phagolysosome biogenesis

    PubMed Central

    Jeschke, Andreas; Zehethofer, Nicole; Lindner, Buko; Krupp, Jessica; Schwudke, Dominik; Haneburger, Ina; Jovic, Marko; Backer, Jonathan M.; Balla, Tamas; Hilbi, Hubert; Haas, Albert

    2015-01-01

    Professional phagocytic cells ingest microbial intruders by engulfing them into phagosomes, which subsequently mature into microbicidal phagolysosomes. Phagosome maturation requires sequential fusion of the phagosome with early endosomes, late endosomes, and lysosomes. Although various phosphoinositides (PIPs) have been detected on phagosomes, it remained unclear which PIPs actually govern phagosome maturation. Here, we analyzed the involvement of PIPs in fusion of phagosomes with various endocytic compartments and identified phosphatidylinositol 4-phosphate [PI(4)P], phosphatidylinositol 3-phosphate [PI(3)P], and the lipid kinases that generate these PIPs, as mediators of phagosome–lysosome fusion. Phagosome–early endosome fusion required PI(3)P, yet did not depend on PI(4)P. Thus, PI(3)P regulates phagosome maturation at early and late stages, whereas PI(4)P is selectively required late in the pathway. PMID:25825728

  5. Properties of microtubule bundles induced by Glyceraldehyde-3-phosphate dehydrogenase

    NASA Astrophysics Data System (ADS)

    Somers, Marijke; Engelborghs, Yves

    1991-05-01

    The binding of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; E.C. 1.2.1.12) to microtubules causes the microtubules to assemble into large bundles. This bundling can be considered as a further step in the assembly of supramolecular structures. The rate of bundle formation, after addition of GAPDH to preformed microtubules, is not dependent on the GAPDH concentration and reflects bundling kinetics. Bundle disassembly can be studied by the addition of 1 mM adenosine 5'-(β, -imidotri-phosphate) (AMPPNP) to bundled microtubules, and is extremely fast. Bundling reduces the rate of association of tubulin dimers to microtubules, as well as the dissocation from the microtubles. Both rates are reduced to the same extent. This is in agreement with the fact that the critical concentration of tubulin is practically not influenced by the binding of the enzyme. Adding microtubule associated proteins (at I=0.1 M) does not appreciably influence the affinity for GAPDH, but reduces bundle formation possibly for sterical reasons.

  6. The reaction of ozone with glyceraldehyde-3-phosphate dehydrogenase

    SciTech Connect

    Knight, K.L.; Mudd, J.B.

    1984-02-15

    Inactivation of glyceraldehyde-3-phosphate dehydrogenase (GPDH) by ozone can be correlated with oxidation of the active-site -SH residue. Oxidation of peripheral -SH groups, and tryptophan, methionine, and histidine residues occurs concomitantly, but loss of activity depends solely on active-site oxidation. Inactivation is only slightly reversible by dithiothreitol. Kinetic studies show that inhibition of GPDH by ozone mimics noncompetitive inhibition and is characterized as irreversible enzyme inactivation. Analysis of products resulting from ozone oxidation of glutathione suggests that cysteic acid is the product of protein-SH oxidation. Despite oxidation of the active-site -SH, no significant decrease in the Racker band absorbance occurs. This is explained by the appearance of a new chromophore in this region of the absorbance spectrum. Increased absorbance at 322 nm following ozone treatment indicates that tryptophan is converted quantitatively to N-formylkynurenine. When the active-site -SH is reversibly blocked by tetrathionate, enzyme activity is completely recoverable following reaction of the derivatized enzyme with a 1.3X excess of ozone over enzyme monomer. Activity is fully recovered despite the oxidation of peripheral -SH, tryptophan, and histidine residues. Circular dichroism spectra of ozone-treated enzyme show that reaction of GPDH with up to a threefold excess of ozone over enzyme monomer results in no significant disruption of protein secondary structure. Spectra in the near-uv show distinct changes that reflect tryptophan oxidation.

  7. Interactions among p22, glyceraldehyde-3-phosphate dehydrogenase and microtubules.

    PubMed

    Andrade, Josefa; Pearce, Sandy Timm; Zhao, Hu; Barroso, Margarida

    2004-12-01

    Previously, we have shown that p22, an EF-hand Ca2+-binding protein, interacts indirectly with microtubules in an N-myristoylation-dependent and Ca2+-independent manner. In the present study, we report that N-myristoylated p22 interacts with several microtubule-associated proteins within the 30-100 kDa range using overlay blots of microtubule pellets containing cytosolic proteins. One of those p22-binding partners, a 35-40 kDa microtubule-binding protein, has been identified by MS as GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Several lines of evidence suggest a functional relationship between GAPDH and p22. First, endogenous p22 interacts with GAPDH by immunoprecipitation. Secondly, p22 and GAPDH align along microtubule tracks in analogous punctate structures in BHK cells. Thirdly, GAPDH facilitates the p22-dependent interactions between microtubules and microsomal membranes, by increasing the ability of p22 to bind microtubules but not membranes. We have also shown a direct interaction between N-myristoylated p22 and GAPDH in vitro with a K(D) of approximately 0.5 microM. The removal of either the N-myristoyl group or the last six C-terminal amino acids abolishes the binding of p22 to GAPDH and reduces the ability of p22 to associate with microtubules. In summary, we report that GAPDH is involved in the ability of p22 to facilitate microtubule-membrane interactions by affecting the p22-microtubule, but not the p22-membrane, association. PMID:15312048

  8. Glycerol-3-Phosphate-Induced Catabolite Repression in Escherichia coli

    PubMed Central

    Eppler, Tanja; Postma, Pieter; Schütz, Alexandra; Völker, Uwe; Boos, Winfried

    2002-01-01

    The formation of glycerol-3-phosphate (G3P) in cells growing on TB causes catabolite repression, as shown by the reduction in malT expression. For this repression to occur, the general proteins of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), in particular EIIAGlc, as well as the adenylate cyclase and the cyclic AMP-catabolite activator protein system, have to be present. We followed the level of EIIAGlc phosphorylation after the addition of glycerol or G3P. In contrast to glucose, which causes a dramatic shift to the dephosphorylated form, glycerol or G3P only slightly increased the amount of dephosphorylated EIIAGlc. Isopropyl-β-d-thiogalactopyranoside-induced overexpression of EIIAGlc did not prevent repression by G3P, excluding the possibility that G3P-mediated catabolite repression is due to the formation of unphosphorylated EIIAGlc. A mutant carrying a C-terminally truncated adenylate cyclase was no longer subject to G3P-mediated repression. We conclude that the stimulation of adenylate cyclase by phosphorylated EIIAGlc is controlled by G3P and other phosphorylated sugars such as d-glucose-6-phosphate and is the basis for catabolite repression by non-PTS compounds. Further metabolism of these compounds is not necessary for repression. Two-dimensional polyacrylamide gel electrophoresis was used to obtain an overview of proteins that are subject to catabolite repression by glycerol. Some of the prominently repressed proteins were identified by peptide mass fingerprinting. Among these were periplasmic binding proteins (glutamine and oligopeptide binding protein, for example), enzymes of the tricarboxylic acid cycle, aldehyde dehydrogenase, Dps (a stress-induced DNA binding protein), and d-tagatose-1,6-bisphosphate aldolase. PMID:12003946

  9. Membrane topology of murine glycerol-3-phosphate acyltransferase 2.

    PubMed

    Nakagawa, Tadahiko; Harada, Nagakatsu; Miyamoto, Aiko; Kawanishi, Yukiko; Yoshida, Masaki; Shono, Masayuki; Mawatari, Kazuaki; Takahashi, Akira; Sakaue, Hiroshi; Nakaya, Yutaka

    2012-02-17

    Glycerol-3-phosphate acyltransferase (GPAT) is a rate-limiting enzyme in mammalian triacylglycerol biosynthesis. GPAT is a target for the treatment of metabolic disorders associated with high lipid accumulation. Although the molecular basis for GPAT1 activation has been investigated extensively, the activation of other isoforms, such as GPAT2, is less well understood. Here the membrane topology of the GPAT2 protein was examined using an epitope-tag-based method. Exogenously expressed GPAT2 protein was present in the membrane fraction of transformed HEK293 cells even in the presence of Na(2)CO(3) (100 mM), indicating that GPAT2 is a membrane-bound protein. Trypsin treatment of the membrane fraction degraded the N-terminal (FLAG) and C-terminal (myc-epitope) protein tags of the GPAT2 protein. Bioinformatic analysis of the GPAT2 protein sequence indicated four hydrophobic sequences as potential membrane-spanning regions (TM1-TM4). Immunoblotting of the myc-epitope tag, which was inserted between each TM region of the GPAT2 protein, showed that the amino acid sequence between TM3 and TM4 was protected from trypsin digestion. These results suggest that the GPAT2 protein has two transmembrane segments and that the N-terminal and C-terminal regions of this protein face the cytoplasm. These results also suggest that the enzymatically active motifs I-III of the GPAT2 protein face the cytosol, while motif IV is within the membrane. It is expected that the use of this topological model of GPAT2 will be essential in efforts to elucidate the molecular mechanisms of GPAT2 activity in mammalian cells. PMID:22285183

  10. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is inactivated by S-sulfuration in vitro.

    PubMed

    Jarosz, Artur P; Wei, Wanlei; Gauld, James W; Auld, Janeen; Özcan, Filiz; Aslan, Mutay; Mutus, Bulent

    2015-12-01

    Hydrogen sulfide (H2S) is produced enzymatically by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), as well as other enzymes in mammalian tissues. These discoveries have led to the crowning of H2S as yet another toxic gas that serves as a gasotransmitter like NO and CO. H2S is thought to exert its biological effects through its reaction with cysteine thiols in proteins, yielding sulfurated thiol (-SSH) derivatives. One of the first proteins shown to be modified by H2S was glyceraldehyde 3-phosphate dehydrogenase (GAPDH) [1] where the S-sulfuration of the active site cysteine (Cys 152) resulted in ~7-fold increase in the activity of the enzyme. In the present study we have attempted to reproduce this result with no success. GAPDH in its reduced, or hydrogen peroxide, or glutathione disulfide, or nitrosonium oxidized forms was reacted with sulfide or polysulfides. Sulfide had no effect on reduced GAPDH activity, while polysulfides inhibited GAPDH to ~42% of control. S-sulfuration of GAPDH occurred at Cys 247 after sulfide treatment, Cys 156 and Cys 247 after polysulfide treatment. No evidence of S-sulfuration at active site Cys 152 was discovered. Both sulfide and polysulfide was able to restore the activity of glutathione disulfide oxidized GAPDH, but not to control untreated levels. Treatment of glutathione disulfide oxidized GAPDH with polysulfide also produced S-sulfuration of Cys 156. Treatment of a C156S mutant of GAPDH with sulfide and polysulfide resulted in S-sulfuration of Cys 152, which also caused a decrease and not an increase in enzymatic activity. Computational chemistry shows S-sulfuration of Cys 156 may affect the position of catalytic Cys 152, raising its pKa by 0.5, which may affect the nucleophilicity of Cys 152. The current study raises significant questions about the reported ability of H2S to activate GAPDH by the sulfuration of its active site thiol, and indicates that polysulfide is a stronger protein S-sulfurating agent

  11. ATP synthase.

    PubMed

    Junge, Wolfgang; Nelson, Nathan

    2015-01-01

    Oxygenic photosynthesis is the principal converter of sunlight into chemical energy. Cyanobacteria and plants provide aerobic life with oxygen, food, fuel, fibers, and platform chemicals. Four multisubunit membrane proteins are involved: photosystem I (PSI), photosystem II (PSII), cytochrome b6f (cyt b6f), and ATP synthase (FOF1). ATP synthase is likewise a key enzyme of cell respiration. Over three billion years, the basic machinery of oxygenic photosynthesis and respiration has been perfected to minimize wasteful reactions. The proton-driven ATP synthase is embedded in a proton tight-coupling membrane. It is composed of two rotary motors/generators, FO and F1, which do not slip against each other. The proton-driven FO and the ATP-synthesizing F1 are coupled via elastic torque transmission. Elastic transmission decouples the two motors in kinetic detail but keeps them perfectly coupled in thermodynamic equilibrium and (time-averaged) under steady turnover. Elastic transmission enables operation with different gear ratios in different organisms. PMID:25839341

  12. Esterification of glycerol 3-phosphate in lactating guinea-pig mammary gland

    PubMed Central

    Kuhn, N. J.

    1967-01-01

    1. The presence of palmitoyl-CoA–l-glycerol 3-phosphate palmitoyltransferase (EC 2.3.1.15) has been demonstrated in a particulate fraction of mammary tissue from lactating guinea pigs. 2. Cell-free preparations also catalysed the activation of palmitate and oleate, and the conversion of enzymically formed phosphatidic acid into glycerides, in accord with the Kennedy pathway of glyceride formation. 3. The properties of the system that esterifies l-glycerol 3-phosphate were studied with respect to substrates and cofactors, and the reaction product was shown to be phosphatidic acid (1,2-diacyl glycerol 3-phosphate). 4. The extent to which newly formed phosphatidic acid was converted into glyceride in a cell-free system was dependent on the nature of the acyl donor, the concentration of subcellular particles, the time of incubation and the concentration of Mg2+. PMID:6070127

  13. Control of glycolysis by glyceraldehyde-3-phosphate dehydrogenase in Streptococcus cremoris and Streptococcus lactis.

    PubMed Central

    Poolman, B; Bosman, B; Kiers, J; Konings, W N

    1987-01-01

    The decreased response of the energy metabolism of lactose-starved Streptococcus cremoris upon readdition of lactose is caused by a decrease of the glycolytic activity (B. Poolman, E. J. Smid, and W. N. Konings, J. Bacteriol. 169:1460-1468, 1987). The decrease in glycolysis is accompanied by a decrease in the activities of glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate mutase. The steady-state levels of pathway intermediates upon refeeding with lactose after various periods of starvation indicate that the decreased glycolysis is primarily due to diminished glyceraldehyde-3-phosphate dehydrogenase activity. Furthermore, quantification of the control strength exerted by glyceraldehyde-3-phosphate dehydrogenase on the overall activity of the glycolytic pathway shows that this enzyme can be significantly rate limiting in nongrowing cells. PMID:2824452

  14. Glycerol-3-phosphate is a critical mobile inducer of systemic immunity in plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Glycerol-3-phosphate (G3P) is an important metabolite that contributes to the growth and disease-related physiologies of prokaryotes, plants, animals and humans alike. Here we show that G3P serves as the inducer of an important form of broad-spectrum immunity in plants, termed systemic acquired resi...

  15. EXPRESSION OF THE SPERMATOGENIC CELL-SPECIFIC GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE (GAPDS) IN RAT TESTIS

    EPA Science Inventory

    The spermatogenic cell-specific variant of glyceraldehyde 3-phosphate dehydrogenase (GAPDS) has been cloned from a rat testis cDNA library and its pattern of expression determined. A 1417 nucleotide cDNA has been found to encode an enzyme with substantial homology to mouse GAPDS...

  16. Chemical and enzymatic methodologies for the synthesis of enantiomerically pure glyceraldehyde 3-phosphates.

    PubMed

    Gauss, Dominik; Schoenenberger, Bernhard; Wohlgemuth, Roland

    2014-05-01

    Glyceraldehyde 3-phosphates are important intermediates of many central metabolic pathways in a large number of living organisms. d-Glyceraldehyde 3-phosphate (d-GAP) is a key intermediate during glycolysis and can as well be found in a variety of other metabolic pathways. The opposite enantiomer, l-glyceraldehyde 3-phosphate (l-GAP), has been found in a few exciting new pathways. Here, improved syntheses of enantiomerically pure glyceraldehyde 3-phosphates are reported. While d-GAP was synthesized by periodate cleavage of d-fructose 6-phosphate, l-GAP was obtained by enzymatic phosphorylation of l-glyceraldehyde. (1)H- and (31)P NMR spectroscopy was applied in order to examine pH-dependent behavior of GAP over time and to identify potential degradation products. It was found that GAP is stable in acidic aqueous solution below pH 4. At pH 7, methylglyoxal is formed, whereas under alkaline conditions, the formation of lactic acid could be observed. PMID:24680504

  17. Crystal structures capture three states in the catalytic cycle of a pyridoxal phosphate (PLP) synthase.

    PubMed

    Smith, Amber Marie; Brown, William Clay; Harms, Etti; Smith, Janet L

    2015-02-27

    PLP synthase (PLPS) is a remarkable single-enzyme biosynthetic pathway that produces pyridoxal 5'-phosphate (PLP) from glutamine, ribose 5-phosphate, and glyceraldehyde 3-phosphate. The intact enzyme includes 12 synthase and 12 glutaminase subunits. PLP synthesis occurs in the synthase active site by a complicated mechanism involving at least two covalent intermediates at a catalytic lysine. The first intermediate forms with ribose 5-phosphate. The glutaminase subunit is a glutamine amidotransferase that hydrolyzes glutamine and channels ammonia to the synthase active site. Ammonia attack on the first covalent intermediate forms the second intermediate. Glyceraldehyde 3-phosphate reacts with the second intermediate to form PLP. To investigate the mechanism of the synthase subunit, crystal structures were obtained for three intermediate states of the Geobacillus stearothermophilus intact PLPS or its synthase subunit. The structures capture the synthase active site at three distinct steps in its complicated catalytic cycle, provide insights into the elusive mechanism, and illustrate the coordinated motions within the synthase subunit that separate the catalytic states. In the intact PLPS with a Michaelis-like intermediate in the glutaminase active site, the first covalent intermediate of the synthase is fully sequestered within the enzyme by the ordering of a generally disordered 20-residue C-terminal tail. Following addition of ammonia, the synthase active site opens and admits the Lys-149 side chain, which participates in formation of the second intermediate and PLP. Roles are identified for conserved Asp-24 in the formation of the first intermediate and for conserved Arg-147 in the conversion of the first to the second intermediate. PMID:25568319

  18. Purification and characterization of glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) from pea seeds.

    PubMed

    Gani, Zahid; Boradia, Vishant Mahendra; Raghu Ram, Janaki; Suryavanshi, Prashant Mohan; Patil, Pravinkumar; Kumar, Santosh; Singh, Ranvir; Raje, Manoj; Raje, Chaaya Iyengar

    2016-11-01

    Glyceraldehyde-3-phosphate dehydrogenase [GAPDH, NAD + oxidoreductase (phosphorylating) 1.2.1.12] catalyzes the conversion of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate coupled with the reduction of NAD(+) to NADH. In addition to its role in glycolysis, this enzyme has numerous alternate functions, in both prokaryotes and eukaryotes. In plants, additional functions have been reported from multiple species including Pisum sativum. A recent study has identified that GAPDH may play an important role in seed ageing and programmed cell death. Despite this the existing purification protocols are almost 40 years old, and only partial characterization of the enzyme has been reported. In the current study, we report a modified method for purification of enzymatically active pea seed GAPDH along with the characterization of the enzyme. Using 2D gel electrophoresis our study also demonstrates that pea seeds contain four isoforms of NAD(+) dependent GAPDH. PMID:27389468

  19. Structure of glycerol-3-phosphate dehydrogenase, an essential monotopic membrane enzyme involved in respiration and metabolism

    SciTech Connect

    Yeh, Joanne I.; Chinte, Unmesh; Du, Shoucheng

    2008-04-02

    Sn-glycerol-3-phosphate dehydrogenase (GlpD) is an essential membrane enzyme, functioning at the central junction of respiration, glycolysis, and phospholipid biosynthesis. Its critical role is indicated by the multitiered regulatory mechanisms that stringently controls its expression and function. Once expressed, GlpD activity is regulated through lipid-enzyme interactions in Escherichia coli. Here, we report seven previously undescribed structures of the fully active E. coli GlpD, up to 1.75 {angstrom} resolution. In addition to elucidating the structure of the native enzyme, we have determined the structures of GlpD complexed with substrate analogues phosphoenolpyruvate, glyceric acid 2-phosphate, glyceraldehyde-3-phosphate, and product, dihydroxyacetone phosphate. These structural results reveal conformational states of the enzyme, delineating the residues involved in substrate binding and catalysis at the glycerol-3-phosphate site. Two probable mechanisms for catalyzing the dehydrogenation of glycerol-3-phosphate are envisioned, based on the conformational states of the complexes. To further correlate catalytic dehydrogenation to respiration, we have additionally determined the structures of GlpD bound with ubiquinone analogues menadione and 2-n-heptyl-4-hydroxyquinoline N-oxide, identifying a hydrophobic plateau that is likely the ubiquinone-binding site. These structures illuminate probable mechanisms of catalysis and suggest how GlpD shuttles electrons into the respiratory pathway. Glycerol metabolism has been implicated in insulin signaling and perturbations in glycerol uptake and catabolism are linked to obesity in humans. Homologs of GlpD are found in practically all organisms, from prokaryotes to humans, with >45% consensus protein sequences, signifying that these structural results on the prokaryotic enzyme may be readily applied to the eukaryotic GlpD enzymes.

  20. Structure of glycerol-3-phosphate dehydrogenase, an essential monotopic membrane enzyme involved in respiration and metabolism

    PubMed Central

    Yeh, Joanne I.; Chinte, Unmesh; Du, Shoucheng

    2008-01-01

    Sn-glycerol-3-phosphate dehydrogenase (GlpD) is an essential membrane enzyme, functioning at the central junction of respiration, glycolysis, and phospholipid biosynthesis. Its critical role is indicated by the multitiered regulatory mechanisms that stringently controls its expression and function. Once expressed, GlpD activity is regulated through lipid-enzyme interactions in Escherichia coli. Here, we report seven previously undescribed structures of the fully active E. coli GlpD, up to 1.75 Å resolution. In addition to elucidating the structure of the native enzyme, we have determined the structures of GlpD complexed with substrate analogues phosphoenolpyruvate, glyceric acid 2-phosphate, glyceraldehyde-3-phosphate, and product, dihydroxyacetone phosphate. These structural results reveal conformational states of the enzyme, delineating the residues involved in substrate binding and catalysis at the glycerol-3-phosphate site. Two probable mechanisms for catalyzing the dehydrogenation of glycerol-3-phosphate are envisioned, based on the conformational states of the complexes. To further correlate catalytic dehydrogenation to respiration, we have additionally determined the structures of GlpD bound with ubiquinone analogues menadione and 2-n-heptyl-4-hydroxyquinoline N-oxide, identifying a hydrophobic plateau that is likely the ubiquinone-binding site. These structures illuminate probable mechanisms of catalysis and suggest how GlpD shuttles electrons into the respiratory pathway. Glycerol metabolism has been implicated in insulin signaling and perturbations in glycerol uptake and catabolism are linked to obesity in humans. Homologs of GlpD are found in practically all organisms, from prokaryotes to humans, with >45% consensus protein sequences, signifying that these structural results on the prokaryotic enzyme may be readily applied to the eukaryotic GlpD enzymes. PMID:18296637

  1. Glyceraldehyde-3-phosphate dehydrogenase-catalyzed chain oxidation of reduced nicotinamide adenine dinucleotide by perhydroxyl radicals

    SciTech Connect

    Chan, P.C.

    1980-02-10

    The chain oxidation of glyceraldehyde-3-phosphate dehydrogenase NADH by perhydroxyl radicals and propagated by molecular oxygen was studied by the xanthine-xanthine oxidase system, /sup 60/Co ..gamma..-ray, and pulse radiolysis. The chain length, amount of NADH oxidized per HO/sub 2/ generated, increases with increasing acidity of the medium and reaches a value of 73 at pH 5.0. The rate constant for the oxidation of the glyceraldehyde-3-phosphate dehydrogenase NADH complex by HO/sub 2/ was estimated to be 2 x 10/sup 7/ m/sup -1/s/sup -1/ at ambient temperatures (23-24/sup 0/C). Rate studies as a function of pH indicate that O/sub 2//sup -/ is unreactive toward the glyceraldehyde-3-phosphate dehydrogenase NADH complex. Other dehydrogenases (malate dehydrogenase, glutamate dehydrogenase, and isocitric dehydrogenase) studied showed no catalytic activity in the oxidation of NADH by HO/sub 2//O/sub 2//sup -/.

  2. Structure of glycerol-3-phosphate dehydrogenase (GPD1) from Saccharomyces cerevisiae at 2.45 Å resolution

    PubMed Central

    Alarcon, David Aparicio; Nandi, Munmun; Carpena, Xavi; Fita, Ignacio; Loewen, Peter C.

    2012-01-01

    The interconversion of glycerol 3-phosphate and dihydroxyacetone phosphate by glycerol-3-phosphate dehydrogenases provides a link between carbohydrate and lipid metabolism and provides Saccharomyces cerevisiae with protection against osmotic and anoxic stress. The first structure of a glycerol-3-phosphate dehydrogenase from S. cerevisiae, GPD1, is reported at 2.45 Å resolution. The asymmetric unit contains two monomers, each of which is organized with N- and C-terminal domains. The N-terminal domain contains a classic Rossmann fold with the (β-α-β-α-β)2 motif typical of many NAD+-dependent enzymes, while the C-terminal domain is mainly α-helical. Structural and phylogenetic comparisons reveal four main structure types among the five families of glycerol-3-phosphate and glycerol-1-phosphate dehydrogenases and reveal that the Clostridium acetobutylican protein with PDB code 3ce9 is a glycerol-1-­phosphate dehydrogenase. PMID:23143232

  3. Selectivity of 3-bromo-isoxazoline inhibitors between human and Plasmodium falciparum glyceraldehyde-3-phosphate dehydrogenases.

    PubMed

    Bruno, Stefano; Margiotta, Marilena; Pinto, Andrea; Cullia, Gregorio; Conti, Paola; De Micheli, Carlo; Mozzarelli, Andrea

    2016-06-15

    Compounds based on the 3-Br-isoxazoline scaffold fully inhibit glyceraldehyde 3-phosphate dehydrogenase from Plasmodium falciparum by selectively alkylating all four catalytic cysteines of the tetramer. Here, we show that, under the same experimental conditions that led to a fast and complete inhibition of the protozoan enzyme, the human ortholog was only 25% inhibited, with the alkylation of a single catalytic cysteine within the tetramer. The partial alkylation seems to produce a slow conformational rearrangement that severely limits the accessibility of the remaining active sites to bulky 3-Br-isoxazoline derivatives, but not to the substrate or smaller alkylating agents. PMID:27137361

  4. Expression, purification and kinetic characterization of His-tagged glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma cruzi.

    PubMed

    Cheleski, Juliana; Freitas, Renato F; Wiggers, Helton José; Rocha, Josmar R; de Araújo, Ana Paula Ulian; Montanari, Carlos A

    2011-04-01

    Trypanosomes are flagellated protozoa responsible for serious parasitic diseases that have been classified by the World Health Organization as tropical sicknesses of major importance. One important drug target receiving considerable attention is the enzyme glyceraldehyde-3-phosphate dehydrogenase from the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease (T. cruzi Glyceraldehyde-3-phosphate dehydrogenase (TcGAPDH); EC 1.2.1.12). TcGAPDH is a key enzyme in the glycolytic pathway of T. cruzi and catalyzes the oxidative phosphorylation of D-glyceraldehyde-3-phosphate (G3P) to 1,3-bisphosphoglycerate (1,3-BPG) coupled to the reduction of oxidized nicotinamide adenine dinucleotide, (NAD(+)) to NADH, the reduced form. Herein, we describe the cloning of the T. cruzi gene for TcGAPDH into the pET-28a(+) vector, its expression as a tagged protein in Escherichia coli, purification and kinetic characterization. The His(6)-tagged TcGAPDH was purified by affinity chromatography. Enzyme activity assays for the recombinant His(6)-TcGAPDH were carried out spectrophotometrically to determine the kinetic parameters. The apparent Michaelis-Menten constant (K(M)(app)) determined for D-glyceraldehyde-3-phosphate and NAD(+) were 352±21 and 272±25 μM, respectively, which were consistent with the values for the untagged enzyme reported in the literature. We have demonstrated by the use of Isothermal Titration Calorimetry (ITC) that this vector modification resulted in activity preserved for a higher period. We also report here the use of response surface methodology (RSM) to determine the region of optimal conditions for enzyme activity. A quadratic model was developed by RSM to describe the enzyme activity in terms of pH and temperature as independent variables. According to the RMS contour plots and variance analysis, the maximum enzyme activity was at 29.1°C and pH 8.6. Above 37°C, the enzyme activity starts to fall, which may be related to previous

  5. Stilbene Synthase and Chalcone Synthase 1

    PubMed Central

    Rolfs, Claus-Henning; Kindl, Helmut

    1984-01-01

    Cultured cells of Picea excelsa capable of forming stilbenes and flavanoids have been established. Unlike needles of intact plants containing piceatannol (3,3′,4′,5-tetrahydroxystilbene) and stilbene glycosides the cultured cells converted phenylalanine and p-coumaric acid primarily into resveratrol monomethyl ether (3,4′-dihydroxy-5-methoxystilbene) and naringenin. Partially purified enzyme preparations were assayed for chalcone synthase as well as for stilbene synthase activity converting malonyl-CoA plus p-coumaroyl-CoA into 3,4′,5-trihydroxystilbene (resveratrol). Although stilbene synthase and chalcone synthase use the same substrates and exhibit similar molecular properties, i.e. molecular weight and subunit molecular weight, they are two different proteins. This difference was demonstrated by gel electrophoresis and by means of monospecific antibodies. PMID:16663649

  6. Expanding the molecular diversity and phenotypic spectrum of glycerol 3-phosphate dehydrogenase 1 deficiency.

    PubMed

    Dionisi-Vici, Carlo; Shteyer, Eyal; Niceta, Marcello; Rizzo, Cristiano; Pode-Shakked, Ben; Chillemi, Giovanni; Bruselles, Alessandro; Semeraro, Michela; Barel, Ortal; Eyal, Eran; Kol, Nitzan; Haberman, Yael; Lahad, Avishai; Diomedi-Camassei, Francesca; Marek-Yagel, Dina; Rechavi, Gideon; Tartaglia, Marco; Anikster, Yair

    2016-09-01

    Transient infantile hypertriglyceridemia (HTGT1; OMIM #614480) is a rare autosomal recessive disorder, which manifests in early infancy with transient hypertriglyceridemia, hepatomegaly, elevated liver enzymes, persistent fatty liver and hepatic fibrosis. This rare clinical entity is caused by inactivating mutations in the GPD1 gene, which encodes the cytosolic isoform of glycerol-3-phosphate dehydrogenase. Here we report on four patients from three unrelated families of diverse ethnic origins, who presented with hepatomegaly, liver steatosis, hypertriglyceridemia, with or without fasting ketotic hypoglycemia. Whole exome sequencing revealed the affected individuals to harbor deleterious biallelic mutations in the GPD1 gene, including the previously undescribed c.806G > A (p.Arg269Gln) and c.640T > C (p.Cys214Arg) mutations. The clinical features in three of our patients showed several differences compared to the original reports. One subject presented with recurrent episodes of fasting hypoglycemia along with hepatomegaly, hypetriglyceridemia, and elevated liver enzymes; the second showed a severe liver disease, with intrahepatic cholestasis associated with kidney involvement; finally, the third presented persistent hypertriglyceridemia at the age of 30 years. These findings expand the current knowledge of this rare disorder, both with regard to the phenotype and molecular basis. The enlarged phenotypic spectrum of glycerol-3-phosphate dehydrogenase 1 deficiency can mimic other inborn errors of metabolism with liver involvement and should alert clinicians to recognize this entity by considering GPD1 mutations in appropriate clinical settings. PMID:27368975

  7. Carbon-13 and deuterium isotope effects on the reaction catalyzed by glyceraldehyde-3-phosphate dehydrogenase

    SciTech Connect

    Canellas, P.F.; Cleland, W.W. )

    1991-09-10

    Carbon-13 and deuterium isotope effects have been measured on the reaction catalyzed by rabbit muscle glyceraldehyde-3-phosphate dehydrogenase in an effort to locate the rate-limiting steps. With D-glyceraldehyde 3-phosphate as substrate, hydride transfer is a major, but not the only, slow step prior to release of the first product, and the intrinsic primary deuterium and {sup 13}C isotope effects on this step are 5-5.5 and 1.034-1.040, and the sum of the commitments to catalysis is {approximately} 3. The {sup 13}C isotope effects on thiohemiacetal formation and thioester phosphorolysis are 1.005 or less. With D-glyceraldehyde as substrate, the isotope effects are similar, but the sum of commitments is {approximately} 1.5, so that hydride transfer is more, but still not solely, rate limiting for this slow substrate. The observed {sup 13}C and deuterium equilibrium isotope effects on the overall reaction from the hydrated aldehyde are 0.995 and 1.145, while the {sup 13}C equilibrium isotope effect for conversion of a thiohemiacetal to a thioester is 0.994, and that for conversion of a thioester to an acyl phosphate is 0.997. Somewhat uncertain values for the {sup 13}C equilibrium isotope effects on aldehyde dehydration and formation of a thiohemiacetal are 1.003 and 1.004.

  8. Cloning and characterization of glyceraldehyde-3-phosphate dehydrogenase encoding gene in Gracilaria/Gracilariopsis lemaneiformis

    NASA Astrophysics Data System (ADS)

    Ren, Xueying; Sui, Zhenghong; Zhang, Xuecheng

    2006-04-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays important roles in various cellular processes. A cytosolic GAPDH encoding gene ( gpd) of Gracilaria/Gracilariopsis lemaneiformis was cloned and characterized. Deduced amino acid sequence of the enzyme of G. lemaneiformis had high homology with those of seven red algae. The 5'-untranslated regions of the GAPDHs encoding genes of these red algae varied greatly. GAPDHs of these red algae shared the highly conserved glyceraldehyde 3-phosphate dehydrogenase active site ASCTTNCL. However, such active site of Cyanidium caldarium was different from those of the other six algae at the last two residues (CL to LF), thus the spatial structure of its GAPDH active center may be different from those of the other six. Phylogenetic analysis indicated that GAPDH of G. lemaneiformis might have undergone an evolution similar to those of Porphyra yezoensis, Chondrus crispus, and Gracilaria verrucosa. C. caldarium had a closer evolutionary relationship with Cyanidioschyzon merolae than with Cyanidium sp. Virtual Northern blot analysis revealed that gpd of G. lemaneiformis expressed constitutively, which suggested that it might be house-keeping and could be adapted as an inner control in gene expression analysis of G. lemaneiformis.

  9. [Use of immobilization in the study of glyceraldehyde 3-phosphate dehydrogenase. Immobilized monomers].

    PubMed

    Muronets, V I; Ashmarina, L I; Asriiants, R A; Nagradova, N K

    1982-06-01

    Active immobilized monomers of glyceraldehyde 3-phosphate dehydrogenase were prepared by means of dissociation of the tetrameric enzyme molecule covalently bound to Sepharose via a single subunit. The conditions were elaborated to achieve the inactivation and solubilization of the non-covalently bound subunits leaving the monomer coupled to the matrix intact. This procedure differs from the previously developed method of matrix-bound oligomeric enzymes dissociation in a detail which was found to be essentially important. The widely used method includes complete denaturation of all subunits during treatment with urea followed by reactivation of the immobilized one, whereas only the non-covalently bound subunits suffer denaturation under the conditions developed in the present work. The immobilized monomers of glyceraldehyde 3-phosphate dehydrogenase exhibit Vmax and Km (for NAD and substrate) values similar to those found for the immobilized tetramer. Reassociation of the immobilized monomers with soluble enzyme subunits obtained in the presence of urea produces matrix-bound tetrameric species. Immobilized trimers ae formed upon incubation of matrix-bound monomers in a diluted apoenzyme solution. The immobilized monomeric, trimeric and tetrameric enzyme species were used to study the role of subunit interactions in cooperative phenomena exhibited by the dehydrogenase. PMID:7115810

  10. Kinetic and Mechanistic Characterization of the Glyceraldehyde 3-Phosphate Dehydrogenase from Mycobacterium tuberculosis

    PubMed Central

    Wolfson-Stofko, Brett; Hadi, Timin; Blanchard, John S.

    2013-01-01

    Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a glycolytic protein responsible for the conversion of glyceraldehyde 3-phosphate (G3P), inorganic phosphate and nicotinamide adenine dinucleotide (NAD+) to 1,3-bisphosphoglycerate (1,3-BPG) and the reduced form of nicotinamide adenine dinucleotide (NADH). Here we report the characterization of GAPDH from Mycobacterium tuberculosis (Mtb). This enzyme exhibits a kinetic mechanism in which first NAD+, then G3P bind to the active site resulting in the formation of a covalently bound thiohemiacetal intermediate. After oxidation of the thiohemiacetal and subsequent nucleotide exchange (NADH off, NAD+ on), the binding of inorganic phosphate and phosphorolysis yields the product 1,3-BPG. Mutagenesis and iodoacetamide (IAM) inactivation studies reveal the conserved C158 to be responsible for nucleophilic catalysis and that the conserved H185 to act as a catalytic base. Primary, solvent and multiple kinetic isotope effects revealed that the first half-reaction is rate limiting and utilizes a step-wise mechanism for thiohemiacetal oxidation via a transient alkoxide to promote hydride transfer and thioester formation. PMID:24161676

  11. Oral streptococcal glyceraldehyde-3-phosphate dehydrogenase mediates interaction with Porphyromonas gingivalis fimbriae.

    PubMed

    Maeda, Kazuhiko; Nagata, Hideki; Nonaka, Aya; Kataoka, Kosuke; Tanaka, Muneo; Shizukuishi, Satoshi

    2004-11-01

    Interaction of Porphyromonas gingivalis with plaque-forming bacteria is necessary for its colonization in periodontal pockets. Participation of Streptococcus oralis glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and P. gingivalis fimbriae in this interaction has been reported. In this investigation, the contribution of various oral streptococcal GAPDHs to interaction with P. gingivalis fimbriae was examined. Streptococcal cell surface GAPDH activity was measured by incubation of a constant number of streptococci with glyceraldehyde-3-phosphate and analysis for the conversion of NAD+ to NADH based on the absorbance at 340 nm. Coaggregation activity was measured by a turbidimetric assay. Cell surface GAPDH activity was correlated with coaggregation activity (r = 0.854, P < 0.01) with Spearman's rank correlation coefficient. S. oralis ATCC 9811 and ATCC 10557, Streptococcus gordonii G9B, Streptococcus sanguinis ATCC 10556, and Streptococcus parasanguinis ATCC 15909 exhibited high cell surface GAPDH activity and coaggregation activity; consequently, their cell surface GAPDHs were extracted with mutanolysin and purified on a Cibacron Blue Sepharose column. Subsequently, their DNA sequences were elucidated. Purified GAPDHs bound P. gingivalis recombinant fimbrillin by Western blot assay, furthermore, their DNA sequences displayed a high degree of homology with one another. Moreover, S. oralis recombinant GAPDH inhibited coaggregation between P. gingivalis and the aforementioned five streptococcal strains in a dose-dependent manner. These results suggest that GAPDHs of various plaque-forming streptococci may be involved in their attachment to P. gingivalis fimbriae and that they may contribute to P. gingivalis colonization. PMID:15488735

  12. Human 1-Acylglycerol-3-phosphate O-Acyltransferase Isoforms 1 and 2

    PubMed Central

    Agarwal, Anil K.; Sukumaran, Suja; Cortés, Víctor A.; Tunison, Katie; Mizrachi, Dario; Sankella, Shireesha; Gerard, Robert D.; Horton, Jay D.; Garg, Abhimanyu

    2011-01-01

    Loss-of-function mutations in 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) 2 in humans and mice result in loss of both the white and brown adipose tissues from birth. AGPAT2 generates precursors for the synthesis of glycerophospholipids and triacylglycerols. Loss of adipose tissue, or lipodystrophy, results in hyperinsulinemia, diabetes mellitus, and severe hepatic steatosis. Here, we analyzed biochemical properties of human AGPAT2 and its close homolog, AGPAT1, and we studied their role in liver by transducing their expression via recombinant adenoviruses in Agpat2−/− mice. The in vitro substrate specificities of AGPAT1 and AGPAT2 are quite similar for lysophosphatidic acid and acyl-CoA. Protein homology modeling of both the AGPATs with glycerol-3-phosphate acyltransferase 1 (GPAT1) revealed that they have similar tertiary protein structure, which is consistent with their similar substrate specificities. When co-expressed, both isoforms co-localize to the endoplasmic reticulum. Despite such similarities, restoring AGPAT activity in liver by overexpression of either AGPAT1 or AGPAT2 in Agpat2−/− mice failed to ameliorate the hepatic steatosis. From these studies, we suggest that the role of AGPAT1 or AGPAT2 in liver lipogenesis is minimal and that accumulation of liver fat is primarily a consequence of insulin resistance and loss of adipose tissue in Agpat2−/− mice. PMID:21873652

  13. Crystal Structures Capture Three States in the Catalytic Cycle of a Pyridoxal Phosphate (PLP) Synthase*♦

    PubMed Central

    Smith, Amber Marie; Brown, William Clay; Harms, Etti; Smith, Janet L.

    2015-01-01

    PLP synthase (PLPS) is a remarkable single-enzyme biosynthetic pathway that produces pyridoxal 5′-phosphate (PLP) from glutamine, ribose 5-phosphate, and glyceraldehyde 3-phosphate. The intact enzyme includes 12 synthase and 12 glutaminase subunits. PLP synthesis occurs in the synthase active site by a complicated mechanism involving at least two covalent intermediates at a catalytic lysine. The first intermediate forms with ribose 5-phosphate. The glutaminase subunit is a glutamine amidotransferase that hydrolyzes glutamine and channels ammonia to the synthase active site. Ammonia attack on the first covalent intermediate forms the second intermediate. Glyceraldehyde 3-phosphate reacts with the second intermediate to form PLP. To investigate the mechanism of the synthase subunit, crystal structures were obtained for three intermediate states of the Geobacillus stearothermophilus intact PLPS or its synthase subunit. The structures capture the synthase active site at three distinct steps in its complicated catalytic cycle, provide insights into the elusive mechanism, and illustrate the coordinated motions within the synthase subunit that separate the catalytic states. In the intact PLPS with a Michaelis-like intermediate in the glutaminase active site, the first covalent intermediate of the synthase is fully sequestered within the enzyme by the ordering of a generally disordered 20-residue C-terminal tail. Following addition of ammonia, the synthase active site opens and admits the Lys-149 side chain, which participates in formation of the second intermediate and PLP. Roles are identified for conserved Asp-24 in the formation of the first intermediate and for conserved Arg-147 in the conversion of the first to the second intermediate. PMID:25568319

  14. Cellulose synthase interacting protein

    PubMed Central

    Somerville, Chris

    2010-01-01

    Cellulose is the most abundant biopolymer on earth. The great abundance of cellulose places it at the forefront as a primary source of biomass for renewable biofuels. However, the knowledge of how plant cells make cellulose remains very rudimentary. Cellulose microfibrils are synthesized at the plasma membrane by hexameric protein complexes, also known as cellulose synthase complexes. The only known components of cellulose synthase complexes are cellulose synthase (CESA) proteins until the recent identification of a novel component. CSI1, which encodes CESA interacting protein 1 (CSI1) in Arabidopsis. CSI1, as the first non-CESA proteins associated with cellulose synthase complexes, opens up many opportunities. PMID:21150290

  15. Pattern Recognition Techniques Applied to the Study of Leishmanial Glyceraldehyde-3-Phosphate Dehydrogenase Inhibition

    PubMed Central

    Lozano, Norka B. H.; Oliveira, Rafael F.; Weber, Karen C.; Honorio, Kathia M.; Guido, Rafael V. C.; Andricopulo, Adriano D.; de Sousa, Alexsandro G.; da Silva, Albérico B. F.

    2014-01-01

    Chemometric pattern recognition techniques were employed in order to obtain Structure-Activity Relationship (SAR) models relating the structures of a series of adenosine compounds to the affinity for glyceraldehyde 3-phosphate dehydrogenase of Leishmania mexicana (LmGAPDH). A training set of 49 compounds was used to build the models and the best ones were obtained with one geometrical and four electronic descriptors. Classification models were externally validated by predictions for a test set of 14 compounds not used in the model building process. Results of good quality were obtained, as verified by the correct classifications achieved. Moreover, the results are in good agreement with previous SAR studies on these molecules, to such an extent that we can suggest that these findings may help in further investigations on ligands of LmGAPDH capable of improving treatment of leishmaniasis. PMID:24566143

  16. Atomic-level characterization of transport cycle thermodynamics in the glycerol-3-phosphate:phosphate antiporter

    NASA Astrophysics Data System (ADS)

    Moradi, Mahmoud; Enkavi, Giray; Tajkhorshid, Emad

    2015-09-01

    Membrane transporters actively translocate their substrate by undergoing large-scale structural transitions between inward- (IF) and outward-facing (OF) states (`alternating-access' mechanism). Despite extensive structural studies, atomic-level mechanistic details of such structural transitions, and as importantly, their coupling to chemical events supplying the energy, remain amongst the most elusive aspects of the function of these proteins. Here we present a quantitative, atomic-level description of the functional thermodynamic cycle for the glycerol-3-phosphate:phosphate antiporter GlpT by using a novel approach in reconstructing the free energy landscape governing the IF<-->OF transition along a cyclic transition pathway involving both apo and substrate-bound states. Our results provide a fully atomic description of the complete transport process, offering a structural model for the alternating-access mechanism and substantiating the close coupling between global structural transitions and local chemical events.

  17. Daily variations in the glycerol-3-phosphate dehydrogenase isoforms expression in Triatoma infestans flight muscles.

    PubMed

    Stroppa, María M; Carriazo, Carlota S; Gerez de Burgos, Nelia M; Garcia, Beatríz A

    2014-08-01

    Triatoma infestans, the main vector of Chagas disease, is a blood-sucking insect. Flight dispersal of adults is the most important mechanism for reinfestation of houses after insecticide spraying. Flight muscles have two glycerol-3-phosphate dehydrogenase (GPDH) isoforms: GPDH-1 is involved in flight metabolism and GPDH-2 provides lipid precursors. In this study, we explored the profile of GPDH expression in females and males adult flight muscles under light/dark cycle, constant light, and constant dark conditions. Under constant dark conditions, GPDH-1 flight muscles of T. infestans showed a rhythmic pattern of transcription synchronous with a rhythmic profile of activity suggesting regulation by the endogenous circadian clock. Otherwise, the GPDH-2 expression analysis showed no regulation by the endogenous clock, but showed that an external factor, such as the dark/light period, was necessary for synchronization of GPDH-2 transcription and activity. PMID:24914000

  18. Mechanistic similarities in docking of the FYVE and PX domains to phosphatidylinositol 3-phosphate containing membranes

    PubMed Central

    Kutateladze, Tatiana G.

    2007-01-01

    Phosphatidylinositol 3-phosphate [PtdIns(3)P], a phospholipid produced by PI 3-kinases in early endosomes and multivesicular bodies, often serves as a marker of endosomal membranes. PtdIns(3)P recruits and activates effector proteins containing the FYVE or PX domain and therefore regulates a variety of biological processes including endo- and exocytosis, membrane trafficking, protein sorting, signal transduction and cytoskeletal rearrangement. Structures and PtdIns(3)P binding modes of several FYVE and PX domains have recently been characterized, unveiling the molecular basis underlying multiple cellular functions of these proteins. Here, structural and functional aspects and current mechanisms of the multivalent membrane anchoring by the FYVE and PX domains are reviewed and compared. PMID:17707914

  19. Atomic-level characterization of transport cycle thermodynamics in the glycerol-3-phosphate:phosphate antiporter

    PubMed Central

    Moradi, Mahmoud; Enkavi, Giray; Tajkhorshid, Emad

    2015-01-01

    Membrane transporters actively translocate their substrate by undergoing large-scale structural transitions between inward- (IF) and outward-facing (OF) states (‘alternating-access' mechanism). Despite extensive structural studies, atomic-level mechanistic details of such structural transitions, and as importantly, their coupling to chemical events supplying the energy, remain amongst the most elusive aspects of the function of these proteins. Here we present a quantitative, atomic-level description of the functional thermodynamic cycle for the glycerol-3-phosphate:phosphate antiporter GlpT by using a novel approach in reconstructing the free energy landscape governing the IF↔OF transition along a cyclic transition pathway involving both apo and substrate-bound states. Our results provide a fully atomic description of the complete transport process, offering a structural model for the alternating-access mechanism and substantiating the close coupling between global structural transitions and local chemical events. PMID:26417850

  20. Molecular cloning and characterization of the glyceraldehyde-3-phosphate dehydrogenase gene from Penicillium expansum PE-12.

    PubMed

    Zhang, T; Qi, Z; Yu, Q S; Tang, K X

    2013-01-01

    Penicillium expansum produces large amounts of lipase, which is widely used in laundry detergent and leather industry. We isolated the glyceraldehyde-3-phosphate dehydrogenase gene (PeGPD) from P. expansum PE-12 through reverse transcriptase PCR and 5'-3' rapid amplification of cDNA ends (RACE-PCR). The gene is 1266 bp long, including an ORF of 1014 bp, encoding a polypeptide chain of 337 amino acids. A phylogenetic tree based on GPD proteins showed that P. expansum is close to Aspergillus species, but comparatively distant from P. marneffei. Southern blot results revealed a single copy of PeGPD, and expression analysis gave evidence of high expression levels. PeGPD genes have potential for genetic engineering of P. expansum for industrial lipase production. PMID:23420404

  1. Daily Variations in the Glycerol-3-Phosphate Dehydrogenase Isoforms Expression in Triatoma infestans Flight Muscles

    PubMed Central

    Stroppa, María M.; Carriazo, Carlota S.; Gerez de Burgos, Nelia M.; Garcia, Beatríz A.

    2014-01-01

    Triatoma infestans, the main vector of Chagas disease, is a blood-sucking insect. Flight dispersal of adults is the most important mechanism for reinfestation of houses after insecticide spraying. Flight muscles have two glycerol-3-phosphate dehydrogenase (GPDH) isoforms: GPDH-1 is involved in flight metabolism and GPDH-2 provides lipid precursors. In this study, we explored the profile of GPDH expression in females and males adult flight muscles under light/dark cycle, constant light, and constant dark conditions. Under constant dark conditions, GPDH-1 flight muscles of T. infestans showed a rhythmic pattern of transcription synchronous with a rhythmic profile of activity suggesting regulation by the endogenous circadian clock. Otherwise, the GPDH-2 expression analysis showed no regulation by the endogenous clock, but showed that an external factor, such as the dark/light period, was necessary for synchronization of GPDH-2 transcription and activity. PMID:24914000

  2. Characterization of a Novel Intestinal Glycerol-3-phosphate Acyltransferase Pathway and Its Role in Lipid Homeostasis.

    PubMed

    Khatun, Irani; Clark, Ronald W; Vera, Nicholas B; Kou, Kou; Erion, Derek M; Coskran, Timothy; Bobrowski, Walter F; Okerberg, Carlin; Goodwin, Bryan

    2016-02-01

    Dietary triglycerides (TG) are absorbed by the enterocytes of the small intestine after luminal hydrolysis into monacylglycerol and fatty acids. Before secretion on chylomicrons, these lipids are reesterified into TG, primarily through the monoacylglycerol pathway. However, targeted deletion of the primary murine monoacylglycerol acyltransferase does not quantitatively affect lipid absorption, suggesting the existence of alternative pathways. Therefore, we investigated the role of the glycerol 3-phosphate pathway in dietary lipid absorption. The expression of glycerol-3-phosphate acyltransferase (GPAT3) was examined throughout the small intestine. To evaluate the role for GPAT3 in lipid absorption, mice harboring a disrupted GPAT3 gene (Gpat3(-/-)) were subjected to an oral lipid challenge and fed a Western-type diet to characterize the role in lipid and cholesterol homeostasis. Additional mechanistic studies were performed in primary enterocytes. GPAT3 was abundantly expressed in the apical surface of enterocytes in the small intestine. After an oral lipid bolus, Gpat3(-/-) mice exhibited attenuated plasma TG excursion and accumulated lipid in the enterocytes. Electron microscopy studies revealed a lack of lipids in the lamina propria and intercellular space in Gpat3(-/-) mice. Gpat3(-/-) enterocytes displayed a compensatory increase in the synthesis of phospholipid and cholesteryl ester. When fed a Western-type diet, hepatic TG and cholesteryl ester accumulation was significantly higher in Gpat3(-/-) mice compared with the wild-type mice accompanied by elevated levels of alanine aminotransferase, a marker of liver injury. Dysregulation of bile acid metabolism was also evident in Gpat3-null mice. These studies identify GPAT3 as a novel enzyme involved in intestinal lipid metabolism. PMID:26644473

  3. Crystallization and preliminary X-ray analysis of the glycerol-3-phosphate 1-acyltransferase from squash (Cucurbita moschata).

    PubMed

    Turnbull, A P; Rafferty, J B; Sedelnikova, S E; Slabas, A R; Schierer, T P; Kroon, J T; Nishida, I; Murata, N; Simon, J W; Rice, D W

    2001-03-01

    Glycerol-3-phosphate 1-acyltransferase (E.C. 2.3.1.15; G3PAT) catalyses the incorporation of an acyl group from either acyl-acyl carrier proteins (acylACPs) or acylCoAs into the sn-1 position of glycerol 3-phosphate to yield 1-acylglycerol 3-phosphate. Crystals of squash G3PAT have been obtained by the hanging-drop method of vapour diffusion using PEG 4000 as the precipitant. These crystals are most likely to belong to space group P2(1)2(1)2(1), with approximate unit-cell parameters a = 61.1, b = 65.1, c = 103.3 A, alpha = beta = gamma = 90 degrees and a monomer in the asymmetric unit. X-ray diffraction data to 1.9 A resolution have been collected in-house using a MAR 345 imaging-plate system. PMID:11223529

  4. Modeling of glycerol-3-phosphate transporter suggests a potential 'tilt' mechanism involved in its function.

    PubMed

    Tsigelny, Igor F; Greenberg, Jerry; Kouznetsova, Valentina; Nigam, Sanjay K

    2008-10-01

    Many major facilitator superfamily (MFS) transporters have similar 12-transmembrane alpha-helical topologies with two six-helix halves connected by a long loop. In humans, these transporters participate in key physiological processes and are also, as in the case of members of the organic anion transporter (OAT) family, of pharmaceutical interest. Recently, crystal structures of two bacterial representatives of the MFS family--the glycerol-3-phosphate transporter (GlpT) and lac-permease (LacY)--have been solved and, because of assumptions regarding the high structural conservation of this family, there is hope that the results can be applied to mammalian transporters as well. Based on crystallography, it has been suggested that a major conformational "switching" mechanism accounts for ligand transport by MFS proteins. This conformational switch would then allow periodic changes in the overall transporter configuration, resulting in its cyclic opening to the periplasm or cytoplasm. Following this lead, we have modeled a possible "switch" mechanism in GlpT, using the concept of rotation of protein domains as in the DynDom program17 and membranephilic constraints predicted by the MAPAS program.(23) We found that the minima of energies of intersubunit interactions support two alternate positions consistent with their transport properties. Thus, for GlpT, a "tilt" of 9 degrees -10 degrees rotation had the most favorable energetics of electrostatic interaction between the two halves of the transporter; moreover, this confirmation was sufficient to suggest transport of the ligand across the membrane. We conducted steered molecular dynamics simulations of the GlpT-ligand system to explore how glycerol-3-phosphate would be handled by the "tilted" structure, and obtained results generally consistent with experimental mutagenesis data. While biochemical data remain most consistent with a single-site alternating access model, our results raise the possibility that, while the

  5. Dengue Virus NS1 Protein Modulates Cellular Energy Metabolism by Increasing Glyceraldehyde-3-Phosphate Dehydrogenase Activity

    PubMed Central

    Allonso, Diego; Andrade, Iamara S.; Conde, Jonas N.; Coelho, Diego R.; Rocha, Daniele C. P.; da Silva, Manuela L.; Ventura, Gustavo T.

    2015-01-01

    ABSTRACT Dengue is one of the main public health concerns worldwide. Recent estimates indicate that over 390 million people are infected annually with the dengue virus (DENV), resulting in thousands of deaths. Among the DENV nonstructural proteins, the NS1 protein is the only one whose function during replication is still unknown. NS1 is a 46- to 55-kDa glycoprotein commonly found as both a membrane-associated homodimer and a soluble hexameric barrel-shaped lipoprotein. Despite its role in the pathogenic process, NS1 is essential for proper RNA accumulation and virus production. In the present study, we identified that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) interacts with intracellular NS1. Molecular docking revealed that this interaction occurs through the hydrophobic protrusion of NS1 and the hydrophobic residues located at the opposite side of the catalytic site. Moreover, addition of purified recombinant NS1 enhanced the glycolytic activity of GAPDH in vitro. Interestingly, we observed that DENV infection promoted the relocalization of GAPDH to the perinuclear region, where NS1 is commonly found. Both DENV infection and expression of NS1 itself resulted in increased GAPDH activity. Our findings indicate that the NS1 protein acts to increase glycolytic flux and, consequently, energy production, which is consistent with the recent finding that DENV induces and requires glycolysis for proper replication. This is the first report to propose that NS1 is an important modulator of cellular energy metabolism. The data presented here provide new insights that may be useful for further drug design and the development of alternative antiviral therapies against DENV. IMPORTANCE Dengue represents a serious public health problem worldwide and is caused by infection with dengue virus (DENV). Estimates indicate that half of the global population is at risk of infection, with almost 400 million cases occurring per year. The NS1 glycoprotein is found in both the

  6. Glycerol-3-Phosphate Levels Are Associated with Basal Resistance to the Hemibiotrophic Fungus Colletotrichum higginsianum in Arabidopsis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Glycerol-3-phosphate (G3P) is an important component of carbohydrate and lipid metabolic processes. In this article, we provide evidence that G3P levels in plants are associated with defense to a hemibiotrophic fungal pathogen Colletotrichum higginsianum. Inoculation of Arabidopsis (Arabidopsis thal...

  7. GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE-S, A SPERM-SPECIFIC GLYCOLYTIC ENZYME, IS REQUIRED FOR SPERM MOTILITY AND MALE FERTILITY

    EPA Science Inventory

    While glycolysis is highly conserved, it is remarkable that several novel isozymes in this central metabolic pathway are found in mammalian sperm. Glyceraldehyde 3-phosphate dehydrogenase-S (GAPDS) is the product of a mouse gene expressed only during spermatogenesis and, like it...

  8. Enhanced resistance in Theobroma cacao against oomycete and fungal pathogens by secretion of phosphatidylinositol-3-phosphate-binding proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The internalization of oomycete and fungal pathogen effectors into host plant cells has been reported to be blocked by proteins that bind to the effectors’ cell entry receptor, phosphatidylinositol-3-phosphate (PI3P). This finding suggested a novel strategy for disease control by engineering plants ...

  9. ROS generation and multiple forms of mammalian mitochondrial glycerol-3-phosphate dehydrogenase.

    PubMed

    Mráček, Tomáš; Holzerová, Eliška; Drahota, Zdeněk; Kovářová, Nikola; Vrbacký, Marek; Ješina, Pavel; Houštěk, Josef

    2014-01-01

    Overproduction of reactive oxygen species (ROS) has been implicated in a range of pathologies. Mitochondrial flavin dehydrogenases glycerol-3-phosphate dehydrogenase (mGPDH) and succinate dehydrogenase (SDH) represent important ROS source, but the mechanism of electron leak is still poorly understood. To investigate the ROS production by the isolated dehydrogenases, we used brown adipose tissue mitochondria solubilized by digitonin as a model. Enzyme activity measurements and hydrogen peroxide production studies by Amplex Red fluorescence, and luminol luminescence in combination with oxygraphy revealed flavin as the most likely source of electron leak in SDH under in vivo conditions, while we propose coenzyme Q as the site of ROS production in the case of mGPDH. Distinct mechanism of ROS production by the two dehydrogenases is also apparent from induction of ROS generation by ferricyanide which is unique for mGPDH. Furthermore, using native electrophoretic systems, we demonstrated that mGPDH associates into homooligomers as well as high molecular weight supercomplexes, which represent native forms of mGPDH in the membrane. By this approach, we also directly demonstrated that isolated mGPDH itself as well as its supramolecular assemblies are all capable of ROS production. PMID:23999537

  10. Oxidatively Modified Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) and Alzheimer Disease: Many Pathways to Neurodegeneration

    PubMed Central

    Butterfield, D. Allan; Hardas, Sarita S.; Bader Lange, Miranda L.

    2009-01-01

    Recently, the oxidoreductase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), has become a subject of interest as more and more studies reveal a surfeit of diverse GAPDH functions, extending beyond traditional aerobic metabolism of glucose. As a result of multiple isoforms and cellular locales, GAPDH is able to come in contact with a variety of small molecules, proteins, membranes, etc. that play important roles in normal and pathologic cell function. Specifically, GAPDH has been shown to interact with neurodegenerative disease-associated proteins, including the β-amyloid precursor protein (AβPP). Studies from our laboratory have shown significant inhibition of GAPDH dehydrogenase activity in Alzheimer disease (AD) brain due to oxidative modification. Although, oxidative stress and damage is a common phenomenon in AD brain, it would seem that inhibition of glycolytic enzyme activity is merely one avenue in which AD pathology affects neuronal cell development and survival, as oxidative modification can also impart a toxic gain-of-function to many proteins, including GAPDH. In this review, we examine the many functions of GAPDH with respect to AD brain; in particular, GAPDH’s apparent role(s) in AD-related apoptotic cell death is emphasized. PMID:20164570

  11. Isolation of a functional, insulin regulatable glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene

    SciTech Connect

    Alexander-Bridges, M.; Ramaika, C.; Lomanto, M.; Florence, B.; Ercolani, L.

    1987-05-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a glycolytic enzyme which is regulated by insulin in 3T3 adipocytes and liver. They have isolated a 10 Kb BAM fragment which contains the entire GAPDH coding sequence and 1 Kb of the 5' flanking region. This clone has been mapped and sequenced to show the presence of a TATAA box; ATG, TAA, ATAA sites; and introns. When transiently expressed in L cells, the gene encodes a full length mRNA and functional protein. Thus, they have isolated a functional gene and not a pseudogene. When the gene is expressed in insulin-sensitive cells, the human GAPDH mRNA level was increased 3-fold in H35 hepatoma cells similar to the fold effect seen for endogenous rat mRNA in the same experiment. A 600 bp fragment of the GAPDH gene 5' flanking sequence was subcloned into a vector containing the chloramphenicol acetyl transferase (CAT) gene and cotransfected with the neomycin-resistant gene in H35 hepatoma cells to select stable lines. RNA isolated from control- and insulin-treated transfected cells show an appropriately sized S1 nuclease-protected fragment indicating that both the control species and insulin-stimulated species are primed off the GAPDH protomer. These studies indicate that insulin regulates GAPDH gene expression through an interaction with specific DNA sequences in rat hepatoma cells.

  12. On the interaction between glyceraldehyde-3-phosphate dehydrogenase and airborne particles: Evidence for electrophilic species

    NASA Astrophysics Data System (ADS)

    Shinyashiki, Masaru; Rodriguez, Chester E.; Di Stefano, Emma W.; Sioutas, Constantinos; Delfino, Ralph J.; Kumagai, Yoshito; Froines, John R.; Cho, Arthur K.

    Many of the adverse health effects of airborne particulate matter (PM) have been attributed to the chemical properties of some of the large number of chemical species present in PM. Some PM component chemicals are capable of generating reactive oxygen species and eliciting a state of oxidative stress. In addition, however, PM can contain chemical species that elicit their effects through covalent bond formation with nucleophilic functions in the cell. In this manuscript, we report the presence of constituents with electrophilic properties in ambient and diesel exhaust particles, demonstrated by their ability to inhibit the thiol enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). GAPDH is irreversibly inactivated by electrophiles under anaerobic conditions by covalent bond formation. This inactivation can be blocked by the prior addition of a high concentration of dithiothreitol (DTT) as an alternate nucleophile. Addition of DTT after the reaction between the electrophile and GAPDH, however, does not reverse the inactivation. This property has been utilized to develop a procedure that provides a quantitative measure of electrophiles present in samples of ambient particles collected in the Los Angeles Basin and in diesel exhaust particles. The toxicity of electrophiles is the result of irreversible changes in biological molecules; recovery is dependent on resynthesis. If the resynthesis is slow, the irreversible effects can be cumulative and manifest themselves after chronic exposure to low levels of electrophiles.

  13. Glyceraldehyde-3-phosphate dehydrogenase gene from Zymomonas mobilis: cloning, sequencing, and identification of promoter region

    SciTech Connect

    Conway, T.; Sewell, G.W.; Ingram, L.O.

    1987-12-01

    The gene encoding glyceraldehyde-3-phosphate dehydrogenase was isolated from a library of Zymomonas mobilis DNA fragments by complementing a deficient strain of Escherichia coli. It contained tandem promoters which were recognized by E. coli but appeared to function less efficiently than the enteric lac promoter in E. coli. The open reading frame for this gene encoded 337 amino acids with an aggregate molecular weight of 36,099 (including the N-terminal methionine). The primary amino acid sequence for this gene had considerable functional homology and amino acid identity with other eukaryotic and bacterial genes. Based on this comparison, the gap gene from Z. mobilis appeared to be most closely related to that of the thermophilic bacteria and to the chloroplast isozymes. Comparison of this gene with other glycolytic enzymes from Z. mobilis revealed a conserved pattern of codon bias and several common features of gene structure. A tentative transcriptional consensus sequence is proposed for Z. mobilis based on comparison of the five known promoters for three glycolytic enzymes.

  14. Glyceraldehyde 3-Phosphate Dehydrogenase-Telomere Association Correlates with Redox Status in Trypanosoma cruzi

    PubMed Central

    Pariona-Llanos, Ricardo; Pavani, Raphael Souza; Reis, Marcelo; Noël, Vincent; Silber, Ariel Mariano; Armelin, Hugo Aguirre; Cano, Maria Isabel Nogueira; Elias, Maria Carolina

    2015-01-01

    Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a classical metabolic enzyme involved in energy production and plays a role in additional nuclear functions, including transcriptional control, recognition of misincorporated nucleotides in DNA and maintenance of telomere structure. Here, we show that the recombinant protein T. cruzi GAPDH (rTcGAPDH) binds single-stranded telomeric DNA. We demonstrate that the binding of GAPDH to telomeric DNA correlates with the balance between oxidized and reduced forms of nicotinamide adenine dinucleotides (NAD+/NADH). We observed that GAPDH-telomere association and NAD+/NADH balance changed throughout the T. cruzi life cycle. For example, in replicative epimastigote forms of T. cruzi, which show similar intracellular concentrations of NAD+ and NADH, GAPDH binds to telomeric DNA in vivo and this binding activity is inhibited by exogenous NAD+. In contrast, in the T. cruzi non-proliferative trypomastigote forms, which show higher NAD+ concentration, GAPDH was absent from telomeres. In addition, NAD+ abolishes physical interaction between recombinant GAPDH and synthetic telomere oligonucleotide in a cell free system, mimicking exogenous NAD+ that reduces GAPDH-telomere interaction in vivo. We propose that the balance in the NAD+/NADH ratio during T. cruzi life cycle homeostatically regulates GAPDH telomere association, suggesting that in trypanosomes redox status locally modulates GAPDH association with telomeric DNA. PMID:25775131

  15. Lactobacillus reuteri glyceraldehyde-3-phosphate dehydrogenase functions in adhesion to intestinal epithelial cells.

    PubMed

    Zhang, Wen-Ming; Wang, Hai-Feng; Gao, Kan; Wang, Cong; Liu, Li; Liu, Jian-Xin

    2015-05-01

    This study was aimed to identify key surface proteins mediating the adhesion of lactobacilli to intestinal epithelial cells. By using Caco-2 and IPEC-J2 cells labeled with sulfo-NHS-biotin in the western blotting, a protein band of an approximately 37 kDa was detected on the surface layer of Lactobacillus reuteri strains ZJ616, ZJ617, ZJ621, and ZJ623 and Lactobacillus rhamnosus GG. Mass spectrometry analysis using the adhesion-related protein from L. reuteri ZJ617 showed that it was 100% homologous to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of L. reuteri JCM 1112 (GenBank: YP_001841377). The ability of L. reuteri ZJ617 to adhere to epithelial cells decreased significantly by treatment with LiCl or by blocking with an anti-GAPDH antibody, in comparison with the untreated strain (p < 0.05). Immunoelectron microscopic and immunofluorescence analyses confirmed that GAPDH is located on the surface layer of L. reuteri ZJ617. The results indicated that the GAPDH protein of L. reuteri ZJ617 acts as an adhesion component that plays an important role in binding to the intestinal epithelial cells. PMID:25867279

  16. Phosphatidylinositol-3-phosphate is light-regulated and essential for survival in retinal rods

    PubMed Central

    He, Feng; Agosto, Melina A.; Anastassov, Ivan A.; Tse, Dennis Y.; Wu, Samuel M.; Wensel, Theodore G.

    2016-01-01

    Phosphoinositides play important roles in numerous intracellular membrane pathways. Little is known about the regulation or function of these lipids in rod photoreceptor cells, which have highly active membrane dynamics. Using new assays with femtomole sensitivity, we determined that whereas levels of phosphatidylinositol-3,4-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate were below detection limits, phosphatidylinositol-3-phosphate (PI(3)P) levels in rod inner/outer segments increased more than 30-fold after light exposure. This increase was blocked in a rod-specific knockout of the PI-3 kinase Vps34, resulting in failure of endosomal and autophagy-related membranes to fuse with lysosomes, and accumulation of abnormal membrane structures. At early ages, rods displayed normal morphology, rhodopsin trafficking, and light responses, but underwent progressive neurodegeneration with eventual loss of both rods and cones by twelve weeks. The degeneration is considerably faster than in rod knockouts of autophagy genes, indicating defects in endosome recycling or other PI(3)P-dependent membrane trafficking pathways are also essential for rod survival. PMID:27245220

  17. Glycolytic flux controls d-serine synthesis through glyceraldehyde-3-phosphate dehydrogenase in astrocytes

    PubMed Central

    Suzuki, Masataka; Sasabe, Jumpei; Miyoshi, Yurika; Kuwasako, Kanako; Muto, Yutaka; Hamase, Kenji; Matsuoka, Masaaki; Imanishi, Nobuaki; Aiso, Sadakazu

    2015-01-01

    d-Serine is an essential coagonist with glutamate for stimulation of N-methyl-d-aspartate (NMDA) glutamate receptors. Although astrocytic metabolic processes are known to regulate synaptic glutamate levels, mechanisms that control d-serine levels are not well defined. Here we show that d-serine production in astrocytes is modulated by the interaction between the d-serine synthetic enzyme serine racemase (SRR) and a glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase (GAPDH). In primary cultured astrocytes, glycolysis activity was negatively correlated with d-serine level. We show that SRR interacts directly with GAPDH, and that activation of glycolysis augments this interaction. Biochemical assays using mutant forms of GAPDH with either reduced activity or reduced affinity to SRR revealed that GAPDH suppresses SRR activity by direct binding to GAPDH and through NADH, a product of GAPDH. NADH allosterically inhibits the activity of SRR by promoting the disassociation of ATP from SRR. Thus, astrocytic production of d-serine is modulated by glycolytic activity via interactions between GAPDH and SRR. We found that SRR is expressed in astrocytes in the subiculum of the human hippocampus, where neurons are known to be particularly vulnerable to loss of energy. Collectively, our findings suggest that astrocytic energy metabolism controls d-serine production, thereby influencing glutamatergic neurotransmission in the hippocampus. PMID:25870284

  18. Reciprocal Phosphorylation of Yeast Glycerol-3-Phosphate Dehydrogenases in Adaptation to Distinct Types of Stress

    PubMed Central

    Lee, Yong Jae; Jeschke, Grace R.; Roelants, Françoise M.; Thorner, Jeremy

    2012-01-01

    Eukaryotic cells have evolved mechanisms for ensuring growth and survival in the face of stress caused by a fluctuating environment. Saccharomyces cerevisiae has two homologous glycerol-3-phosphate dehydrogenases, Gpd1 and Gpd2, that are required to endure various stresses, including hyperosmotic shock and hypoxia. These enzymes are only partially redundant, and their unique functions were attributed previously to differential transcriptional regulation and localization. We find that Gpd1 and Gpd2 are negatively regulated through phosphorylation by distinct kinases under reciprocal conditions. Gpd2 is phosphorylated by the AMP-activated protein kinase Snf1 to curtail glycerol production when nutrients are limiting. Gpd1, in contrast, is a target of TORC2-dependent kinases Ypk1 and Ypk2. Inactivation of Ypk1 by hyperosmotic shock results in dephosphorylation and activation of Gpd1, accelerating recovery through increased glycerol production. Gpd1 dephosphorylation acts synergistically with its transcriptional upregulation, enabling long-term growth at high osmolarity. Phosphorylation of Gpd1 and Gpd2 by distinct kinases thereby enables rapid adaptation to specific stress conditions. Introduction of phosphorylation motifs targeted by distinct kinases provides a general mechanism for functional specialization of duplicated genes during evolution. PMID:22988299

  19. Phosphatidylinositol-3-phosphate is light-regulated and essential for survival in retinal rods.

    PubMed

    He, Feng; Agosto, Melina A; Anastassov, Ivan A; Tse, Dennis Y; Wu, Samuel M; Wensel, Theodore G

    2016-01-01

    Phosphoinositides play important roles in numerous intracellular membrane pathways. Little is known about the regulation or function of these lipids in rod photoreceptor cells, which have highly active membrane dynamics. Using new assays with femtomole sensitivity, we determined that whereas levels of phosphatidylinositol-3,4-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate were below detection limits, phosphatidylinositol-3-phosphate (PI(3)P) levels in rod inner/outer segments increased more than 30-fold after light exposure. This increase was blocked in a rod-specific knockout of the PI-3 kinase Vps34, resulting in failure of endosomal and autophagy-related membranes to fuse with lysosomes, and accumulation of abnormal membrane structures. At early ages, rods displayed normal morphology, rhodopsin trafficking, and light responses, but underwent progressive neurodegeneration with eventual loss of both rods and cones by twelve weeks. The degeneration is considerably faster than in rod knockouts of autophagy genes, indicating defects in endosome recycling or other PI(3)P-dependent membrane trafficking pathways are also essential for rod survival. PMID:27245220

  20. Mitochondrial FAD-linked Glycerol-3-phosphate Dehydrogenase: A Target for Cancer Therapeutics

    PubMed Central

    Singh, Gurmit

    2014-01-01

    Imbalances in cellular redox state are frequently observed in cancer cells, and contribute significantly to cancer progression and apoptotic resistance. Hydrogen peroxide (H2O2) is one reactive oxygen species (ROS) that is produced in excess within cancer cells. In this study, we investigated the mitochondrial glycerol-3-phosphate-dependent (GPD2) ROS production in PC-3 cells and demonstrated the importance of excessive H2O2 production on their survival. By exploiting the abnormal H2O2 production of PC-3 cells, we initiated a high-throughput screening of the Canadian Compound Collection, composed of 29,586 small molecules, targeting the glycerophosphate-dependent H2O2 formation in PC-3 cells. Eighteen compounds were identified to have significant inhibitory activity. These compounds have not been previously characterized as inhibitors of the enzyme. Six of these compounds were further analyzed in PC-3 cells and dose response studies displayed an inhibitory and anti-oxidative potency that ranged from 1 µM to 30 µM. The results presented here demonstrate that inhibitors of mitochondrial GPD2 activity elicit anti-proliferative effects on cancer cells. PMID:24521925

  1. Chemical Synthesis and Molecular Recognition of Phosphatase-Resistant Analogues of Phosphatidylinositol-3-phosphate

    PubMed Central

    Xu, Yong; Lee, Stephanie A.; Kutateladze, Tatiana G.; Sbrissa, Diego; Shisheva, Assia; Prestwich, Glenn D.

    2008-01-01

    The remodeling of phosphatidylinositol polyphosphates in cellular membranes by phosphatases and kinases orchestrates the signaling by these lipids in space and time. In order to provide chemical tools to study of the changes in cell physiology mediated by these lipids, three new metabolically-stabilized (ms) analogues of phosphatidylinositol-3-phosphate (PtdIns(3)P were synthesized. We describe herein the total asymmetric synthesis of 3-methylphosphonate, 3-monofluoromethylphosphonate and 3-phosphorothioate analogues of PtdIns(3)P. From differentially protected D-myo-inositol key intermediates, a versatile phosphoramidite reagent was employed in the synthesis of PtdIns(3)P analogues with diacylglyceryl moieties containing dioleoyl, dipalmitoyl and dibutyryl chains. In addition, we introduce a new phosphorlyation reagent, monofluoromethylphosphonyl chloride, which has general applications for the preparation of “pKa-matched” monofluorophosphonates. These ms-PtdIns(3)P analogues exhibited reduced binding activities with 15N-labelled FYVE and PX domains, as significant 1H and 15N chemical shift changes in the FYVE domain were induced by titrating ms-PtdIns(3)Ps into membrane-mimetic dodecylphosphocholine (DPC) micelles. In addition, the PtdIns(3)P analogues with dioleyl and dipalmitoyl chains were substrates for the 5-kinase enzyme PIKfyve; the corresponding phosphorylated ms-PI(3,5)P2 products were detected by radio-TLC analysis. PMID:16417379

  2. U(VI) Sequestration in Hydroxyapatite Produced by Microbial Glycerol 3-Phosphate Metabolism▿ †

    PubMed Central

    Shelobolina, Evgenya S.; Konishi, Hiromi; Xu, Huifang; Roden, Eric E.

    2009-01-01

    Previous studies have demonstrated the potential for removal of U(VI) from solution via precipitation of U(VI)-bearing calcium-phosphate (Ca-P) minerals coupled to microbial hydrolysis of glycerol phosphate compounds. We evaluated this process in circumneutral-pH groundwater from Area 2 of the U.S. Department of Energy Field Research Center at Oak Ridge National Laboratory. Area 2 groundwater contains high concentrations of dissolved calcium (ca. 4 mM), and thus, release of phosphate during glycerol phosphate metabolism has the potential to create conditions favorable for U(VI) sequestration in Ca-P minerals. Microbial enumeration and isolation studies verified the presence of aerobic and nitrate-reducing glycerol 3-phosphate (G3P)-metabolizing microorganisms in Area 2 sediments. Coprecipitation of U(VI) with Ca-P minerals coupled to microbial G3P hydrolysis was demonstrated in artificial groundwater under aerobic and nitrate-reducing conditions. Transmission electron microscopy analysis and mineral-washing experiments demonstrated that U(VI) was incorporated into the structure of the insoluble Ca-P mineral hydroxyapatite [Ca5(PO4)3OH]. Our results support the idea that U(VI) can be effectively removed from solution in contaminated aquifers through stimulation of microbial organophosphate metabolism. PMID:19633115

  3. Glyceraldehyde 3-phosphate dehydrogenase-telomere association correlates with redox status in Trypanosoma cruzi.

    PubMed

    Pariona-Llanos, Ricardo; Pavani, Raphael Souza; Reis, Marcelo; Noël, Vincent; Silber, Ariel Mariano; Armelin, Hugo Aguirre; Cano, Maria Isabel Nogueira; Elias, Maria Carolina

    2015-01-01

    Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a classical metabolic enzyme involved in energy production and plays a role in additional nuclear functions, including transcriptional control, recognition of misincorporated nucleotides in DNA and maintenance of telomere structure. Here, we show that the recombinant protein T. cruzi GAPDH (rTcGAPDH) binds single-stranded telomeric DNA. We demonstrate that the binding of GAPDH to telomeric DNA correlates with the balance between oxidized and reduced forms of nicotinamide adenine dinucleotides (NAD+/NADH). We observed that GAPDH-telomere association and NAD+/NADH balance changed throughout the T. cruzi life cycle. For example, in replicative epimastigote forms of T. cruzi, which show similar intracellular concentrations of NAD+ and NADH, GAPDH binds to telomeric DNA in vivo and this binding activity is inhibited by exogenous NAD+. In contrast, in the T. cruzi non-proliferative trypomastigote forms, which show higher NAD+ concentration, GAPDH was absent from telomeres. In addition, NAD+ abolishes physical interaction between recombinant GAPDH and synthetic telomere oligonucleotide in a cell free system, mimicking exogenous NAD+ that reduces GAPDH-telomere interaction in vivo. We propose that the balance in the NAD+/NADH ratio during T. cruzi life cycle homeostatically regulates GAPDH telomere association, suggesting that in trypanosomes redox status locally modulates GAPDH association with telomeric DNA. PMID:25775131

  4. Cloning, characterization and application of a glyceraldehyde-3-phosphate dehydrogenase promoter from Aspergillus terreus.

    PubMed

    Huang, Xuenian; Lu, Xuefeng; Li, Jian-Jun

    2014-03-01

    It is important to develop native and highly efficient promoters for effective genetic engineering of filamentous fungi. Although Aspergillus terreus is an important industrial fungus for the production of itaconic acid and lovastatin, the available genetic toolbox for this microorganism is still rather limited. We have cloned the 5' upstream region of the glyceraldehyde-3-phosphate dehydrogenase gene (gpd; 2,150 bp from the start codon) from A. terreus CICC 40205 and subsequently confirmed its promoter function using sgfp (synthetic green fluorescent protein) as the reporter. The sequence of the promoter PgpdAt was further analysed by systematic deletion to obtain an effective and compact functional promoter. Two truncated versions of PgpdAt (1,081 and 630 bp) were also able to drive sgfp expression in A. terreus. The activities of these three PgpdAt promoters of varying different lengths were further confirmed by fluorescence, western blot and transcription. The shortest one (630 bp) was successfully applied as a driver of vgb expression in the genetic engineering of A. terreus. The function of expressed haemoglobin was demonstrated by the CO (carbon monoxide)-difference spectrum and enhanced oxygen uptake rate, glucose consumption and itaconic acid titer. Our study was successful in developing and validating an efficient and compact native promoter for genetic engineering of A. terreus. PMID:24306453

  5. Assisted folding of D-glyceraldehyde-3-phosphate dehydrogenase by trigger factor.

    PubMed Central

    Huang, G. C.; Li, Z. Y.; Zhou, J. M.; Fischer, G.

    2000-01-01

    The Escherichia coli trigger factor is a peptidyl-prolyl cis-trans isomerase that catalyzes proline-limited protein folding extremely well. Here, refolding of D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the presence of trigger factor was investigated. The regain of activity of GAPDH was markedly increased by trigger factor after either long- or short-term denaturation, and detectable aggregation of GAPDH intermediates was prevented. In both cases, time courses of refolding of GAPDH were decelerated by trigger factor. The reactivation yield of GAPDH showed a slow down-turn when molar ratios of trigger factor to GAPDH were above 5, due to tight binding between trigger factor and GAPDH intermediates. Such inactive bound GAPDH could be partially rescued from trigger factor by addition of reduced alphaLA as competitor, by further diluting the refolding mixture, or by disrupting hydrophobic interactions in the complexes. A model for trigger factor assisted refolding of GAPDH is proposed. We also suggest that assisted refolding of GAPDH is due mainly to the chaperone function of trigger factor. PMID:10892818

  6. Modulation of glyceraldehyde-3-phosphate dehydrogenase activity by surface functionalized quantum dots.

    PubMed

    Ghosh, Srabanti; Ray, Manju; Das, Mahua Rani; Chakrabarti, Adrita; Khan, Ali Hossain; Sarma, D D; Acharya, Somobrata

    2014-03-21

    Enzymatic regulation is a fast and reliable diagnosis tool via identification and design of inhibitors for modulation of enzyme function. Previous reports on quantum dots (QDs)-enzyme interactions reveal a protein-surface recognition ability leading to promising applications in protein stabilization, protein delivery, bio-sensing and detection. However, the direct use of QDs to control enzyme inhibition has never been revealed to date. Here we show that a series of biocompatible surface-functionalized metal-chalcogenide QDs can be used as potent inhibitors for malignant cells through the modulation of enzyme activity, while normal cells remain unaffected. The in vitro activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an enzyme involved critically in the glycolysis of cancer cells, is inactivated selectively in a controlled way by the QDs at a significantly low concentration (nM). Cumulative kinetic studies delineate that the QDs undergo both reversible and irreversible inhibition mechanisms owing to the site-specific interactions, enabling control over the inhibition kinetics. These complementary loss-of-function probes may offer a novel route for rapid clinical diagnosis of malignant cells and biomedical applications. PMID:24496476

  7. Structural and functional properties of glycerol-3-phosphate dehydrogenase from a mammalian hibernator.

    PubMed

    de la Roche, Marc; Tessier, Shannon N; Storey, Kenneth B

    2012-02-01

    Glycerol-3-phosphate dehydrogenase (G3PDH; E.C.1.1.1.8) was purified from liver and skeletal muscle of black-tailed prairie dogs (Cynomys ludivicianus), a hibernating species. Native and subunit molecular masses of the dimeric enzyme were 77 and 40 kD, respectively, and both tissues contained a single isozyme with a pI of 6.4. Kinetic parameters of purified G3PDH from prairie dog liver and muscle were characterized at 22 and 5 °C and compared with rabbit muscle G3PDH. Substrate affinities for hibernator muscle G3PDH were stable (NAD) or increased significantly (K(m) G3P and DHAP decreased) at low temperature whereas K(m) NAD and DHAP of rabbit G3PDH increased. Prairie dog G3PDH showed greater conservation of K(m) G3P over a wide temperature range as well as greater thermal stability and resistance to chemical denaturation by guanidine hydrochloride than the rabbit enzyme. In addition, using the protein sequence of the hibernating thirteen-lined ground squirrel (Ictidomys tridecemlineatus) and bioinformatics tools, the deduced protein structure of G3PDH was compared between heterothermic and homeothermic mammals. Structural and functional characteristics of G3PDH from the hibernating species would support enzyme function over a wide range of core body temperatures over cycles of torpor and arousal. PMID:22180227

  8. Isolation and some properties of glycated D-glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle.

    PubMed Central

    He, R Q; Yang, M D; Zheng, X; Zhou, J X

    1995-01-01

    Glycated D-glyceraldehyde-3-phosphate dehydrogenases (GAPDH) from rabbit muscle and human erythrocytes have been investigated. The specific activity of the non-glycated GAPDH from rabbit muscle is approx. 180 units. (One unit is defined as the specific activity required to convert 1 microM of substrate/min per mg of enzyme.) The activity of the glycated enzyme, consisting of two sugars per tetramer, is lower than that of the non-glycated GAPDH. Non-enzymic transamination of the N-termini of glycated GAPDH (gGAPDH) indicates that they are not blocked by glycation. The rate of modification of thiols (Cys-149) with 5,5'-dithiobis-(2-nitrobenzoic acid) was greater for the glycated than the non-glycated enzymes. The rate of modification of amino groups of Lys residues of gGAPDH with o-phthalaldehyde was greater for the non-glycated enzyme. In 0.18 M guanidine-HC1 solution, the emission intensity at 410 nm of a fluorescent NAD+ derivative introduced into the active site decreased to 80%, whereas that of gGAPDH decreased to 50%. This suggests that the glycated sites are near the active site; glycation of the enzyme leads to a change of the microenvironment of Cys-149, alters the conformation of the active site and decreases the activity. Images Figure 1 PMID:7619048

  9. Negative regulation of phosphatidylinositol 3-phosphate levels in early-to-late endosome conversion

    PubMed Central

    Liu, Kai; Jian, Youli; Sun, Xiaojuan; Yang, Chengkui; Gao, Zhiyang; Zhang, Zhili; Liu, Xuezhao; Li, Yang; Xu, Jing; Jing, Yudong; Mitani, Shohei; He, Sudan

    2016-01-01

    Phosphatidylinositol 3-phosphate (PtdIns3P) plays a central role in endosome fusion, recycling, sorting, and early-to-late endosome conversion, but the mechanisms that determine how the correct endosomal PtdIns3P level is achieved remain largely elusive. Here we identify two new factors, SORF-1 and SORF-2, as essential PtdIns3P regulators in Caenorhabditis elegans. Loss of sorf-1 or sorf-2 leads to greatly elevated endosomal PtdIns3P, which drives excessive fusion of early endosomes. sorf-1 and sorf-2 function coordinately with Rab switching genes to inhibit synthesis of PtdIns3P, allowing its turnover for endosome conversion. SORF-1 and SORF-2 act in a complex with BEC-1/Beclin1, and their loss causes elevated activity of the phosphatidylinositol 3-kinase (PI3K) complex. In mammalian cells, inactivation of WDR91 and WDR81, the homologs of SORF-1 and SORF-2, induces Beclin1-dependent enlargement of PtdIns3P-enriched endosomes and defective degradation of epidermal growth factor receptor. WDR91 and WDR81 interact with Beclin1 and inhibit PI3K complex activity. These findings reveal a conserved mechanism that controls appropriate PtdIns3P levels in early-to-late endosome conversion. PMID:26783301

  10. Generation of insect-resistant and glyphosate-tolerant rice by introduction of a T-DNA containing two Bt insecticidal genes and an EPSPS gene*

    PubMed Central

    ZHAO, Qi-chao; LIU, Ming-hong; ZHANG, Xian-wen; LIN, Chao-yang; ZHANG, Qing; SHEN, Zhi-cheng

    2015-01-01

    Insect resistance and glyphosate tolerance have been two of the most important traits in the genetic improvement of various crops. In this study, two Bacillus thuringiensis (Bt) insecticidal genes, Cry1Ac and Cry1Ig, and a modified glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene (G10) were combined into a single transferred DNA (T-DNA) fragment and introduced into rice by Agrobacterium-mediated transformation. A transgenic line with single-copy T-DNA insertion named GAI-14 was found to be highly resistant to striped stem borer and rice leaf roller, and tolerant to glyphosate. Analysis of T-DNA border sequence suggested that the transgenes were inserted at the chromosome 3 and appeared to have not interrupted any known or putative genes. A field trial observed no significant difference in the basic agronomic traits between GAI-14 and the recipient rice. PMID:26465130

  11. Cloning and characterization of the aroA gene from Mycobacterium tuberculosis.

    PubMed Central

    Garbe, T; Jones, C; Charles, I; Dougan, G; Young, D

    1990-01-01

    The aroA gene from Mycobacterium tuberculosis has been cloned by complementation of an aroA mutant of Escherichia coli after lysogenization with a recombinant DNA library in the lambda gt11 vector. Detailed characterization of the M. tuberculosis aroA gene by nucleotide sequencing and by immunochemical analysis of the expressed product indicates that it encodes a 5-enolpyruvylshikimate-3-phosphate synthase that is structurally related to analogous enzymes from other bacterial, fungal, and plant sources. The potential use of the cloned gene in construction of genetically defined mutant strains of M. tuberculosis by gene replacement is proposed as a novel approach to the rational attenuation of mycobacterial pathogens and the possible development of new antimycobacterial vaccines. Images PMID:2123856

  12. Characterization of the Amaranthus palmeri Physiological Response to Glyphosate in Susceptible and Resistant Populations.

    PubMed

    Fernández-Escalada, Manuel; Gil-Monreal, Miriam; Zabalza, Ana; Royuela, Mercedes

    2016-01-13

    The herbicide glyphosate inhibits the plant enzyme 5-enolpyruvylshikimate3-phosphate synthase (EPSPS) in the aromatic amino acid (AAA) biosynthetic pathway. The physiologies of an Amaranthus palmeri population exhibiting resistance to glyphosate by EPSPS gene amplification (NC-R) and a susceptible population (NC-S) were compared. The EPSPS copy number of NC-R plants was 47.5-fold the copy number of NC-S plants. Although the amounts of EPSPS protein and activity were higher in NC-R plants than in NC-S plants, the AAA concentrations were similar. The increases in total free amino acid and in AAA contents induced by glyphosate were more evident in NC-S plants. In both populations, the EPSPS protein increased after glyphosate exposure, suggesting regulation of gene expression. EPSPS activity seems tightly controlled in vivo. Carbohydrate accumulation and a slight induction of ethanol fermentation were detected in both populations. PMID:26652930

  13. Generation of insect-resistant and glyphosate-tolerant rice by introduction of a T-DNA containing two Bt insecticidal genes and an EPSPS gene.

    PubMed

    Zhao, Qi-chao; Liu, Ming-hong; Zhang, Xian-wen; Lin, Chao-yang; Zhang, Qing; Shen, Zhi-cheng

    2015-10-01

    Insect resistance and glyphosate tolerance have been two of the most important traits in the genetic improvement of various crops. In this study, two Bacillus thuringiensis (Bt) insecticidal genes, Cry1Ac and Cry1Ig, and a modified glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene (G10) were combined into a single transferred DNA (T-DNA) fragment and introduced into rice by Agrobacterium-mediated transformation. A transgenic line with single-copy T-DNA insertion named GAI-14 was found to be highly resistant to striped stem borer and rice leaf roller, and tolerant to glyphosate. Analysis of T-DNA border sequence suggested that the transgenes were inserted at the chromosome 3 and appeared to have not interrupted any known or putative genes. A field trial observed no significant difference in the basic agronomic traits between GAI-14 and the recipient rice. PMID:26465130

  14. Real-Time PCR-Based Quantitation Method for the Genetically Modified Soybean Line GTS 40-3-2.

    PubMed

    Kitta, Kazumi; Takabatake, Reona; Mano, Junichi

    2016-01-01

    This chapter describes a real-time PCR-based method for quantitation of the relative amount of genetically modified (GM) soybean line GTS 40-3-2 [Roundup Ready(®) soybean (RRS)] contained in a batch. The method targets a taxon-specific soybean gene (lectin gene, Le1) and the specific DNA construct junction region between the Petunia hybrida chloroplast transit peptide sequence and the Agrobacterium 5-enolpyruvylshikimate-3-phosphate synthase gene (epsps) sequence present in GTS 40-3-2. The method employs plasmid pMulSL2 as a reference material in order to quantify the relative amount of GTS 40-3-2 in soybean samples using a conversion factor (Cf) equal to the ratio of the RRS-specific DNA to the taxon-specific DNA in representative genuine GTS 40-3-2 seeds. PMID:26614294

  15. Glutathione conjugates recognize the Rossmann fold of glyceraldehyde-3-phosphate dehydrogenase.

    PubMed

    Puder, M; Soberman, R J

    1997-04-18

    Leukotriene (LT) C4 and other glutathione conjugates are synthesized intracellularly and then move to the plasma membrane for export. The intracellular proteins that bind these molecules and the significance of these interactions are poorly understood. To identify the binding sites of membrane-associated proteins that recognize these molecules, we utilized photoaffinity probes to label the inner leaflet of erythrocytes. The predominant molecule labeled with S-(p-nitrobenzyl)glutathione-[125I]4-azidosalicylic acid (PNBG-[125I]ASA) or LTC4-[125I]4-azidosalicylic acid (LTC4-[125I]ASA) was 38 kDa. The protein was labeled with PNBG-[125I]ASA, electroblotted to polyvinylidene difluoride membranes, digested in situ with lysyl endopeptidase, and two radiolabeled peptides isolated by reverse phase-high performance liquid chromatography. These contained an identity of 7/11 with amino acids 119-129, and 11/11 with amino acids 67-77 of human liver glyceraldehyde-3-phosphate dehydrogenase (GAPDH), respectively. Photoaffinity labeling with PNBG-[125I]ASA was blocked completely by 100 microM ATP and greater than 50% with 100 microM NAD+. LTC4-[125I]ASA binding to the NAD+ site was confirmed by V8 protease digestion of purified GAPDH labeled with LTC4-[125I]ASA or PNBG-[125I]ASA, with both labels localized to the 6.8-kDa N-terminal fragment. Photoaffinity labeling of HL-60 cells with LTC4-125I-ASA identified GAPDH as the predominant cytoplasmic binding protein in these cells. These data indicate that GAPDH is a membrane-associated and cytoplasmic protein which binds glutathione conjugates including LTC4. PMID:9099752

  16. Disruption of NAD+ binding site in glyceraldehyde 3-phosphate dehydrogenase affects its intranuclear interactions

    PubMed Central

    Phadke, Manali; Krynetskaia, Natalia; Mishra, Anurag; Barrero, Carlos; Merali, Salim; Gothe, Scott A; Krynetskiy, Evgeny

    2015-01-01

    AIM: To characterize phosphorylation of human glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and mobility of GAPDH in cancer cells treated with chemotherapeutic agents. METHODS: We used proteomics analysis to detect and characterize phosphorylation sites within human GAPDH. Site-specific mutagenesis and alanine scanning was then performed to evaluate functional significance of phosphorylation sites in the GAPDH polypeptide chain. Enzymatic properties of mutated GAPDH variants were assessed using kinetic studies. Intranuclear dynamics parameters (diffusion coefficient and the immobile fraction) were estimated using fluorescence recovery after photobleaching (FRAP) experiments and confocal microscopy. Molecular modeling experiments were performed to estimate the effects of mutations on NAD+ cofactor binding. RESULTS: Using MALDI-TOF analysis, we identified novel phosphorylation sites within the NAD+ binding center of GAPDH at Y94, S98, and T99. Using polyclonal antibody specific to phospho-T99-containing peptide within GAPDH, we demonstrated accumulation of phospho-T99-GAPDH in the nuclear fractions of A549, HCT116, and SW48 cancer cells after cytotoxic stress. We performed site-mutagenesis, and estimated enzymatic properties, intranuclear distribution, and intranuclear mobility of GAPDH mutated variants. Site-mutagenesis at positions S98 and T99 in the NAD+ binding center reduced enzymatic activity of GAPDH due to decreased affinity to NAD+ (Km = 741 ± 257 μmol/L in T99I vs 57 ± 11.1 µmol/L in wild type GAPDH. Molecular modeling experiments revealed the effect of mutations on NAD+ binding with GAPDH. FRAP (fluorescence recovery after photo bleaching) analysis showed that mutations in NAD+ binding center of GAPDH abrogated its intranuclear interactions. CONCLUSION: Our results suggest an important functional role of phosphorylated amino acids in the NAD+ binding center in GAPDH interactions with its intranuclear partners. PMID:26629320

  17. Glyceraldehyde-3-phosphate Dehydrogenase Aggregates Accelerate Amyloid-β Amyloidogenesis in Alzheimer Disease.

    PubMed

    Itakura, Masanori; Nakajima, Hidemitsu; Kubo, Takeya; Semi, Yuko; Kume, Satoshi; Higashida, Shusaku; Kaneshige, Akihiro; Kuwamura, Mitsuru; Harada, Naoki; Kita, Akinori; Azuma, Yasu-Taka; Yamaji, Ryoichi; Inui, Takashi; Takeuchi, Tadayoshi

    2015-10-23

    Alzheimer disease (AD) is a progressive neurodegenerative disorder characterized by loss of neurons and formation of pathological extracellular deposits induced by amyloid-β peptide (Aβ). Numerous studies have established Aβ amyloidogenesis as a hallmark of AD pathogenesis, particularly with respect to mitochondrial dysfunction. We have previously shown that glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forms amyloid-like aggregates upon exposure to oxidative stress and that these aggregates contribute to neuronal cell death. Here, we report that GAPDH aggregates accelerate Aβ amyloidogenesis and subsequent neuronal cell death both in vitro and in vivo. Co-incubation of Aβ40 with small amounts of GAPDH aggregates significantly enhanced Aβ40 amyloidogenesis, as assessed by in vitro thioflavin-T assays. Similarly, structural analyses using Congo red staining, circular dichroism, and atomic force microscopy revealed that GAPDH aggregates induced Aβ40 amyloidogenesis. In PC12 cells, GAPDH aggregates augmented Aβ40-induced cell death, concomitant with disruption of mitochondrial membrane potential. Furthermore, mice injected intracerebroventricularly with Aβ40 co-incubated with GAPDH aggregates exhibited Aβ40-induced pyramidal cell death and gliosis in the hippocampal CA3 region. These observations were accompanied by nuclear translocation of apoptosis-inducing factor and cytosolic release of cytochrome c from mitochondria. Finally, in the 3×Tg-AD mouse model of AD, GAPDH/Aβ co-aggregation and mitochondrial dysfunction were consistently detected in an age-dependent manner, and Aβ aggregate formation was attenuated by GAPDH siRNA treatment. Thus, this study suggests that GAPDH aggregates accelerate Aβ amyloidogenesis, subsequently leading to mitochondrial dysfunction and neuronal cell death in the pathogenesis of AD. PMID:26359500

  18. Glycerol-3-phosphate metabolism plays a role in stress response in the red alga Pyropia haitanensis.

    PubMed

    Lai, Xiao-Juan; Yang, Rui; Luo, Qi-Jun; Chen, Juan-Juan; Chen, Hai-Min; Yan, Xiao-Jun

    2015-04-01

    Glycerol-3-phosphate (G3P) has been suggested as a novel regulator of plant defense signaling, however, its role in algal resistance remains largely unknown. The glycerol kinase (also designated as NHO1) and NAD-dependent G3P dehydrogenase (GPDH) are two key enzymes involved in the G3P biosynthesis. In our study, we cloned the full-length cDNA of NHO1 (NHO1Ph ) and GPDH (GPDHP h ) from the red alga Pyropia haitanensis (denoted as NHO1Ph and GPDHP h ) and examined their expression level under flagellin peptide 22 (flg22) stimulation or heat stress. We also measured the level of G3P and floridoside (a downstream product of G3P in P. haitanensis) under flg22 stimulation or heat stress. Both NHO1Ph and GPDHP h shared high sequence identity and structural conservation with their orthologs from different species, especially from red algae. Phylogenetic analysis showed that NHO1s and GPDHs from red algae were closely related to those from animals. Under flg22 stimulation or heat stress, the expression levels of NHO1Ph and GPDHP h were up-regulated, G3P levels increased, and the contents of floridoside decreased. But the floridoside level increased in the recovery period after heat stress. Taken together, we found that G3P metabolism was associated with the flg22-induced defense response and heat stress response in P. haitanensis, indicating the general conservation of defense response in angiosperms and algae. Furthermore, floridoside might also participate in the stress resistance of P. haitanensis. PMID:26986527

  19. Cellular recovery of glyceraldehyde-3-phosphate dehydrogenase activity and thiol status after exposure to hydroperoxides

    SciTech Connect

    Brodie, A.E.; Reed, D.J. )

    1990-01-01

    The activity of the thiol-dependent enzyme glyceraldehyde-3-phosphate dehydrogenase (GPD), in vertebrate cells, was modulated by a change in the intracellular thiol:disulfide redox status. Human lung carcinoma cells (A549) were incubated with 1-120 mM H2O2, 1-120 mM t-butyl hydroperoxide, 1-6 mM ethacrynic acid, or 0.1-10 mM N-ethylmaleimide for 5 min. Loss of reduced protein thiols, as measured by binding of the thiol reagent iodoacetic acid to GPD, and loss of GPD enzymatic activity occurred in a dose-dependent manner. Incubation of the cells, following oxidative treatment, in saline for 30 min or with 20 mM dithiothreitol (DTT) partially reversed both changes in GPD. The enzymatic recovery of GPD activity was observed either without addition of thiols to the medium or by incubation of a sonicated cell mixture with 2 mM cysteine, cystine, cysteamine, or glutathione (GSH); GSSG had no effect. Treatment of cells with buthionine sulfoximine (BSO) to decrease cellular GSH by varying amounts caused a dose-related increase in sensitivity of GPD activity to inactivation by H2O2 and decreased cellular ability for subsequent recovery. GPD responded in a similar fashion with oxidative treatment of another lung carcinoma cell line (A427) as well as normal lung tissue from human and rat. These findings indicate that the cellular thiol redox status can be important in determining GPD enzymatic activity.

  20. Vaccine efficacy of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Edwardsiella ictaluri against E. tarda in tilapia.

    PubMed

    Trung Cao, Thanh; Tsai, Ming-An; Yang, Chung-Da; Wang, Pei-Chyi; Kuo, Tsun-Yung; Gabriel Chen, Hsu-Chung; Chen, Shih-Chu

    2014-01-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), derived from the outer-membrane protein (OMP) fraction, has been used as a potential candidate for vaccine development. The gene-encoding 37 kDa GAPDH outer membrane protein (OMP) from Edwardsiella ictaluri was amplified using polymerase chain reaction (PCR) and was cloned and expressed in Escherichia coli BL21 (DE3). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, and nucleotide and amino acid sequencing were used to analyze the expressed antigenic protein and gene encoding this protein. Comparative DNA and protein sequence analysis of GAPDH from E. ictaluri GAPDHs from several Gram-negative bacterial species within the Enterobacteriaceae family revealed that the GAPDHs within this group are highly conserved and share a sequence similarity of 75-100% with E. ictaluri GDPDH. Rabbit antiserum raised against the E. ictaluri recombinant GAPDH (rGAPDH) protein recognized purified GADPH, indicating that it has a strong immunogenicity. Tilapia fish were intraperitoneally immunized with formalin-killed E. ictaluri whole cells, and rGAPDH (30 μg fish(-1)) from E. ictaluri, both of which were emulsified in ISA 763A adjuvant. At 3 months after immunization, fish were challenged with the E. tarda strain to assess vaccine efficacy; the relative percent survival (RPS) values were found to exceed 71.4%. The specific mean antibody titer log2 level of groups vaccinated with rGAPDH at 3 months was significantly higher than that of non-vaccinated fish (control group). Therefore, this recombinant protein can be considered a multi-purpose candidate vaccine against several pathogenic bacteria. PMID:25742975

  1. Identification of some ectomycorrhizal basidiomycetes by PCR amplification of their gpd (glyceraldehyde-3-phosphate dehydrogenase) genes.

    PubMed

    Kreuzinger, N; Podeu, R; Gruber, F; Göbl, F; Kubicek, C P

    1996-09-01

    Degenerated oligonucleotide primers designed to flank an approximately 1.2-kb fragment of the gene encoding glyceraldehyde-3-phosphate dehydrogenase (gpd) from ascomycetes and basidiomycetes were used to amplify the corresponding gpd fragments from several species of the ectomycorrhizal fungal taxa Boletus, Amanita, and Lactarius. Those from B. edulis, A. muscaria, and L. deterrimus were cloned and sequenced. The respective nucleotide sequences of these gene fragments showed a moderate degree of similarity (72 to 76%) in the protein-encoding regions and only a low degree of similarity in the introns (56 to 66%). Introns, where present, occurred at conserved positions, but the respective positions and numbers of introns in a given taxon varied. The amplified fragment from a given taxon could be distinguished from that of others by both restriction nuclease cleavage analysis and Southern hybridization. A procedure for labeling DNA probes with fluorescein-12-dUTP by PCR was developed. These probes were used in a nonradioactive hybridization assay, with which the gene could be detected in 2 ng of chromosomal DNA of L. deterrimus on slot blots. Taxon-specific amplification was achieved by the design of specific oligonucleotide primers. The application of the gpd gene for the identification of mycorrhizal fungi under field conditions was demonstrated, with Picea abies (spruce) mycorrhizal roots harvested from a northern alpine forest area as well as from a plant-breeding nursery. The interference by inhibitory substances, which sometimes occurred in the DNA extracted from the root-fungus mixture, could be overcome by using very diluted concentrations of template DNA for a first round of PCR amplification followed by a second round with nested oligonucleotide primers. We conclude that gpd can be used to detect ectomycorrhizal fungi during symbiotic interaction. PMID:8795234

  2. MOLECULAR MECHANISM OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE (GAPDH) INACTIVATION BY α,β-UNSATURATED CARBONYL DERIVATIVES

    PubMed Central

    Martyniuk, Christopher J.; Fang, Bin; Koomen, John M.; Gavin, Terrence; Zhang, Lihai; Barber, David S.; LoPachin, Richard M.

    2011-01-01

    α,β-Unsaturated carbonyls are an important class of chemicals involved in environmental toxicity and disease processes. Whereas adduction of cysteine residues on proteins is a well-documented reaction of these chemicals, such a generic effect cannot explain the molecular mechanism of cytotoxicity. Instead, more detailed information is needed regarding the possible specificity and kinetics of cysteine targeting and the quantitative relationship between adduct burden and protein dysfunction. To address these datagaps, purified human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was incubated with acrylamide (ACR), acrolein or methylvinyl ketone (MVK). Results show that these α,β-unsaturated carbonyl toxicants inhibited GAPDH activity in a concentration-and time-dependent manner. The rank order of enzyme inhibition (KI); i.e., ACR << MVK < acrolein, was related to the calculated electrophilic reactivity of each compound and to the corresponding kinetics of cysteine adduct formation. Tandem mass spectrometry revealed that adduct formation was selective at lower concentrations; i.e., ACR preferentially formed adducts with Cys152 (residues 146-162). At higher concentrations, ACR also formed adducts with Cys156 and Cys247 (residues 235-248). Adduct formation at Cys152 was correlated to enzyme inhibition, which is consistent with the regulatory role of this residue in enzyme function and its location within the GAPDH active site. Further analyses indicated that Cys152 was present in a pKa-lowering microenvironment (pKa = 6.03) and, at physiological pH, the corresponding sulfhydryl group exists in the highly reactive nucleophilic thiolate-state. These data suggest a general cytotoxic mechanism where electrophilic α,β-unsaturated carbonyls selectively form adducts with reactive nucleophilic cysteine residues specifically associated with the active sites of proteins. These specialized cysteine residues are toxicologically relevant molecular targets, since chemical

  3. Cloning and characterization of two novel chloroplastic glycerol-3-phosphate dehydrogenases from Dunaliella viridis.

    PubMed

    He, Yunxia; Meng, Xiangzong; Fan, Qianlan; Sun, Xiaoliang; Xu, Zhengkai; Song, Rentao

    2009-09-01

    Dunaliella, a unicellular green alga, has the unusual ability to survive dramatic osmotic stress by accumulating high concentrations of intracellular glycerol as a compatible solute. The chloroplastic glycerol-3-phosphate dehydrogenase (GPDH) has been considered to be the key enzyme that produces glycerol for osmoregulation in Dunaliella. In this study, we cloned the two most prominent GPDH cDNAs (DvGPDH1 and DvGPDH2) from Dunaliella viridis, which encode two polypeptides of 695 and 701 amino acids, respectively. Unlike higher plant GPDHs, both proteins contained extra phosphoserine phosphatase (SerB) domains at their N-termini in addition to C-terminal GPDH domains. Such bi-domain GPDHs represent a novel type of GPDH and are found exclusively in the chlorophyte lineage. Transient expression of EGFP fusion proteins in tobacco leaf cells demonstrated that both DvGPDH1 and DvGPDH2 are localized in the chloroplast. Overexpression of DvGPDH1 or DvGPDH2 could complement a yeast GPDH mutant (gpd1Delta), but not a yeast SerB mutant (ser2Delta). In vitro assays with purified DvGPDH1 and DvGPDH2 also showed apparent GPDH activity for both, but no SerB activity was detected. Surprisingly, unlike chloroplastic GPDHs from plants, DvGPDH1 and DvGPDH2 could utilize both NADH and NADPH as coenzymes and exhibited significantly higher GPDH activities when NADH was used as the coenzyme. Q-PCR analysis revealed that both genes exhibited transient transcriptional induction of gene expression upon hypersalinity shock, followed by a negative feedback of gene expression. These results shed light on the regulation of glycerol synthesis during salt stress in Dunaliella. PMID:19551475

  4. Glyceraldehyde-3-Phosphate Dehydrogenase Interacts with Proapoptotic Kinase Mst1 to Promote Cardiomyocyte Apoptosis

    PubMed Central

    You, Bei; Huang, Shengdong; Qin, Qing; Yi, Bing; Yuan, Yang; Xu, Zhiyun; Sun, Jianxin

    2013-01-01

    Mammalian sterile 20-like kinase 1 (Mst1) is a critical component of the Hippo signaling pathway, which regulates a variety of biological processes ranging from cell contact inhibition, organ size control, apoptosis and tumor suppression in mammals. Mst1 plays essential roles in the heart disease since its activation causes cardiomyocyte apoptosis and dilated cardiomyopathy. However, the mechanism underlying Mst1 activation in the heart remains unknown. In a yeast two-hybrid screen of a human heart cDNA library with Mst1 as bait, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified as an Mst1-interacting protein. The interaction of GAPDH with Mst1 was confirmed by co-immunoprecipitation in both co-transfected HEK293 cells and mouse heart homogenates, in which GAPDH interacted with the kinase domain of Mst1, whereas the C-terminal catalytic domain of GAPDH mediated its interaction with Mst1. Moreover, interaction of Mst1 with GAPDH caused a robust phosphorylation of GAPDH and markedly increased the Mst1 activity in cells. Chelerythrine, a potent inducer of apoptosis, substantially increased the nuclear translocation and interaction of GAPDH and Mst1 in cardiomyocytes. Overexpression of GAPDH significantly augmented the Mst1 mediated apoptosis, whereas knockdown of GAPDH markedly attenuated the Mst1 activation and cardiomyocyte apoptosis in response to either chelerythrine or hypoxia/reoxygenation. These findings reveal a novel function of GAPDH in Mst1 activation and cardiomyocyte apoptosis and suggest that disruption of GAPDH interaction with Mst1 may prevent apoptosis related heart diseases such as heart failure and ischemic heart disease. PMID:23527007

  5. Diacylglycerol pyrophosphate binds and inhibits the glyceraldehyde-3-phosphate dehydrogenase in barley aleurone.

    PubMed

    Astorquiza, Paula Luján; Usorach, Javier; Racagni, Graciela; Villasuso, Ana Laura

    2016-04-01

    The aleurona cell is a model that allows the study of the antagonistic effect of gibberellic acid (GA) and abscisic acid (ABA). Previous results of our laboratory demonstrated the involvement of phospholipids during the response to ABA and GA. ABA modulates the levels of diacylglycerol, phosphatidic acid and diacylglycerol pyrophosphate (DAG, PA, DGPP) through the activities of phosphatidate phosphatases, phospholipase D, diacylglycerol kinase and phosphatidate kinase (PAP, PLD, DGK and PAK). PA and DGPP are key phospholipids in the response to ABA, since both are capable of modifying the hydrolitic activity of the aleurona. Nevertheless, little is known about the mechanism of action of these phospholipids during the ABA signal. DGPP is an anionic phospholipid with a pyrophosphate group attached to diacylglycerol. The ionization of the pyrophosphate group may be important to allow electrostatic interactions between DGPP and proteins. To understand how DGPP mediates cell functions in barley aleurone, we used a DGPP affinity membrane assay to isolate DGPP-binding proteins from Hordeum vulgare, followed by mass spectrometric sequencing. A cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) was identified for being bound to DGPP. To validate our method, the relatively abundant GAPDH was characterized with respect to its lipid-binding properties, by fat western blot. GAPDH antibody interacts with proteins that only bind to DGPP and PA. We also observed that ABA treatment increased GAPDH abundance and enzyme activity. The presence of phospholipids during GAPDH reaction modulated the GAPDH activity in ABA treated aleurone. These data suggest that DGPP binds to GAPDH and this DGPP and GAPDH interaction provides new evidences in the study of DGPP-mediated ABA responses in barley aleurone. PMID:26866974

  6. Glyceraldehyde-3-phosphate Dehydrogenase Aggregate Formation Participates in Oxidative Stress-induced Cell Death*

    PubMed Central

    Nakajima, Hidemitsu; Amano, Wataru; Kubo, Takeya; Fukuhara, Ayano; Ihara, Hideshi; Azuma, Yasu-Taka; Tajima, Hisao; Inui, Takashi; Sawa, Akira; Takeuchi, Tadayoshi

    2009-01-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)2 is a classic glycolytic enzyme that also mediates cell death by its nuclear translocation under oxidative stress. Meanwhile, we previously presented that oxidative stress induced disulfide-bonded GAPDH aggregation in vitro. Here, we propose that GAPDH aggregate formation might participate in oxidative stress-induced cell death both in vitro and in vivo. We show that human GAPDH amyloid-like aggregate formation depends on the active site cysteine-152 (Cys-152) in vitro. In SH-SY5Y neuroblastoma, treatment with dopamine decreases the cell viability concentration-dependently (IC50 = 202 μm). Low concentrations of dopamine (50–100 μm) mainly cause nuclear translocation of GAPDH, whereas the levels of GAPDH aggregates correlate with high concentrations of dopamine (200–300 μm)-induced cell death. Doxycycline-inducible overexpression of wild-type GAPDH in SH-SY5Y, but not the Cys-152-substituted mutant (C152A-GAPDH), accelerates cell death accompanying both endogenous and exogenous GAPDH aggregate formation in response to high concentrations of dopamine. Deprenyl, a blocker of GAPDH nuclear translocation, fails to inhibit the aggregation both in vitro and in cells but reduced cell death in SH-SY5Y treated with only a low concentration of dopamine (100 μm). These results suggest that GAPDH participates in oxidative stress-induced cell death via an alternative mechanism in which aggregation but not nuclear translocation of GAPDH plays a role. Moreover, we observe endogenous GAPDH aggregate formation in nigra-striatum dopaminergic neurons after methamphetamine treatment in mice. In transgenic mice overexpressing wild-type GAPDH, increased dopaminergic neuron loss and GAPDH aggregate formation are observed. These data suggest a critical role of GAPDH aggregates in oxidative stress-induced brain damage. PMID:19837666

  7. Glycerolipid biosynthesis in Saccharomyces cerevisiae: sn-glycerol-3-phosphate and dihydroxyacetone phosphate acyltransferase activities.

    PubMed Central

    Schlossman, D M; Bell, R M

    1978-01-01

    Yeast acyl-coenzyme A:dihydroxyacetone-phosphate O-acyltransferase (DHAP acyltransferase; EC 2.3.1.42) was investigated to (i) determine whether its activity and that of acyl-coenzyme A:sn-glycerol-3-phosphate O-acyltransferase (glycerol-P acyltransferase; EC 2.3.1.15) represent dual catalytic functions of a single membranous enzyme, (ii) estimate the relative contributions of the glycerol-P and DHAP pathways for yeast glycerolipid synthesis, and (iii) evaluate the suitability of yeast for future genetic investigations of the eucaryotic glycerol-P and DHAP acyltransferase activities. The membranous DHAP acyltransferase activity showed an apparent Km of 0.79 mM for DHAP, with a Vmax of 5.3 nmol/min per mg, whereas the glycerol-P acyltransferase activity showed an apparent Km of 0.05 mM for glycerol-P, with a Vmax of 3.4 nmol/min per mg. Glycerol-P was a competitive inhibitor (Ki, 0.07 mM) of the DHAP acyltransferase activity, and DHAP was a competitive inhibitor (Ki, 0.91 mM) of the glycerol-P acyltransferase activity. The two acyltransferase activities exhibited marked similarities in their pH dependence, acyl-coenzyme A chain length preference and substrate concentration dependencies, thermolability, and patterns of inactivation by N-ethylmaleimide, trypsin, and detergents. Thus, the data strongly suggest that yeast glycerol-P and DHAP acyltransferase activities represent dual catalytic functions of a single membrane-bound enzyme. Furthermore, since no acyl-DHAP oxidoreductase activity could be detected in yeast membranes, the DHAP pathway for glycerolipid synthesis may not operate in yeast. PMID:25265

  8. An Arabidopsis callose synthase.

    PubMed

    Ostergaard, Lars; Petersen, Morten; Mattsson, Ole; Mundy, John

    2002-08-01

    Beta-1,3-glucan polymers are major structural components of fungal cell walls, while cellulosic beta-1,4-glucan is the predominant polysaccharide in plant cell walls. Plant beta-1,3-glucan, called callose, is produced in pollen and in response to pathogen attack and wounding, but it has been unclear whether callose synthases can also produce cellulose and whether plant cellulose synthases may also produce beta-1,3-glucans. We describe here an Arabidopsis gene, AtGsl5, encoding a plasma membrane-localized protein homologous to yeast beta-1,3-glucan synthase whose expression partially complements a yeast beta-1,3-glucan synthase mutant. AtGsl5 is developmentally expressed at highest levels in flowers, consistent with flowers having high beta-1,3-glucan synthase activities for deposition of callose in pollen. A role for AtGsl5 in callose synthesis is also indicated by AtGsl5 expression in the Arabidopsis mpk4 mutant which exhibits systemic acquired resistance (SAR), elevated beta-1,3-glucan synthase activity, and increased callose levels. In addition, AtGsl5 is a likely target of salicylic acid (SA)-dependent SAR, since AtGsl5 mRNA accumulation is induced by SA in wild-type plants, while expression of the nahG salicylate hydroxylase reduces AtGsl5 mRNA levels in the mpk4 mutant. These results indicate that AtGsl5 is likely involved in callose synthesis in flowering tissues and in the mpk4 mutant. PMID:12081364

  9. Purification and characterisation of acyl-CoA: glycerol 3-phosphate acyltransferase from oil palm (Elaeis guineensis) tissues.

    PubMed

    Manaf, A M; Harwood, J L

    2000-01-01

    Glycerol 3-phosphate acyltransferase (GPAT, EC 2.3.15) catalyses the first step of the Kennedy pathway for acyl lipid formation. This enzyme was studied using high-speed particulate fractions from oil palm (Elaeis guineensis Jacq.) tissue cultures and mesocarp acetone powders. The fractions were incubated with [(14)C]glycerol 3-phosphate and incorporation of radioactivity into Kennedy pathway intermediates studied. Optimal conditions were broadly similar between the two preparations but those from fruit mesocarp clearly contained more active enzymes for the subsequent stages of the Kennedy pathway - as exemplified by the appreciable accumulation of radioactivity in triacylglycerol. Experiments with different acyl-CoA substrates showed that the GPAT in both high-speed particulate preparations had a significant preference for palmitate. Glycerol 3-phosphate acyltransferase was solubilised from both preparations with optimal solubilisation being achieved at 0.5% (w/v) CHAPS concentrations. Solubilised GPATs were purified further using DE52 ion-exchange chromatography and Sephadex G-100 molecular exclusion chromatography. Purifications of up to about 70-fold were achieved. The purified GPATs showed a strong preference for palmitoyl-CoA compared to other acyl-CoA donors, in keeping with the importance of palmitate in palm oil. PMID:10664139

  10. The Glycerol-3-Phosphate Acyltransferase TbGAT is Dispensable for Viability and the Synthesis of Glycerolipids in Trypanosoma brucei.

    PubMed

    Patel, Nipul; Pirani, Karim A; Zhu, Tongtong; Cheung-See-Kit, Melanie; Lee, Sungsu; Chen, Daniel G; Zufferey, Rachel

    2016-09-01

    Glycerolipids are the main constituents of biological membranes in Trypanosoma brucei, which causes sleeping sickness in humans. Importantly, they occur as a structural component of the glycosylphosphatidylinositol lipid anchor of the abundant cell surface glycoproteins procyclin in procyclic forms and variant surface glycoprotein in bloodstream form, that play crucial roles for the development of the parasite in the insect vector and the mammalian host, respectively. The present work reports the characterization of the glycerol-3-phosphate acyltransferase TbGAT that initiates the biosynthesis of ester glycerolipids. TbGAT restored glycerol-3-phosphate acyltransferase activity when expressed in a Leishmania major deletion strain lacking this activity and exhibited preference for medium length, unsaturated fatty acyl-CoAs. TbGAT localized to the endoplasmic reticulum membrane with its N-terminal domain facing the cytosol. Despite that a TbGAT null mutant in T. brucei procyclic forms lacked glycerol-3-phosphate acyltransferase activity, it remained viable and exhibited similar growth rate as the wild type. TbGAT was dispensable for the biosynthesis of phosphatidylcholine, phosphatidylinositol, phosphatidylserine, and GPI-anchored protein procyclin. However, the null mutant exhibited a slight decrease in phosphatidylethanolamine biosynthesis that was compensated with a modest increase in production of ether phosphatidylcholine. Our data suggest that an alternative initial acyltransferase takes over TbGAT's function in its absence. PMID:26909872

  11. Sulfur mustard induced nuclear translocation of glyceraldehyde-3-phosphate-dehydrogenase (GAPDH).

    PubMed

    Steinritz, Dirk; Weber, Jana; Balszuweit, Frank; Thiermann, Horst; Schmidt, Annette

    2013-12-01

    Sulfur Mustard (SM) is a vesicant chemical warfare agent, which is acutely toxic to a variety of organ systems including skin, eyes, respiratory system and bone marrow. The underlying molecular pathomechanism was mainly attributed to the alkylating properties of SM. However, recent studies have revealed that cellular responses to SM exposure are of more complex nature and include increased protein expression and protein modifications that can be used as biomarkers. In order to confirm already known biomarkers, to detect potential new ones and to further elucidate the pathomechanism of SM, we conducted large-scale proteomic experiments based on a human keratinocyte cell line (HaCaT) exposed to SM. Surprisingly, our analysis identified glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) as one of the up-regulated proteins after exposure of HaCaT cells to SM. In this paper we demonstrate the sulfur mustard induced nuclear translocation of GAPDH in HaCaT cells by 2D gel-electrophoresis (2D GE), immunocytochemistry (ICC), Western Blot (WB) and a combination thereof. 2D GE in combination with MALDI-TOF MS/MS analysis identified GAPDH as an up-regulated protein after SM exposure. Immunocytochemistry revealed a distinct nuclear translocation of GAPDH after exposure to 300μM SM. This finding was confirmed by fractionated WB analysis. 2D GE and subsequent immunoblot staining of GAPDH demonstrated two different spot locations of GAPH (pI 7.0 and pI 8.5) that are related to cytosolic or nuclear GAPDH respectively. After exposure to 300μM SM a significant increase of nuclear GAPDH at pI 8.5 occurred. Nuclear GAPDH has been associated with apoptosis, detection of structural DNA alterations, DNA repair and regulation of genomic integrity and telomere structure. The results of our study add new aspects to the pathophysiology of sulfur mustard toxicity, yet further studies will be necessary to reveal the specific function of nuclear GAPDH in the pathomechanism of sulfur mustard

  12. Geranyl diphosphate synthase from mint

    SciTech Connect

    Croteau, Rodney Bruce; Wildung, Mark Raymond; Burke, Charles Cullen; Gershenzon, Jonathan

    1999-01-01

    A cDNA encoding geranyl diphosphate synthase from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID No:1) is provided which codes for the expression of geranyl diphosphate synthase (SEQ ID No:2) from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase or for a base sequence sufficiently complementary to at least a portion of the geranyl diphosphate synthase DNA or RNA to enable hybridization therewith (e.g., antisense geranyl diphosphate synthase RNA or fragments of complementary geranyl diphosphate synthase DNA which are useful as polymerase chain reaction primers or as probes for geranyl diphosphate synthase or related genes). In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant geranyl diphosphate synthase for subsequent use, to obtain expression or enhanced expression of geranyl diphosphate synthase in plants in order to enhance the production of monoterpenoids, to produce geranyl diphosphate in cancerous cells as a precursor to monoterpenoids having anti-cancer properties or may be otherwise employed for the regulation or expression of geranyl diphosphate synthase or the production of geranyl diphosphate.

  13. Geranyl diphosphate synthase from mint

    SciTech Connect

    Croteau, R.B.; Wildung, M.R.; Burke, C.C.; Gershenzon, J.

    1999-03-02

    A cDNA encoding geranyl diphosphate synthase from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID No:1) is provided which codes for the expression of geranyl diphosphate synthase (SEQ ID No:2) from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase or for a base sequence sufficiently complementary to at least a portion of the geranyl diphosphate synthase DNA or RNA to enable hybridization therewith (e.g., antisense geranyl diphosphate synthase RNA or fragments of complementary geranyl diphosphate synthase DNA which are useful as polymerase chain reaction primers or as probes for geranyl diphosphate synthase or related genes). In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant geranyl diphosphate synthase for subsequent use, to obtain expression or enhanced expression of geranyl diphosphate synthase in plants in order to enhance the production of monoterpenoids, to produce geranyl diphosphate in cancerous cells as a precursor to monoterpenoids having anti-cancer properties or may be otherwise employed for the regulation or expression of geranyl diphosphate synthase or the production of geranyl diphosphate. 5 figs.

  14. Substrate-dependent utilization of the glycerol 3-phosphate or malate/aspartate redox shuttles by Ehrlich ascites cells.

    PubMed Central

    Grivell, A R; Korpelainen, E I; Williams, C J; Berry, M N

    1995-01-01

    The rate of transfer of reducing equivalents from cytoplasm to mitochondria has been examined in Ehrlich ascites tumour cells incubated in the presence of lactate. The flux of reducing equivalents was determined from the rate of metabolism of reduced intermediates that are oxidized within the cytosol. The magnitude of the flux of reducing equivalents was dependent on both the concentration of added lactate and the presence of carbohydrate. The rate of flux was twice as great in the presence of glucose and four times as high when glucose and lactate were added together as when lactate was the only added substrate. Fructose was less effective than glucose in stimulating reducing equivalent flux. In the presence of glucose or fructose, there was a substantial accumulation of hexose phosphates, dihydroxyacetone phosphate and glycerol 3-phosphate. Rotenone, an inhibitor of NADH dehydrogenase, and amino-oxyacetate, which inhibits the malate/aspartate shuttle, were powerful suppressors of reducing equivalent flux from lactate as sole substrate, but were much less potent in the presence of carbohydrate. Antimycin substantially inhibited reducing equivalent flux from all combinations of added substrates, consistent with its ability to block oxidation of reducing equivalents transferred by both the malate/aspartate and glycerol 3-phosphate shuttles. The glycerol 3-phosphate shuttle represents around 80% of the maximum total observed activity but is active only while glycolytic intermediates are present to provide the necessary substrates of the shuttle. This Ehrlich ascites cell line has an essentially similar total reducing equivalent shuttle capacity to that of isolated hepatocytes. PMID:7654209

  15. Hybrid polyketide synthases

    DOEpatents

    Fortman, Jeffrey L.; Hagen, Andrew; Katz, Leonard; Keasling, Jay D.; Poust, Sean; Zhang, Jingwei; Zotchev, Sergey

    2016-05-10

    The present invention provides for a polyketide synthase (PKS) capable of synthesizing an even-chain or odd-chain diacid or lactam or diamine. The present invention also provides for a host cell comprising the PKS and when cultured produces the even-chain diacid, odd-chain diacid, or KAPA. The present invention also provides for a host cell comprising the PKS capable of synthesizing a pimelic acid or KAPA, and when cultured produces biotin.

  16. A human nuclear uracil DNA glycosylase is the 37-kDa subunit of glyceraldehyde-3-phosphate dehydrogenase

    SciTech Connect

    Meyer-Siegler, K.; Mauro, D.J.; Seal, G.; Wurzer, J.; DeRiel, J.K.; Sirover, M.A. )

    1991-10-01

    The authors have isolated and characterized a plasmid (pChug 20.1) that contains the cDNA of a nuclear uracil DNA glycosylase (UDG) gene isolated from normal human placenta. This cDNA directed the synthesis of a fusion protein that exhibited UDG activity. The enzymatic activity was specific for a uracil-containing polynucleotide substrate and was inhibited by a glycosylase antibody or a {beta}-galactosidase antibody. Sequence analysis demonstrated an open reading frame that encoded a protein of 335 amino acids of calculated M{sub r} 36,050 and pI 8.7, corresponding to the M{sub r} 37,000 and pI 8.1 of purified human placental UDG. Surprisingly, a search of the GenBank data base revealed that the cDNA of UDG was completely homologous with the 378-kDa subunit of human glyceraldehyde-3-phosphate dehydrogenase. Human erythrocyte glyceraldehyde-3-phosphate dehydrogenase was obtained commercially in its tetrameric form. A 37-kDa subunit was isolated form it and shown to possess UDG activity equivalent to that seen for the purified human placental UDG. The multiple functions of this 37-kDa protein as here and previously reported indicate that it possesses a series of activities, depending on its oligomeric state. Accordingly, mutation(s) in the gene of this multifunctional protein may conceivably result in the diverse cellular phenotypes of Bloom syndrome.

  17. Metabolic engineering of enhanced glycerol-3-phosphate synthesis to increase lipid production in Synechocystis sp. PCC 6803.

    PubMed

    Wang, Xi; Xiong, Xiaochao; Sa, Na; Roje, Sanja; Chen, Shulin

    2016-07-01

    With the growing attention to global warming and energy sustainability, biosynthesis of lipids by photosynthetic microorganisms has attracted more interest for the production of renewable transportation fuels. Recently, the cyanobacterium Synechocystis sp. PCC 6803 has been widely used for biofuel production through metabolic engineering because of its efficient photosynthesis and well-developed genetic tools. In lipid biosynthesis, glycerol-3-phosphate (G3P) is a key node for both CO2 fixation and lipid metabolism in cyanobacteria. However, few studies have explored the use of G3P synthesis to improve photosynthetic lipid production. In this study, metabolic engineering combined with flux balance analysis (FBA) was conducted to reveal the effect of G3P synthesis on lipid production. Heterologous genes that encoded glycerol-3-phosphate dehydrogenase (GPD) and diacylglycerol acyltransferase (DGAT) were engineered into Synechocystis sp. PCC 6803 to enhance G3P supply and lipid production. The resultant recombinant Synechocystis produced higher levels of lipids without a significant reduction in cell growth. Compared with the wild-type strain, lipid content and productivity of the engineered cyanobacteria increased by up to 36 and 31 %, respectively, under autotrophic conditions. Lipid production under mixotrophic conditions of the engineered cyanobacteria was also investigated. This work demonstrated that enhanced G3P synthesis was an important factor in photosynthetic lipid production and that introducing heterologous GPD and DGAT genes was an effective strategy to increase lipid production in Synechocystis sp. PCC 6803. PMID:27154348

  18. Cloning, heterologous expression and biochemical characterization of plastidial sn-glycerol-3-phosphate acyltransferase from Helianthus annuus.

    PubMed

    Payá-Milans, Miriam; Venegas-Calerón, Mónica; Salas, Joaquín J; Garcés, Rafael; Martínez-Force, Enrique

    2015-03-01

    The acyl-[acyl carrier protein]:sn-1-glycerol-3-phosphate acyltransferase (GPAT; E.C. 2.3.1.15) catalyzes the first step of glycerolipid assembly within the stroma of the chloroplast. In the present study, the sunflower (Helianthus annuus, L.) stromal GPAT was cloned, sequenced and characterized. We identified a single ORF of 1344base pairs that encoded a GPAT sharing strong sequence homology with the plastidial GPAT from Arabidopsis thaliana (ATS1, At1g32200). Gene expression studies showed that the highest transcript levels occurred in green tissues in which chloroplasts are abundant. The corresponding mature protein was heterologously overexpressed in Escherichia coli for purification and biochemical characterization. In vitro assays using radiolabelled acyl-ACPs and glycerol-3-phosphate as substrates revealed a strong preference for oleic versus palmitic acid, and weak activity towards stearic acid. The positional fatty acid composition of relevant chloroplast phospholipids from sunflower leaves did not reflect the in vitro GPAT specificity, suggesting a more complex scenario with mixed substrates at different concentrations, competition with other acyl-ACP consuming enzymatic reactions, etc. In summary, this study has confirmed the affinity of this enzyme which would partly explain the resistance to cold temperatures observed in sunflower plants. PMID:25618244

  19. Synergistic interaction of glyceraldehydes-3-phosphate dehydrogenase and ArsJ, a novel organoarsenical efflux permease, confers arsenate resistance.

    PubMed

    Chen, Jian; Yoshinaga, Masafumi; Garbinski, Luis D; Rosen, Barry P

    2016-06-01

    Microbial biotransformations are major contributors to the arsenic biogeocycle. In parallel with transformations of inorganic arsenic, organoarsenicals pathways have recently been recognized as important components of global cycling of arsenic. The well-characterized pathway of resistance to arsenate is reduction coupled to arsenite efflux. Here, we describe a new pathway of arsenate resistance involving biosynthesis and extrusion of an unusual pentavalent organoarsenical. A number of arsenic resistance (ars) operons have two genes of unknown function that are linked in these operons. One, gapdh, encodes the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase. The other, arsJ, encodes a major facilitator superfamily (MFS) protein. The two genes were cloned from the chromosome of Pseudomonas aeruginosa. When expressed together, but not alone, in Escherichia coli, gapdh and arsJ specifically conferred resistance to arsenate and decreased accumulation of As(V). Everted membrane vesicles from cells expressing arsJ accumulated As(V) in the presence of purified GAPDH, D-glceraldehylde 3-phosphate (G3P) and NAD(+) . GAPDH forms the unstable organoarsenical 1-arseno-3-phosphoglycerate (1As3PGA). We propose that ArsJ is an efflux permease that extrudes 1As3PGA from cells, where it rapidly dissociates into As(V) and 3-phosphoglycerate (3PGA), creating a novel pathway of arsenate resistance. PMID:26991003

  20. Monoterpene synthases from common sage (Salvia officinalis)

    DOEpatents

    Croteau, Rodney Bruce; Wise, Mitchell Lynn; Katahira, Eva Joy; Savage, Thomas Jonathan

    1999-01-01

    cDNAs encoding (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase from common sage (Salvia officinalis) have been isolated and sequenced, and the corresponding amino acid sequences has been determined. Accordingly, isolated DNA sequences (SEQ ID No:1; SEQ ID No:3 and SEQ ID No:5) are provided which code for the expression of (+)-bornyl diphosphate synthase (SEQ ID No:2), 1,8-cineole synthase (SEQ ID No:4) and (+)-sabinene synthase SEQ ID No:6), respectively, from sage (Salvia officinalis). In other aspects, replicable recombinant cloning vehicles are provided which code for (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase, or for a base sequence sufficiently complementary to at least a portion of (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant monoterpene synthases that may be used to facilitate their production, isolation and purification in significant amounts. Recombinant (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase may be used to obtain expression or enhanced expression of (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase in plants in order to enhance the production of monoterpenoids, or may be otherwise employed for the regulation or expression of (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase, or the production of their products.

  1. Structure of Insoluble Rat Sperm Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) via Heterotetramer Formation with Escherichia coli GAPDH Reveals Target for Contraceptive Design*

    PubMed Central

    Frayne, Jan; Taylor, Abby; Cameron, Gus; Hadfield, Andrea T.

    2009-01-01

    Sperm glyceraldehyde-3-phosphate dehydrogenase has been shown to be a successful target for a non-hormonal contraceptive approach, but the agents tested to date have had unacceptable side effects. Obtaining the structure of the sperm-specific isoform to allow rational inhibitor design has therefore been a goal for a number of years but has proved intractable because of the insoluble nature of both native and recombinant protein. We have obtained soluble recombinant sperm glyceraldehyde-3-phosphate dehydrogenase as a heterotetramer with the Escherichia coli glyceraldehyde-3-phosphate dehydrogenase in a ratio of 1:3 and have solved the structure of the heterotetramer which we believe represents a novel strategy for structure determination of an insoluble protein. A structure was also obtained where glyceraldehyde 3-phosphate binds in the Ps pocket in the active site of the sperm enzyme subunit in the presence of NAD. Modeling and comparison of the structures of human somatic and sperm-specific glyceraldehyde-3-phosphate dehydrogenase revealed few differences at the active site and hence rebut the long presumed structural specificity of 3-chlorolactaldehyde for the sperm isoform. The contraceptive activity of α-chlorohydrin and its apparent specificity for the sperm isoform in vivo are likely to be due to differences in metabolism to 3-chlorolactaldehyde in spermatozoa and somatic cells. However, further detailed analysis of the sperm glyceraldehyde-3-phosphate dehydrogenase structure revealed sites in the enzyme that do show significant difference compared with published somatic glyceraldehyde-3-phosphate dehydrogenase structures that could be exploited by structure-based drug design to identify leads for novel male contraceptives. PMID:19542219

  2. Targeting DXP synthase in human pathogens: enzyme inhibition and antimicrobial activity of butylacetylphosphonate

    PubMed Central

    Smith, Jessica M.; Warrington, Nicole V.; Vierling, Ryan J.; Kuhn, Misty L.; Anderson, Wayne F.; Koppisch, Andrew T.; Freel Meyers, Caren L.

    2013-01-01

    The unique methylerythritol phosphate (MEP) pathway for isoprenoid biosynthesis is essential in most bacterial pathogens. The first enzyme in this pathway, 1-deoxy-D-xylulose 5-phosphate (DXP) synthase, catalyzes a distinct thiamin diphosphate (ThDP)-dependent reaction to form DXP from D-glyceraldehyde 3-phosphate (D-GAP) and pyruvate and represents a potential anti-infective drug target. We have previously demonstrated that the unnatural bisubstrate analog, butylacetylphosphonate (BAP), exhibits selective inhibition of Escherichia coli DXP synthase over mammalian ThDP-dependent enzymes. Here, we report the selective inhibition by BAP against recombinant DXP synthase homologs from Mycobacterium tuberculosis, Yersinia pestis, and Salmonella enterica. We also demonstrate antimicrobial activity of BAP against both Gram-negative and Gram-positive strains (including E. coli, S. enterica, Bacillus anthracis), and several clinically isolated pathogens. Our results suggest a mechanism of action involving inhibition of DXP synthase and show that BAP acts synergistically with established antimicrobial agents, highlighting a potential strategy to combat emerging resistance in bacterial pathogens. PMID:24169798

  3. Characterization of the highly active fragment of glyceraldehyde-3-phosphate dehydrogenase gene promoter for recombinant protein expression in Pleurotus ostreatus.

    PubMed

    Yin, Chaomin; Zheng, Liesheng; Zhu, Jihong; Chen, Liguo; Ma, Aimin

    2015-03-01

    Developing efficient native promoters is important for improving recombinant protein expression by fungal genetic engineering. The promoter region of glyceraldehyde-3-phosphate dehydrogenase gene in Pleurotus ostreatus (Pogpd) was isolated and optimized by upstream truncation. The activities of these promoters with different lengths were further confirmed by fluorescence, quantitative real-time PCR and Western blot analysis. A truncated Pogpd-P2 fragment (795 bp) drove enhanced green fluorescence protein (egfp) gene expression in P. ostreatus much more efficiently than full-length Pogpd-P1. Further truncating Pogpd-P2 to 603, 403 and 231 bp reduced the eGFP expression significantly. However, the 403-bp fragment between -356 bp and the start codon was the minimal but sufficient promoter element for eGFP expression. Compact native promoters for genetic engineering of P. ostreatus were successfully developed and validated in this study. This will broaden the preexisting repertoire of fungal promoters for biotechnology application. PMID:25743073

  4. Cytoplastic Glyceraldehyde-3-Phosphate Dehydrogenases Interact with ATG3 to Negatively Regulate Autophagy and Immunity in Nicotiana benthamiana.

    PubMed

    Han, Shaojie; Wang, Yan; Zheng, Xiyin; Jia, Qi; Zhao, Jinping; Bai, Fan; Hong, Yiguo; Liu, Yule

    2015-04-01

    Autophagy as a conserved catabolic pathway can respond to reactive oxygen species (ROS) and plays an important role in degrading oxidized proteins in plants under various stress conditions. However, how ROS regulates autophagy in response to oxidative stresses is largely unknown. Here, we show that autophagy-related protein 3 (ATG3) interacts with the cytosolic glyceraldehyde-3-phosphate dehydrogenases (GAPCs) to regulate autophagy in Nicotiana benthamiana plants. We found that oxidative stress inhibits the interaction of ATG3 with GAPCs. Silencing of GAPCs significantly activates ATG3-dependent autophagy, while overexpression of GAPCs suppresses autophagy in N. benthamiana plants. Moreover, silencing of GAPCs enhances N gene-mediated cell death and plant resistance against both incompatible pathogens Tobacco mosaic virus and Pseudomonas syringae pv tomato DC3000, as well as compatible pathogen P. syringae pv tabaci. These results indicate that GAPCs have multiple functions in the regulation of autophagy, hypersensitive response, and plant innate immunity. PMID:25829441

  5. Cytoplastic Glyceraldehyde-3-Phosphate Dehydrogenases Interact with ATG3 to Negatively Regulate Autophagy and Immunity in Nicotiana benthamiana

    PubMed Central

    Han, Shaojie; Wang, Yan; Zheng, Xiyin; Jia, Qi; Zhao, Jinping; Bai, Fan; Hong, Yiguo; Liu, Yule

    2015-01-01

    Autophagy as a conserved catabolic pathway can respond to reactive oxygen species (ROS) and plays an important role in degrading oxidized proteins in plants under various stress conditions. However, how ROS regulates autophagy in response to oxidative stresses is largely unknown. Here, we show that autophagy-related protein 3 (ATG3) interacts with the cytosolic glyceraldehyde-3-phosphate dehydrogenases (GAPCs) to regulate autophagy in Nicotiana benthamiana plants. We found that oxidative stress inhibits the interaction of ATG3 with GAPCs. Silencing of GAPCs significantly activates ATG3-dependent autophagy, while overexpression of GAPCs suppresses autophagy in N. benthamiana plants. Moreover, silencing of GAPCs enhances N gene-mediated cell death and plant resistance against both incompatible pathogens Tobacco mosaic virus and Pseudomonas syringae pv tomato DC3000, as well as compatible pathogen P. syringae pv tabaci. These results indicate that GAPCs have multiple functions in the regulation of autophagy, hypersensitive response, and plant innate immunity. PMID:25829441

  6. Homocysteine induces glyceraldehyde-3-phosphate dehydrogenase acetylation and apoptosis in the neuroblastoma cell line Neuro2a.

    PubMed

    Fang, M; Jin, A; Zhao, Y; Liu, X

    2016-02-01

    High plasma levels of homocysteine (Hcy) promote the progression of neurodegenerative diseases. However, the mechanism by which Hcy mediates neurotoxicity has not been elucidated. We observed that upon incubation with Hcy, the viability of a neuroblastoma cell line Neuro2a declined in a dose-dependent manner, and apoptosis was induced within 48 h. The median effective concentration (EC50) of Hcy was approximately 5 mM. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) nuclear translocation and acylation has been implicated in the regulation of apoptosis. We found that nuclear translocation and acetylation of GAPDH increased in the presence of 5 mM Hcy and that higher levels of acetyltransferase p300/CBP were detected in Neuro2a cells. These findings implicate the involvement of GAPDH in the mechanism whereby Hcy induces apoptosis in neurons. This study highlights a potentially important pathway in neurodegenerative disorders, and a novel target pathway for neuroprotective therapy. PMID:26785692

  7. Endosymbiotic origin and codon bias of the nuclear gene for chloroplast glyceraldehyde-3-phosphate dehydrogenase from maize.

    PubMed

    Brinkmann, H; Martinez, P; Quigley, F; Martin, W; Cerff, R

    1987-01-01

    The nuclei of plant cells harbor genes for two types of glyceraldehyde-3-phosphate dehydrogenases (GAPDH) displaying a sequence divergence corresponding to the prokaryote/eukaryote separation. This strongly supports the endosymbiotic theory of chloroplast evolution and in particular the gene transfer hypothesis suggesting that the gene for the chloroplast enzyme, initially located in the genome of the endosymbiotic chloroplast progenitor, was transferred during the course of evolution into the nuclear genome of the endosymbiotic host. Codon usage in the gene for chloroplast GAPDH of maize is radically different from that employed by present-day chloroplasts and from that of the cytosolic (glycolytic) enzyme from the same cell. This reveals the presence of subcellular selective pressures which appear to be involved in the optimization of gene expression in the economically important graminaceous monocots. PMID:3131533

  8. Homocysteine induces glyceraldehyde-3-phosphate dehydrogenase acetylation and apoptosis in the neuroblastoma cell line Neuro2a

    PubMed Central

    Fang, M.; Jin, A.; Zhao, Y.; Liu, X.

    2016-01-01

    High plasma levels of homocysteine (Hcy) promote the progression of neurodegenerative diseases. However, the mechanism by which Hcy mediates neurotoxicity has not been elucidated. We observed that upon incubation with Hcy, the viability of a neuroblastoma cell line Neuro2a declined in a dose-dependent manner, and apoptosis was induced within 48 h. The median effective concentration (EC50) of Hcy was approximately 5 mM. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) nuclear translocation and acylation has been implicated in the regulation of apoptosis. We found that nuclear translocation and acetylation of GAPDH increased in the presence of 5 mM Hcy and that higher levels of acetyltransferase p300/CBP were detected in Neuro2a cells. These findings implicate the involvement of GAPDH in the mechanism whereby Hcy induces apoptosis in neurons. This study highlights a potentially important pathway in neurodegenerative disorders, and a novel target pathway for neuroprotective therapy. PMID:26785692

  9. In silico cloning and characterization of the glycerol-3-phosphate dehydrogenase (GPDH) gene family in the green microalga Chlamydomonas reinhardtii.

    PubMed

    Herrera-Valencia, Virginia A; Macario-González, Laura A; Casais-Molina, Melissa L; Beltran-Aguilar, Anayeli G; Peraza-Echeverría, Santy

    2012-05-01

    Glycerol-3-phosphate dehydrogenase (GPDH) catalyzes the conversion of dihydroxyacetone phosphate (DHAP) and NADH to glycerol-3-phosphate (G3P) and NAD(+). G3P is important as a precursor for glycerol and glycerolipid synthesis in microalgae. A GPDH enzyme has been previously purified from the green microalga Chlamydomonas reinhardtii, however, no genes coding for GPDH have been characterized before. In this study, we report the in silico characterization of three putative GPDH genes from C. reinhardtii: CrGPDH1, CrGPDH2, and CrGPDH3. These sequences showed a significant similarity to characterized GPDH genes from the microalgae Dunaliella salina and Dunaliella viridis. The prediction of the three-dimensional structure of the proteins showed the characteristic fold topology of GPDH enzymes. Furthermore, the phylogenetic analysis showed that the three CrGPDHs share the same clade with characterized GPDHs from Dunaliella suggesting a common evolutionary origin and a similar catalytic function. In addition, the K(a)/K(s) ratios of these sequences suggested that they are under purifying selection. Moreover, the expression analysis showed a constitutive expression of CrGPDH1, while CrGPDH2 and CrGPDH3 were induced in response to osmotic stress, suggesting a possible role for these two sequences in the synthesis of glycerol as a compatible solute in osmoregulation, and perhaps also in lipid synthesis in C. reinhardtii. This study has provided a foundation for further biochemical and genetic studies of the GPDH family in this model microalga, and also opportunities to assess the potential of these genes to enhance the synthesis of TAGs for biodiesel production. PMID:22358185

  10. Purification and characterization of thiol-reagent-sensitive glycerol-3-phosphate acyltransferase from the membrane fraction of an oleaginous fungus.

    PubMed Central

    Mishra, S; Kamisaka, Y

    2001-01-01

    Glycerol-3-phosphate acyltransferase (GPAT), responsible for the first committed, rate-limiting, step of glycerolipid synthesis, was purified to homogeneity from the membrane fraction of an oleaginous fungus, Mortierella ramanniana var. angulispora. The enzyme was solubilized from the membrane fraction by pretreatment with 0.05% Triton X-100 and treatment of the resulting pellet with 0.3% Triton X-100. The enzyme was subsequently purified by column chromatography on heparin-Sepharose, Yellow 86 agarose, a second heparin-Sepharose column, Superdex-200 and hydroxylapatite Bio-Gel. Enzyme activity was finally enriched 1308-fold over that of the starting membrane fraction. SDS/PAGE of the purified fraction revealed a single band with a molecular mass of 45 kDa. Native PAGE showed a major band that corresponded to GPAT activity. Enzyme activity was inhibited by thiol reagents, suggesting that it originated from microsomes rather than mitochondria. Purified GPAT depended on exogenous oleoyl-CoA and sn-glycerol-3-phosphate, with the highest activity at approx. 50 and 250 microM, respectively, and preferred oleoyl-CoA 5.4-fold over palmitoyl-CoA as an acyl donor. Anionic phospholipids, such as phosphatidic acid and phosphatidylserine, were absolutely required for activity of the purified enzyme, and their ability to activate GPAT was influenced by the purity of the GPAT preparation. Bivalent cations, such as Mg(2+) and Ca(2+), inhibited purified GPAT activity, whereas 5 mM Mn(2+) elevated activity approx. 2-fold. These results provide new insights into the molecular characterization of microsomal GPAT, which has not been well characterized compared with mitochondrial and plastidic GPAT. PMID:11284717

  11. Expression, purification, crystallization and preliminary X-ray analysis of an NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Helicobacter pylori

    SciTech Connect

    Elliott, Paul R.; Evans, Daniel; Greenwood, Jacqueline A.; Moody, Peter C. E.

    2008-08-01

    Glyceraldehyde-3-phosphate dehydrogenase A has been cloned, expressed and purified. Apoprotein crystals have been grown which diffracted to 1.75 Å resolution and belonged to space group P2{sub 1}; holo crystals were grown in the presence of NADP, diffracted to 2.6 Å resolution and belonged to space group P3{sub 2}. The classical glycolytic pathway contains an NAD-dependent glyceraldehyde-3-phosphate dehydrogenase, with NADP-dependent forms reserved for photosynthetic organisms and archaea. Here, the cloning, expression, purification, crystallization and preliminary X-ray analysis of an NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Helicobacter pylori is reported; crystals of the protein were grown both in the presence and the absence of NADP.

  12. The role of glycerol-3-phosphate dehydrogenase 1 in the progression of fatty liver after acute ethanol administration in mice

    SciTech Connect

    Sato, Tomoki; Morita, Akihito; Mori, Nobuko; Miura, Shinji

    2014-02-21

    Highlights: • Ethanol administration increased GPD1 mRNA expression. • Ethanol administration increased glucose incorporation into TG glycerol moieties. • No increase in hepatic TG levels was observed in ethanol-injected GPD1 null mice. • We propose that GPD1 is required for ethanol-induced TG accumulation in the liver. - Abstract: Acute ethanol consumption leads to the accumulation of triglycerides (TGs) in hepatocytes. The increase in lipogenesis and reduction of fatty acid oxidation are implicated as the mechanisms underlying ethanol-induced hepatic TG accumulation. Although glycerol-3-phosphate (Gro3P), formed by glycerol kinase (GYK) or glycerol-3-phosphate dehydrogenase 1 (GPD1), is also required for TG synthesis, the roles of GYK and GPD1 have been the subject of some debate. In this study, we examine (1) the expression of genes involved in Gro3P production in the liver of C57BL/6J mice in the context of hepatic TG accumulation after acute ethanol intake, and (2) the role of GPD1 in the progression of ethanol-induced fatty liver using GPD1 null mice. As a result, in C57BL/6J mice, ethanol-induced hepatic TG accumulation began within 2 h and was 1.7-fold greater than that observed in the control group after 6 h. The up-regulation of GPD1 began 2 h after administering ethanol, and significantly increased 6 h later with the concomitant escalation in the glycolytic gene expression. The incorporation of {sup 14}C-labelled glucose into TG glycerol moieties increased during the same period. On the other hand, in GPD1 null mice carrying normal GYK activity, no significant increase in hepatic TG level was observed after acute ethanol intake. In conclusion, GPD1 and glycolytic gene expression is up-regulated by ethanol, and GPD1-mediated incorporation of glucose into TG glycerol moieties together with increased lipogenesis, is suggested to play an important role in ethanol-induced hepatic TG accumulation.

  13. Thymidylate synthase inhibitors.

    PubMed

    Danenberg, P V; Malli, H; Swenson, S

    1999-12-01

    Thymidylate synthase (TS) is a critical enzyme for DNA replication and cell growth because it is the only de novo source of thymine nucleotide precursors for DNA synthesis. TS is the primary target of 5-fluorouracil (5-FU), which has been used for cancer treatment for more than 40 years. However, dissatisfaction with the overall activity of 5-FU against the major cancers, and the recognition that TS still remains an attractive target for anticancer drugs because of its central position in the pathway of DNA synthesis, led to a search for new inhibitors of TS structurally analogous to 5,10-methylenetetrahydrofolate, the second substrate of TS. TS inhibitory antifolates developed to date that are in various stages of clinical evaluation are ZD 1694 and ZD9331 (Astra-Zeneca, London, UK), (Eli Lilly, Indianapolis, IN), LY231514 (BW1843U89 (Glaxo-Wellcome, Research Triangle Park, NC), and AG337 and AG331 (Agouron, La Jolla, CA). Although each of these compounds has TS as its major intracellular site of action, they differ in propensity for polyglutamylation and for transport by the reduced folate carrier. LY231514 also has secondary target enzymes. As a result, each compound is likely to have a different spectrum of antitumor activity and toxicity. This review will summarize the development and properties of this new class of TS inhibitors. PMID:10606255

  14. Morphological and metabolic changes in transgenic wheat with altered glycerol-3-phosphate acyltransferase or acyl-acyl carrier protein (ACP) thioesterase activities.

    PubMed

    Edlin, D A; Kille, P; Wilkinson, M D; Jones, H D; Harwood, J L

    2000-12-01

    We have transformed varieties of wheat with a Pisum sativum glycerol-3-phosphate acyltransferase gene, and also with an Arabidopsis thaliana acyl-ACP thioesterase gene. Morphological (growth, organelle development) and metabolic changes (fatty acid labelling of chloroplast and non-chloroplast lipids) have been observed in transgenics with altered gene expression for either enzyme. PMID:11171169

  15. An unexpected phosphate binding site in Glyceraldehyde 3-Phosphate Dehydrogenase: Crystal structures of apo, holo and ternary complex of Cryptosporidium parvum enzyme

    SciTech Connect

    Cook, William J; Senkovich, Olga; Chattopadhyay, Debasish

    2009-06-08

    The structure, function and reaction mechanism of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) have been extensively studied. Based on these studies, three anion binding sites have been identified, one 'Ps' site (for binding the C-3 phosphate of the substrate) and two sites, 'Pi' and 'new Pi', for inorganic phosphate. According to the original flip-flop model, the substrate phosphate group switches from the 'Pi' to the 'Ps' site during the multistep reaction. In light of the discovery of the 'new Pi' site, a modified flip-flop mechanism, in which the C-3 phosphate of the substrate binds to the 'new Pi' site and flips to the 'Ps' site before the hydride transfer, was proposed. An alternative model based on a number of structures of B. stearothermophilus GAPDH ternary complexes (non-covalent and thioacyl intermediate) proposes that in the ternary Michaelis complex the C-3 phosphate binds to the 'Ps' site and flips from the 'Ps' to the 'new Pi' site during or after the redox step. We determined the crystal structure of Cryptosporidium parvum GAPDH in the apo and holo (enzyme + NAD) state and the structure of the ternary enzyme-cofactor-substrate complex using an active site mutant enzyme. The C. parvum GAPDH complex was prepared by pre-incubating the enzyme with substrate and cofactor, thereby allowing free movement of the protein structure and substrate molecules during their initial encounter. Sulfate and phosphate ions were excluded from purification and crystallization steps. The quality of the electron density map at 2{angstrom} resolution allowed unambiguous positioning of the substrate. In three subunits of the homotetramer the C-3 phosphate group of the non-covalently bound substrate is in the 'new Pi' site. A concomitant movement of the phosphate binding loop is observed in these three subunits. In the fourth subunit the C-3 phosphate occupies an unexpected site not seen before and the phosphate binding loop remains in the substrate-free conformation

  16. SIRT1 interacts with and protects glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from nuclear translocation: Implications for cell survival after irradiation

    SciTech Connect

    Joo, Hyun-Yoo; Woo, Seon Rang; Shen, Yan-Nan; Yun, Mi Yong; Shin, Hyun-Jin; Park, Eun-Ran; Kim, Su-Hyeon; Park, Jeong-Eun; Ju, Yeun-Jin; Hong, Sung Hee; Hwang, Sang-Gu; Cho, Myung-Haing; Kim, Joon; Lee, Kee-Ho

    2012-08-10

    Highlights: Black-Right-Pointing-Pointer SIRT1 serves to retain GAPDH in the cytosol, preventing GAPDH nuclear translocation. Black-Right-Pointing-Pointer When SIRT1 is depleted, GAPDH translocation occurs even in the absence of stress. Black-Right-Pointing-Pointer Upon irradiation, SIRT1 interacts with GAPDH. Black-Right-Pointing-Pointer SIRT1 prevents irradiation-induced nuclear translocation of GAPDH. Black-Right-Pointing-Pointer SIRT1 presence rather than activity is essential for inhibiting GAPDH translocation. -- Abstract: Upon apoptotic stimulation, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a cytosolic enzyme normally active in glycolysis, translocates into the nucleus and activates an apoptotic cascade therein. In the present work, we show that SIRT1 prevents nuclear translocation of GAPDH via interaction with GAPDH. SIRT1 depletion triggered nuclear translocation of cytosolic GAPDH even in the absence of apoptotic stress. Such translocation was not, however, observed when SIRT1 enzymatic activity was inhibited, indicating that SIRT1 protein per se, rather than the deacetylase activity of the protein, is required to inhibit GAPDH translocation. Upon irradiation, SIRT1 prevented irradiation-induced nuclear translocation of GAPDH, accompanied by interaction of SIRT1 and GAPDH. Thus, SIRT1 functions to retain GAPDH in the cytosol, protecting the enzyme from nuclear translocation via interaction with these two proteins. This serves as a mechanism whereby SIRT1 regulates cell survival upon induction of apoptotic stress by means that include irradiation.

  17. Identification of a mammalian glycerol-3-phosphate phosphatase: Role in metabolism and signaling in pancreatic β-cells and hepatocytes.

    PubMed

    Mugabo, Yves; Zhao, Shangang; Seifried, Annegrit; Gezzar, Sari; Al-Mass, Anfal; Zhang, Dongwei; Lamontagne, Julien; Attane, Camille; Poursharifi, Pegah; Iglesias, José; Joly, Erik; Peyot, Marie-Line; Gohla, Antje; Madiraju, S R Murthy; Prentki, Marc

    2016-01-26

    Obesity, and the associated disturbed glycerolipid/fatty acid (GL/FA) cycle, contribute to insulin resistance, islet β-cell failure, and type 2 diabetes. Flux through the GL/FA cycle is regulated by the availability of glycerol-3-phosphate (Gro3P) and fatty acyl-CoA. We describe here a mammalian Gro3P phosphatase (G3PP), which was not known to exist in mammalian cells, that can directly hydrolyze Gro3P to glycerol. We identified that mammalian phosphoglycolate phosphatase, with an uncertain function, acts in fact as a G3PP. We found that G3PP, by controlling Gro3P levels, regulates glycolysis and glucose oxidation, cellular redox and ATP production, gluconeogenesis, glycerolipid synthesis, and fatty acid oxidation in pancreatic islet β-cells and hepatocytes, and that glucose stimulated insulin secretion and the response to metabolic stress, e.g., glucolipotoxicity, in β-cells. In vivo overexpression of G3PP in rat liver lowers body weight gain and hepatic glucose production from glycerol and elevates plasma HDL levels. G3PP is expressed at various levels in different tissues, and its expression varies according to the nutritional state in some tissues. As Gro3P lies at the crossroads of glucose, lipid, and energy metabolism, control of its availability by G3PP adds a key level of metabolic regulation in mammalian cells, and G3PP offers a potential target for type 2 diabetes and cardiometabolic disorders. PMID:26755581

  18. Enhanced resistance in Theobroma cacao against oomycete and fungal pathogens by secretion of phosphatidylinositol-3-phosphate-binding proteins.

    PubMed

    Helliwell, Emily E; Vega-Arreguín, Julio; Shi, Zi; Bailey, Bryan; Xiao, Shunyuan; Maximova, Siela N; Tyler, Brett M; Guiltinan, Mark J

    2016-03-01

    The internalization of some oomycete and fungal pathogen effectors into host plant cells has been reported to be blocked by proteins that bind to the effectors' cell entry receptor, phosphatidylinositol-3-phosphate (PI3P). This finding suggested a novel strategy for disease control by engineering plants to secrete PI3P-binding proteins. In this study, we tested this strategy using the chocolate tree Theobroma cacao. Transient expression and secretion of four different PI3P-binding proteins in detached leaves of T. cacao greatly reduced infection by two oomycete pathogens, Phytophthora tropicalis and Phytophthora palmivora, which cause black pod disease. Lesion size and pathogen growth were reduced by up to 85%. Resistance was not conferred by proteins lacking a secretory leader, by proteins with mutations in their PI3P-binding site, or by a secreted PI4P-binding protein. Stably transformed, transgenic T. cacao plants expressing two different PI3P-binding proteins showed substantially enhanced resistance to both P. tropicalis and P. palmivora, as well as to the fungal pathogen Colletotrichum theobromicola. These results demonstrate that secretion of PI3P-binding proteins is an effective way to increase disease resistance in T. cacao, and potentially in other plants, against a broad spectrum of pathogens. PMID:26214158

  19. Human and pneumococcal cell surface glyceraldehyde-3-phosphate dehydrogenase (GAPDH) proteins are both ligands of human C1q protein.

    PubMed

    Terrasse, Rémi; Tacnet-Delorme, Pascale; Moriscot, Christine; Pérard, Julien; Schoehn, Guy; Vernet, Thierry; Thielens, Nicole M; Di Guilmi, Anne Marie; Frachet, Philippe

    2012-12-14

    C1q, a key component of the classical complement pathway, is a major player in the response to microbial infection and has been shown to detect noxious altered-self substances such as apoptotic cells. In this work, using complementary experimental approaches, we identified the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a C1q partner when exposed at the surface of human pathogenic bacteria Streptococcus pneumoniae and human apoptotic cells. The membrane-associated GAPDH on HeLa cells bound the globular regions of C1q as demonstrated by pulldown and cell surface co-localization experiments. Pneumococcal strains deficient in surface-exposed GAPDH harbored a decreased level of C1q recognition when compared with the wild-type strains. Both recombinant human and pneumococcal GAPDHs interacted avidly with C1q as measured by surface plasmon resonance experiments (K(D) = 0.34-2.17 nm). In addition, GAPDH-C1q complexes were observed by transmission electron microscopy after cross-linking. The purified pneumococcal GAPDH protein activated C1 in an in vitro assay unlike the human form. Deposition of C1q, C3b, and C4b from human serum at the surface of pneumococcal cells was dependent on the presence of surface-exposed GAPDH. This ability of C1q to sense both human and bacterial GAPDHs sheds new insights on the role of this important defense collagen molecule in modulating the immune response. PMID:23086952

  20. Secreted multifunctional Glyceraldehyde-3-phosphate dehydrogenase sequesters lactoferrin and iron into cells via a non-canonical pathway

    PubMed Central

    Chauhan, Anoop S.; Rawat, Pooja; Malhotra, Himanshu; Sheokand, Navdeep; Kumar, Manoj; Patidar, Anil; Chaudhary, Surbhi; Jakhar, Priyanka; Raje, Chaaya I.; Raje, Manoj

    2015-01-01

    Lactoferrin is a crucial nutritionally important pleiotropic molecule and iron an essential trace metal for all life. The current paradigm is that living organisms have evolved specific membrane anchored receptors along with iron carrier molecules for regulated absorption, transport, storage and mobilization of these vital nutrients. We present evidence for the existence of non-canonical pathway whereby cells actively forage these vital resources from beyond their physical boundaries, by secreting the multifunctional housekeeping enzyme Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) into the extracellular milieu. This effect’s an autocrine/paracrine acquisition of target ligand into the cell. Internalization by this route is extensively favoured even by cells that express surface receptors for lactoferrin and involves urokinase plasminogen activator receptor (uPAR). We also demonstrate the operation of this phenomenon during inflammation, as an arm of the innate immune response where lactoferrin denies iron to invading microorganisms by chelating it and then itself being sequestered into surrounding host cells by GAPDH. PMID:26672975

  1. Over-expression of PsGPD, a mushroom glyceraldehyde-3-phosphate dehydrogenase gene, enhances salt tolerance in rice plants.

    PubMed

    Cho, Jung-Il; Lim, Hye-Min; Siddiqui, Zamin Shaheed; Park, Sung-Han; Kim, A-Ram; Kwon, Taek-Ryoun; Lee, Seong-Kon; Park, Soo-Chul; Jeong, Mi-Jeong; Lee, Gang-Seob

    2014-08-01

    Transgenic potatoes expressing glyceraldehyde-3-phosphate dehydrogenase (GPD), isolated from the oyster mushroom, Pleurotus sajor-caju, had increased tolerance to salt stress (Jeong et al. Biochem Biophys Res Commun 278:192-196, 2000). To examine the physiological mechanisms enhancing salt tolerance in GPD-transgenic rice plants, the salt tolerance of five GPD transgenic rice lines (T1-T5) derived from Dongjin rice cultivar were evaluated in a fixed 150 mM saline environment in comparison to two known wild-type rice cultivars, Dongjin (salt sensitive) and Pokali (salt tolerant). Transgenic lines, T2, T3, and T5, had a substantial increase in biomass and relative water content compared to Dongjin. Stomatal conductance and osmotic potential were higher in the GPD transgenic lines and were similar to those in Pokali. The results are discussed based on the comparative physiological response of GPD transgenic lines with those of the salt-sensitive and salt-tolerant rice cultivars. PMID:24737077

  2. Cloning, expression, purification, crystallization and preliminary X-ray analysis of human liver glyceraldehyde-3-phosphate dehydrogenase.

    PubMed

    Warizaya, Masaichi; Kinoshita, Takayoshi; Kato, Akemi; Nakajima, Hidenori; Fujii, Takashi

    2004-03-01

    Human liver glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purified and crystallized using PEG 3350 as a precipitant. However, the crystals were extremely fragile towards osmotic shock. A 1% change in PEG 3350 content causes destruction of the crystals. After many trials for freezing the crystals, X-ray diffraction data from a native crystal were collected at 2.8 A resolution using as a cryoprotectant a mixture consisting of paraffin oil and Paratone-N in a 3:1 ratio and a cryoloop covered with Formver film. Crystals belong to space group P2(1), with unit-cell parameters a = 63.23, b = 97.84, c = 84.23 A, beta = 104.1 degrees. Molecular replacement with a starting model consisting of a homology model based on the low-resolution structure of human skeletal muscle GAPDH, which has 90% identical residues with the liver protein, led to a solution. Most of the current model was assigned properly in the electron-density map, but the map corresponding to some important regions containing the phosphate-binding loop was ambiguous. It is planned to crystallize human liver GAPDH in the presence of phosphate ions and/or some kind of inhibitor in order to fix the flexible region. PMID:14993695

  3. The influence of oxygen on radiation-induced structural and functional changes in glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase

    NASA Astrophysics Data System (ADS)

    Rodacka, Aleksandra; Serafin, Eligiusz; Bubinski, Michal; Krokosz, Anita; Puchala, Mieczyslaw

    2012-07-01

    Proteins are major targets for oxidative damage due to their abundance in cells and high reactivity with free radicals. In the present study we examined the influence of oxygen on radiation-induced inactivation and structural changes of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and lactate dehydrogenase (LDH). We chose these two enzymes because they occur at high concentrations and participate in the most important processes in organisms; furthermore, they show considerable similarity in their structure. Protein solutions were irradiated with X-rays in doses ranging from 0.1 to 0.7 kGy, in air and N2O. The much higher radiation inactivation of GAPDH as compared to LDH is correlated with substantially greater structural changes in this protein, mainly involving the loss of free thiol groups (-SH). Of lesser importance in the differentiation of the radiosensitivity of the studied enzymes are tryptophan residues. Molecular oxygen, present during irradiation, increased to a significantly greater extent the inactivation and structural changes of GAPDH than that of LDH. The results suggest that the greater effect of oxygen on GAPDH is due to the higher efficiency of the superoxide radical, the higher amount of hydroperoxides generated, and the higher degree of unfolding of this protein.

  4. Insulin regulation of protein biosynthesis in differentiated 3T3 adipocytes. Regulation of glyceraldehyde-3-phosphate dehydrogenase

    SciTech Connect

    Alexander, M.; Curtis, G.; Avruch, J.; Goodman, H.M.

    1985-10-05

    The effect of insulin on protein biosynthesis was examined in differentiated 3T3-L1 and 3T3-F442A adipocytes. Insulin altered the relative rate of synthesis of specific proteins independent of its ability to hasten conversion of the fibroblast (preadipocyte) phenotype to the adipocyte phenotype. Although more than one pattern of response to insulin was observed, the authors focused on the induction of a Mr 33,000 protein which was identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Exposure of 3T3 adipocytes to insulin throughout differentiation specifically increased GAPDH activity and protein content by 2- to 3-fold as compared to 3T3 adipocytes differentiated in the absence of insulin. These changes in enzyme activity and content could be accounted for by a 4-fold increase in the relative rate of synthesis of GAPDH and a 9-fold increase in hybridizable mRNA levels. Within 2 h of insulin addition to 3T3 adipocytes differentiated in the absence of hormone, hybridizable GAPDH mRNA levels increased 3-fold, and within 24 h GAPDH mRNA levels increased 8-fold, and (TVS) methionine incorporation into GAPDH protein increased 5-fold. These studies demonstrate that insulin, as the sole hormonal perturbant, can increase the synthesis of certain 3T3 adipocyte proteins by altering the cellular content of a specific mRNA.

  5. Export of malaria proteins requires co-translational processing of the PEXEL motif independent of phosphatidylinositol-3-phosphate binding

    PubMed Central

    Boddey, Justin A.; O'Neill, Matthew T.; Lopaticki, Sash; Carvalho, Teresa G.; Hodder, Anthony N.; Nebl, Thomas; Wawra, Stephan; van West, Pieter; Ebrahimzadeh, Zeinab; Richard, Dave; Flemming, Sven; Spielmann, Tobias; Przyborski, Jude; Babon, Jeff J.; Cowman, Alan F.

    2016-01-01

    Plasmodium falciparum exports proteins into erythrocytes using the Plasmodium export element (PEXEL) motif, which is cleaved in the endoplasmic reticulum (ER) by plasmepsin V (PMV). A recent study reported that phosphatidylinositol-3-phosphate (PI(3)P) concentrated in the ER binds to PEXEL motifs and is required for export independent of PMV, and that PEXEL motifs are functionally interchangeable with RxLR motifs of oomycete effectors. Here we show that the PEXEL does not bind PI(3)P, and that this lipid is not concentrated in the ER. We find that RxLR motifs cannot mediate export in P. falciparum. Parasites expressing a mutated version of KAHRP, with the PEXEL motif repositioned near the signal sequence, prevented PMV cleavage. This mutant possessed the putative PI(3)P-binding residues but is not exported. Reinstatement of PEXEL to its original location restores processing by PMV and export. These results challenge the PI(3)P hypothesis and provide evidence that PEXEL position is conserved for co-translational processing and export. PMID:26832821

  6. Glyceraldehyde-3-phosphate dehydrogenase antisense oligodeoxynucleotides protect against cytosine arabinonucleoside-induced apoptosis in cultured cerebellar neurons.

    PubMed Central

    Ishitani, R; Chuang, D M

    1996-01-01

    Cytosine arabinonucleoside (AraC) is a pyrimidine antimetabolite that kills proliferating cells by inhibiting DNA synthesis and, importantly, is also an inducer of apoptosis. We recently reported that age-induced apoptotic cell death of cultured cerebellar neurons is directly associated with an over-expression of a particulate 38-kDa protein, identified by us as glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12). We now show that the AraC-induced neuronal death of immature cerebellar granule cells in culture is effectively delayed by actinomycin-D, cycloheximide, or aurintricarboxylic acid (a DNase inhibitor). Furthermore, two GAPDH antisense, but not their corresponding sense, oligodeoxyribonucleotides markedly arrested AraC-induced apoptosis. This protection was more effective than that induced by the above-mentioned classical inhibitors of apoptosis. Prior to AraC-induced neuronal death, GAPDH mRNA levels increased by approximately 2.5-fold, and this mRNA accumulation was blocked by actinomycin-D and the GAPDH antisense (but not sense) oligonucleotide. Like actinomycin-D, a GAPDH antisense oligonucleotide also suppressed the AraC-induced over-expression of the 38-kDa particulate protein (i.e., GAPDH), while the corresponding sense oligonucleotide was totally ineffective. Thus, the present results show that GAPDH over-expression is involved in AraC-induced apoptosis of cultured cerebellar granule cells. Images Fig. 2 Fig. 3 Fig. 4 PMID:8790435

  7. Glycerol-3-phosphate acyltransferase 4 is essential for the normal development of reproductive organs and the embryo in Brassica napus

    PubMed Central

    Chen, Xue; Chen, Guanqun; Truksa, Martin; Snyder, Crystal L.; Shah, Saleh; Weselake, Randall J.

    2014-01-01

    The enzyme sn-glycerol-3-phosphate acyltransferase 4 (GPAT4) is involved in the biosynthesis of plant lipid poly-esters. The present study further characterizes the enzymatic activities of three endoplasmic reticulum-bound GPAT4 isoforms of Brassica napus and examines their roles in the development of reproductive organs and the embryo. All three BnGPAT4 isoforms exhibited sn-2 acyltransferase and phosphatase activities with dicarboxylic acid-CoA as acyl donor. When non-substituted acyl-CoA was used as acyl donor, the rate of acylation was considerably lower and phosphatase activity was not manifested. RNA interference (RNAi)-mediated down-regulation of all GPAT4 homologues in B. napus under the control of the napin promoter caused abnormal development of several reproductive organs and reduced seed set. Microscopic examination and reciprocal crosses revealed that both pollen grains and developing embryo sacs of the B. napus gpat4 lines were affected. The gpat4 mature embryos showed decreased cutin content and altered monomer composition. The defective embryo development further affected the oil body morphology, oil content, and fatty acid composition in gpat4 seeds. These results suggest that GPAT4 has a critical role in the development of reproductive organs and the seed of B. napus. PMID:24821955

  8. Autophagy and endosomal trafficking inhibition by Vibrio cholerae MARTX toxin phosphatidylinositol-3-phosphate-specific phospholipase A1 activity

    PubMed Central

    Agarwal, Shivani; Kim, Hyunjin; Chan, Robin B.; Agarwal, Shivangi; Williamson, Rebecca; Cho, Wonhwa; Paolo, Gilbert D.; Satchell, Karla J. F.

    2015-01-01

    Vibrio cholerae, responsible for acute gastroenteritis secretes a large multifunctional-autoprocessing repeat-in-toxin (MARTX) toxin linked to evasion of host immune system, facilitating colonization of small intestine. Unlike other effector domains of the multifunctional toxin that target cytoskeleton, the function of alpha-beta hydrolase (ABH) remained elusive. This study demonstrates that ABH is an esterase/lipase with catalytic Ser–His–Asp triad. ABH binds with high affinity to phosphatidylinositol-3-phosphate (PtdIns3P) and cleaves the fatty acid in PtdIns3P at the sn1 position in vitro making it the first PtdIns3P-specific phospholipase A1 (PLA1). Expression of ABH in vivo reduces intracellular PtdIns3P levels and its PtdIns3P-specific PLA1 activity blocks endosomal and autophagic pathways. In accordance with recent studies acknowledging the potential of extracellular pathogens to evade or exploit autophagy to prevent their clearance and facilitate survival, this is the first report highlighting the role of ABH in inhibiting autophagy and endosomal trafficking induced by extracellular V. cholerae. PMID:26498860

  9. Glycerol-3-Phosphate Acyltransferase Contributes to Triacylglycerol Biosynthesis, Lipid Droplet Formation, and Host Invasion in Metarhizium robertsii

    PubMed Central

    Gao, Qiang; Shang, Yanfang; Huang, Wei

    2013-01-01

    Enzymes involved in the triacylglycerol (TAG) biosynthesis have been well studied in the model organisms of yeasts and animals. Among these, the isoforms of glycerol-3-phosphate acyltransferase (GPAT) redundantly catalyze the first and rate-limiting step in glycerolipid synthesis. Here, we report the functions of mrGAT, a GPAT ortholog, in an insect-pathogenic fungus, Metarhizium robertsii. Unlike in yeasts and animals, a single copy of the mrGAT gene is present in the fungal genome and the gene deletion mutant is viable. Compared to the wild type and the gene-rescued mutant, the ΔmrGAT mutant demonstrated reduced abilities to produce conidia and synthesize TAG, glycerol, and total lipids. More importantly, we found that mrGAT is localized to the endoplasmic reticulum and directly linked to the formation of lipid droplets (LDs) in fungal cells. Insect bioassay results showed that mrGAT is required for full fungal virulence by aiding fungal penetration of host cuticles. Data from this study not only advance our understanding of GPAT functions in fungi but also suggest that filamentous fungi such as M. robertsii can serve as a good model to elucidate the role of the glycerol phosphate pathway in fungal physiology, particularly to determine the mechanistic connection of GPAT to LD formation. PMID:24077712

  10. Improved purification of sn-glycerol-3-phosphate dehydrogenase of Saccharomyces cerevisiae and its inhibition by ethanol

    SciTech Connect

    Merkel, J.R.; Chen, S.M.; Osinchak, J.; Trumbore, M.

    1986-05-01

    An improved purification procedure yielded a homogeneous preparation of sn-glycerol-3-phosphate dehydrogenase (GPD) from commercially available baker's yeast. The enzyme had an apparent molecular weight of 42,000 by SDS-polyacrylamide gel electrophoresis. This differs from the 31,000 reported earlier on the basis of its elution from a calibrated Sepharose 6B column. When denatured by guanidine (6M) and chromatographed on a Sephadex G-100 column with 6M guanidine in 0.1M phosphate buffer, pH 6.5, containing 0.1M ..beta..-mercaptoethanol, GPD eluted with the approximately 42,000 mw proteins. S. cerevisiae GPD is an NAD-dependent oxidoreductase. With NADH as the variable substrate the GPD-catalyzed reduction of dihydroxacetone phosphate (DHAP) had a K/sub M/ of 0.018 mM and was competitively inhibited by ethanol. With DHAP as the variable substrate and NADH constant GPD catalyzed the reduction with a K/sub M/ of 0.37 mM and was noncompetitively inhibited by ethanol. The calculated K/sub i/ for the non-competitive inhibition was 3.4M. K/sub i/ for the competitive inhibition of NADH by ethanol varied with increasing concentrations of ethanol indicating a more complex mechanism than a truly competitive one.

  11. Sequence analysis and structural characterization of a glyceraldehyde-3-phosphate dehydrogenase gene from the phytopathogenic fungus Eremothecium ashbyi.

    PubMed

    Sengupta, Sudeshna; Chandra, T S

    2011-02-01

    Eremothecium ashbyi is a phytopathogenic fungus infesting cotton, soybeans and several other plants. This highly flavinogenic fungus has been phylogenetically characterized, but the genetic aspects of its central metabolic and riboflavin biosynthetic pathways are unknown. An ORF of 996 bp was obtained from E. ashbyi by using degenerate primers for glyceraldehyde-3-phosphate dehydrogenase (GPD) through reverse transcriptase polymerase chain reaction (RT-PCR) and 5'-3' rapid amplification of cDNA ends (RACE-PCR). This nucleotide sequence had a high similarity of 88% with GPD sequence of Ashbya gossypii. The putative GPD peptide of 331-aa had a high similarity of 85% with the GPD sequence from other ascomycetes. The ORF had an unusually strong codon bias with 5 amino acids showing strict preference of a single codon. The theoretical molecular weight for the putative peptide was 35.58 kDa with an estimated pI of 5.7. A neighbor-joining tree showed that the putative peptide from E. ashbyi displayed the highest similarity to GPD of A. gossypii. The gene sequence is available at the GenBank, accession number EU717696. Homology modeling done with Kluyveromyces marxianus GPD (PDB: 2I5P) as template indicated high structural similarity. PMID:20820924

  12. Altered chloroplast structure and function in a mutant of Arabidopsis deficient in plastid glycerol-3-phosphate acyltransferase activity

    SciTech Connect

    Kunst, L.; Somerville, C. ); Browse, J. )

    1989-07-01

    Mutants of Arabidopsis thaliana deficient in plastid glycerol-3-phosphate acyltransferase activity have altered chloroplast membrane lipid composition. This caused an increase in the number of regions of appressed membrane per chloroplast and a decrease in the average number of thylakoid membranes in the appressed regions. The net effect was a significant decrease in the ratio of appressed to nonappressed membranes. A comparison of 77 K fluorescence emission spectra of thylakoid membranes from the mutant and wild type indicated that the ultrastructural changes were associated with an altered distribution of excitation energy transfer from antenna chlorophyll to photosystem II and photosystem I in the mutant. The changes in leaf lipid composition did not significantly affect growth or development of the mutant under standard conditions. However, at temperatures above 28{degree}C the mutant grew slightly more rapidly than the wild type, and measurements of temperature-induced fluorescence yield enhancement suggested an increased thermal stability of the photosynthetic apparatus of the mutant. These effects are consistent with other evidence suggesting that membrane lipid composition is an important determinant of chloroplast structure but has relatively minor direct effects on the function of the membrane proteins associated with photosynthetic electron transport.

  13. Structural basis for the NAD binding cooperativity and catalytic characteristics of sperm-specific glyceraldehyde-3-phosphate dehydrogenase.

    PubMed

    Kuravsky, M L; Barinova, K V; Asryants, R A; Schmalhausen, E V; Muronetz, V I

    2015-08-01

    Catalytic properties of enzymes used in biotechnology can be improved by eliminating those regulatory mechanisms that are not absolutely required for their functioning. We exploited mammalian glyceraldehyde-3-phosphate dehydrogenase as a model protein and examined the structural basis of the NAD(+) cooperative binding exhibited by its homologous isoenzymes: the somatic enzyme (GAPD) and the recombinant sperm-specific enzyme (dN-GAPDS). Moreover, we obtained a mutant dN-GAPDS, which misses the cooperativity, but exhibits a twofold increase in the specific activity instead (92 and 45 μmol NADH/min per mg protein for the mutant and the wild type proteins, respectively). Such an effect was caused by the disruption of the interdomain salt bridge D311-H124, which is located close to the active site of the enzyme. The thermal stability of the mutant protein also increased compared to the wild type form (heat absorption peak values were 70.4 and 68.6 °C, respectively). We expect our findings to be of importance for the purposes of biotechnological applications. PMID:25936797

  14. The plasmin-binding protein Plr of group A streptococci is identified as glyceraldehyde-3-phosphate dehydrogenase.

    PubMed

    Winram, S B; Lottenberg, R

    1996-08-01

    Group A streptococci bind the serine protease plasmin with high affinity. Previously, a 41 kDa protein was identified as a candidate plasmin receptor protein (Plr) from group A streptococcal strain 64/14. The plr gene encoding Plr was cloned and the deduced amino acid sequence of Plr had significant similarity to glyceraldehyde-3-phosphate dehydrogenases (GAPDHs). In this study we have isolated cytoplasmic GAPDH of streptococcal strain 64/14. This enzyme was examined, on both structural and functional levels, for its relatedness to the Plr of strain 64/14 purified from mutanolysin extract and to recombinant Plr. We report here that no differences were detected between streptococcal Plr and cytoplasmic GAPDH on the basis of antibody reactivity, plasmin-binding activity, GAPDH activity, N-terminal amino acid sequence, peptide map analysis by V8 protease digestion and amino acid composition analysis. Furthermore, the plr gene appears to be present as a single copy in group A streptococci. PMID:8760943

  15. A Dimer Interface Mutation in Glyceraldehyde-3-Phosphate Dehydrogenase Regulates Its Binding to AU-rich RNA*

    PubMed Central

    White, Michael R.; Khan, Mohd M.; Deredge, Daniel; Ross, Christina R.; Quintyn, Royston; Zucconi, Beth E.; Wysocki, Vicki H.; Wintrode, Patrick L.; Wilson, Gerald M.; Garcin, Elsa D.

    2015-01-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an enzyme best known for its role in glycolysis. However, extra-glycolytic functions of GAPDH have been described, including regulation of protein expression via RNA binding. GAPDH binds to numerous adenine-uridine rich elements (AREs) from various mRNA 3′-untranslated regions in vitro and in vivo despite its lack of a canonical RNA binding motif. How GAPDH binds to these AREs is still unknown. Here we discovered that GAPDH binds with high affinity to the core ARE from tumor necrosis factor-α mRNA via a two-step binding mechanism. We demonstrate that a mutation at the GAPDH dimer interface impairs formation of the second RNA-GAPDH complex and leads to changes in the RNA structure. We investigated the effect of this interfacial mutation on GAPDH oligomerization by crystallography, small-angle x-ray scattering, nano-electrospray ionization native mass spectrometry, and hydrogen-deuterium exchange mass spectrometry. We show that the mutation does not significantly affect GAPDH tetramerization as previously proposed. Instead, the mutation promotes short-range and long-range dynamic changes in regions located at the dimer and tetramer interface and in the NAD+ binding site. These dynamic changes are localized along the P axis of the GAPDH tetramer, suggesting that this region is important for RNA binding. Based on our results, we propose a model for sequential GAPDH binding to RNA via residues located at the dimer and tetramer interfaces. PMID:25451934

  16. Phosphatidylcholine is essential for efficient functioning of the mitochondrial glycerol-3-phosphate dehydrogenase Gut2 in Saccharomyces cerevisiae.

    PubMed

    Rijken, Pieter J; De Kruijff, Ben; De Kroon, Anton I P M

    2007-01-01

    Gut2, the mitochondrial glycerol-3-phosphate dehydrogenase, was previously shown to become preferentially labelled with photoactivatable phosphatidylcholine (PC), pointing to a functional relation between these molecules. In the present study we analyzed whether Gut2 functioning depends on the PC content of yeast cells, using PC biosynthetic mutants in which the PC content was lowered. PC depletion was found to reduce growth on glycerol and to increase glycerol excretion, both indicating that PC is needed for optimal Gut2 functioning in vivo. Using several in vitro approaches the nature of the dependence of Gut2 functioning on cellular PC contents was investigated. The results of these experiments suggest that it is unlikely that the effects observed in vivo are due to changes in cellular Gut2 content, in specific activity of Gut2 in isolated mitochondria, or in the membrane association of Gut2, upon lowering the PC level. The in vivo effects are more likely an indirect result of PC depletion-induced changes in the cellular context in which Gut2 functions, that are not manifested in the in vitro systems used. PMID:17520483

  17. Identification of a mammalian glycerol-3-phosphate phosphatase: Role in metabolism and signaling in pancreatic β-cells and hepatocytes

    PubMed Central

    Mugabo, Yves; Zhao, Shangang; Seifried, Annegrit; Gezzar, Sari; Al-Mass, Anfal; Zhang, Dongwei; Lamontagne, Julien; Attane, Camille; Poursharifi, Pegah; Iglesias, José; Joly, Erik; Peyot, Marie-Line; Gohla, Antje; Madiraju, S. R. Murthy; Prentki, Marc

    2016-01-01

    Obesity, and the associated disturbed glycerolipid/fatty acid (GL/FA) cycle, contribute to insulin resistance, islet β-cell failure, and type 2 diabetes. Flux through the GL/FA cycle is regulated by the availability of glycerol-3-phosphate (Gro3P) and fatty acyl-CoA. We describe here a mammalian Gro3P phosphatase (G3PP), which was not known to exist in mammalian cells, that can directly hydrolyze Gro3P to glycerol. We identified that mammalian phosphoglycolate phosphatase, with an uncertain function, acts in fact as a G3PP. We found that G3PP, by controlling Gro3P levels, regulates glycolysis and glucose oxidation, cellular redox and ATP production, gluconeogenesis, glycerolipid synthesis, and fatty acid oxidation in pancreatic islet β-cells and hepatocytes, and that glucose stimulated insulin secretion and the response to metabolic stress, e.g., glucolipotoxicity, in β-cells. In vivo overexpression of G3PP in rat liver lowers body weight gain and hepatic glucose production from glycerol and elevates plasma HDL levels. G3PP is expressed at various levels in different tissues, and its expression varies according to the nutritional state in some tissues. As Gro3P lies at the crossroads of glucose, lipid, and energy metabolism, control of its availability by G3PP adds a key level of metabolic regulation in mammalian cells, and G3PP offers a potential target for type 2 diabetes and cardiometabolic disorders. PMID:26755581

  18. Glycerol-3-phosphate acyltransferase 4 is essential for the normal development of reproductive organs and the embryo in Brassica napus.

    PubMed

    Chen, Xue; Chen, Guanqun; Truksa, Martin; Snyder, Crystal L; Shah, Saleh; Weselake, Randall J

    2014-08-01

    The enzyme sn-glycerol-3-phosphate acyltransferase 4 (GPAT4) is involved in the biosynthesis of plant lipid poly-esters. The present study further characterizes the enzymatic activities of three endoplasmic reticulum-bound GPAT4 isoforms of Brassica napus and examines their roles in the development of reproductive organs and the embryo. All three BnGPAT4 isoforms exhibited sn-2 acyltransferase and phosphatase activities with dicarboxylic acid-CoA as acyl donor. When non-substituted acyl-CoA was used as acyl donor, the rate of acylation was considerably lower and phosphatase activity was not manifested. RNA interference (RNAi)-mediated down-regulation of all GPAT4 homologues in B. napus under the control of the napin promoter caused abnormal development of several reproductive organs and reduced seed set. Microscopic examination and reciprocal crosses revealed that both pollen grains and developing embryo sacs of the B. napus gpat4 lines were affected. The gpat4 mature embryos showed decreased cutin content and altered monomer composition. The defective embryo development further affected the oil body morphology, oil content, and fatty acid composition in gpat4 seeds. These results suggest that GPAT4 has a critical role in the development of reproductive organs and the seed of B. napus. PMID:24821955

  19. Export of malaria proteins requires co-translational processing of the PEXEL motif independent of phosphatidylinositol-3-phosphate binding.

    PubMed

    Boddey, Justin A; O'Neill, Matthew T; Lopaticki, Sash; Carvalho, Teresa G; Hodder, Anthony N; Nebl, Thomas; Wawra, Stephan; van West, Pieter; Ebrahimzadeh, Zeinab; Richard, Dave; Flemming, Sven; Spielmann, Tobias; Przyborski, Jude; Babon, Jeff J; Cowman, Alan F

    2016-01-01

    Plasmodium falciparum exports proteins into erythrocytes using the Plasmodium export element (PEXEL) motif, which is cleaved in the endoplasmic reticulum (ER) by plasmepsin V (PMV). A recent study reported that phosphatidylinositol-3-phosphate (PI(3)P) concentrated in the ER binds to PEXEL motifs and is required for export independent of PMV, and that PEXEL motifs are functionally interchangeable with RxLR motifs of oomycete effectors. Here we show that the PEXEL does not bind PI(3)P, and that this lipid is not concentrated in the ER. We find that RxLR motifs cannot mediate export in P. falciparum. Parasites expressing a mutated version of KAHRP, with the PEXEL motif repositioned near the signal sequence, prevented PMV cleavage. This mutant possessed the putative PI(3)P-binding residues but is not exported. Reinstatement of PEXEL to its original location restores processing by PMV and export. These results challenge the PI(3)P hypothesis and provide evidence that PEXEL position is conserved for co-translational processing and export. PMID:26832821

  20. Exogenous nitric oxide (NO) generation or IL-1[beta]-induced intracellular NO production stimulates inhibitory auto-ADP-ribosylation of glyceraldehyde-3-phosphate dehydrogenase in RINm5F cells

    SciTech Connect

    Dimmeler, S.; Bruene, B. ); Ankarcrona, M.; Nicotera, P. )

    1993-04-01

    Nitric oxide (NO) stimulates the auto-ADP-ribosylation of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) which results in the inhibition of enzyme activity. In the present work the authors show that addition of exogenous NO or IL-1[beta]-induced intracellular NO generation cause GAPDH ADP-ribosylation and inhibition of enzyme activity. Incubation of RINm5F cells with sodium nitroprusside (SNP) for 18 h caused a time- and dose-dependent inhibition of GAPDH activity. Half-maximal inhibition of GAPDH activity was observed with 80 [mu]M of the NO donor, with maximal inhibition after roughly 6 h of incubation. In parallel, SNP induced endogenous ADP-ribosylation of GAPDH measured by a decreased incorporation of [[sup 32]P]ADP-ribose from [[sup 32]P]NAD[sup +] in the cytosol of the SNP-treated cells. Stimulation of endogenous NO production by inducing the NO synthase by exposure to the cytokine IL-1[beta] results in decreased GAPDH activity. IL-1[beta] (10[sup [minus]9] M) inhibited GAPDH activity about 55%, compared with control values. Production of nitrite and inhibition of GAPDH was reversed by the NAD[sup +] synthease inhibitor N[sub G]-monomethyl-L-arginine, indicating the endogenous generated NO was the effective molecule. Again, GAPDH inhibition was associated with NO-stimulated endogenous ADP-ribosylation of the enzyme Western blot analysis of GAPDH excluded degradation of GAPDH by NO. NO-stimulated auto-ADP-ribosylation resulted in inhibition of the glycolytic enzyme GAPDH and may be relevant as a cytotoxic effect of NO. In concert with its inhibitory actions on iron-sulfur enzymes like aconitase and electron transport proteins of the respiratory chain, NO may mediate autocytotoxic effect in [beta]-cells. 40 refs., 7 figs.

  1. Investigation of the complex between rabbit muscle glyceraldehyde-3-phosphate dehydrogenase and aldolase using fluorescence laser spectroscopy

    NASA Astrophysics Data System (ADS)

    Orstan, Aydin; Gafni, Ari

    1990-05-01

    The interaction of rabbit muscle aldolase with glyceraldehyde-3-phosphate dehydrogenase (GPDH) labeled with fluorescein-5-isothiocyanate (FITC) has been investigated at 25°C in Tris buffer, pH 7. 5. The addition of 5 to1 0 fold excess of aldolase to 0.1-1 micron GPDH labeled with FITC (GPDHFITC) causes a large increase in both the fluorescence and polarization of FITC over a period of several hours, reflecting the formation ofa complex between the enzymes. When GPDH-FITC is incubated with either 1 mM NAD or ADP, the fluorescence of FITC increases while the polarization decreases, indicating that these nucleotides may increase the degree of dissociation of tetrameric GPDH. The rate of approach to equilibrium during the formation of the complex between the two enzymes increases in the presence of either NAD or ADP but decreases with increasing concentrations ofGPDH. Therefore, the interaction of the enzymes may involve the dissociation of tetrameric GPDH into smaller units. Aldolase causes no changes in the fluorescence properties of probes that are attacted to the active site cysteine residues of GPDH, such as fluorescein acrylamide, indicating that when the active site of GPDH is blocked the formation of the complex between the two enzymes is prevented. When GPDH-FITC is incubated with either excess NAD or aldolase, the average fluorescence lifetime of FITC slowly increases and approaches that of free FITC. Since fluorescein is quenched by tryptophan, it is likely that the increases observed both in the fluorescence intensities and lifetimes of GPDH-FITC during its interaction with aldolase arise from the removal of tryptophan residues from the vicinity of the FITC groups as a result of changes either in the conformation or in the degree of dissociation of tetrameric GPDH.

  2. The Glycerol-3-Phosphate Acyltransferase GPAT6 from Tomato Plays a Central Role in Fruit Cutin Biosynthesis.

    PubMed

    Petit, Johann; Bres, Cécile; Mauxion, Jean-Philippe; Tai, Fabienne Wong Jun; Martin, Laetitia B B; Fich, Eric A; Joubès, Jérôme; Rose, Jocelyn K C; Domergue, Frédéric; Rothan, Christophe

    2016-06-01

    The thick cuticle covering and embedding the epidermal cells of tomato (Solanum lycopersicum) fruit acts not only as a protective barrier against pathogens and water loss but also influences quality traits such as brightness and postharvest shelf-life. In a recent study, we screened a mutant collection of the miniature tomato cultivar Micro-Tom and isolated several glossy fruit mutants in which the abundance of cutin, the polyester component of the cuticle, was strongly reduced. We employed a newly developed mapping-by-sequencing strategy to identify the causal mutation underlying the cutin deficiency in a mutant thereafter named gpat6-a (for glycerol-3-phosphate acyltransferase6). To this end, a backcross population (BC1F2) segregating for the glossy trait was phenotyped. Individuals displaying either a wild-type or a glossy fruit trait were then pooled into bulked populations and submitted to whole-genome sequencing prior to mutation frequency analysis. This revealed that the causal point mutation in the gpat6-a mutant introduces a charged amino acid adjacent to the active site of a GPAT6 enzyme. We further showed that this mutation completely abolished the GPAT activity of the recombinant protein. The gpat6-a mutant showed perturbed pollen formation but, unlike a gpat6 mutant of Arabidopsis (Arabidopsis thaliana), was not male sterile. The most striking phenotype was observed in the mutant fruit, where cuticle thickness, composition, and properties were altered. RNA sequencing analysis highlighted the main processes and pathways that were affected by the mutation at the transcriptional level, which included those associated with lipid, secondary metabolite, and cell wall biosynthesis. PMID:27208295

  3. Crystal Structure of CTP: Glycerol-3-Phosphate Cytidylyl Tranferase from Staphylococcus Aurues: Examination of Structural Basis for Kinetic Mechanism

    SciTech Connect

    Fong,D.; Yim, V.; D'elia, M.; Brown, E.; Berghuis, A.

    2006-01-01

    Integrity of the cell wall is essential for bacterial survival, and as a consequence components involved in its biosynthesis can potentially be exploited as targets for antibiotics. One such potential target is CTP:glycerol-3-phosphate cytidylyltransferase. This enzyme (TarD{sub Sa} in Staphylococcus aureus and TagD{sub Bs} in Bacillus subtilis) catalyzes the formation of CDP-glycerol, which is used for the assembly of linkages between peptidoglycan and teichoic acid polymer in Gram-positive bacteria. Intriguingly, despite the high sequence identity between TarD{sub Sa} and TagD{sub Bs} (69% identity), kinetic studies show that these two enzymes differ markedly in their kinetic mechanism and activity. To examine the basis for the disparate enzymological properties, we have determined the crystal structure of TarD{sub Sa} in the apo state to 3 Angstroms resolution, and performed equilibrium sedimentation analysis. Comparison of the structure with that of CTP- and CDP-glycerol-bound TagD{sub Bs} crystal structures reveals that the overall structure of TarD{sub Sa} is essentially the same as that of TagD{sub Bs}, except in the C-terminus, where it forms a helix in TagD{sub Bs} but is disordered in the apo TarDSa structure. In addition, TarD{sub Sa} can exist both as a tetramer and as a dimer, unlike TagD{sub Bs}, which is a dimer. These observations shed light on the structural basis for the differing kinetic characteristics between TarD{sub Sa} and TagD{sub Bs}.

  4. Glycerol-3-phosphate Acyltransferase Isoform-4 (GPAT4) Limits Oxidation of Exogenous Fatty Acids in Brown Adipocytes.

    PubMed

    Cooper, Daniel E; Grevengoed, Trisha J; Klett, Eric L; Coleman, Rosalind A

    2015-06-12

    Glycerol-3-phosphate acyltransferase-4 (GPAT4) null pups grew poorly during the suckling period and, as adults, were protected from high fat diet-induced obesity. To determine why Gpat4(-/-) mice failed to gain weight during these two periods of high fat feeding, we examined energy metabolism. Compared with controls, the metabolic rate of Gpat4(-/-) mice fed a 45% fat diet was 12% higher. Core body temperature was 1 ºC higher after high fat feeding. Food intake, fat absorption, and activity were similar in both genotypes. Impaired weight gain in Gpat4(-/-) mice did not result from increased heat loss, because both cold tolerance and response to a β3-adrenergic agonist were similar in both genotypes. Because GPAT4 comprises 65% of the total GPAT activity in brown adipose tissue (BAT), we characterized BAT function. A 45% fat diet increased the Gpat4(-/-) BAT expression of peroxisome proliferator-activated receptor α (PPAR) target genes, Cpt1α, Pgc1α, and Ucp1, and BAT mitochondria oxidized oleate and pyruvate at higher rates than controls, suggesting that fatty acid signaling and flux through the TCA cycle were enhanced. To assess the role of GPAT4 directly, neonatal BAT preadipocytes were differentiated to adipocytes. Compared with controls, Gpat4(-/-) brown adipocytes incorporated 33% less fatty acid into triacylglycerol and 46% more into the pathway of β-oxidation. The increased oxidation rate was due solely to an increase in the oxidation of exogenous fatty acids. These data suggest that in the absence of cold exposure, GPAT4 limits excessive fatty acid oxidation and the detrimental induction of a hypermetabolic state. PMID:25918168

  5. A land-plant-specific glycerol-3-phosphate acyltransferase family in Arabidopsis: substrate specificity, sn-2 preference, and evolution.

    PubMed

    Yang, Weili; Simpson, Jeffrey P; Li-Beisson, Yonghua; Beisson, Fred; Pollard, Mike; Ohlrogge, John B

    2012-10-01

    Arabidopsis (Arabidopsis thaliana) has eight glycerol-3-phosphate acyltransferase (GPAT) genes that are members of a plant-specific family with three distinct clades. Several of these GPATs are required for the synthesis of cutin or suberin. Unlike GPATs with sn-1 regiospecificity involved in membrane or storage lipid synthesis, GPAT4 and -6 are unique bifunctional enzymes with both sn-2 acyltransferase and phosphatase activity resulting in 2-monoacylglycerol products. We present enzymology, pathway organization, and evolutionary analysis of this GPAT family. Within the cutin-associated clade, GPAT8 is demonstrated as a bifunctional sn-2 acyltransferase/phosphatase. GPAT4, -6, and -8 strongly prefer C16:0 and C18:1 ω-oxidized acyl-coenzyme As (CoAs) over unmodified or longer acyl chain substrates. In contrast, suberin-associated GPAT5 can accommodate a broad chain length range of ω-oxidized and unsubstituted acyl-CoAs. These substrate specificities (1) strongly support polyester biosynthetic pathways in which acyl transfer to glycerol occurs after oxidation of the acyl group, (2) implicate GPAT specificities as one major determinant of cutin and suberin composition, and (3) argue against a role of sn-2-GPATs (Enzyme Commission 2.3.1.198) in membrane/storage lipid synthesis. Evidence is presented that GPAT7 is induced by wounding, produces suberin-like monomers when overexpressed, and likely functions in suberin biosynthesis. Within the third clade, we demonstrate that GPAT1 possesses sn-2 acyltransferase but not phosphatase activity and can utilize dicarboxylic acyl-CoA substrates. Thus, sn-2 acyltransferase activity extends to all subbranches of the Arabidopsis GPAT family. Phylogenetic analyses of this family indicate that GPAT4/6/8 arose early in land-plant evolution (bryophytes), whereas the phosphatase-minus GPAT1 to -3 and GPAT5/7 clades diverged later with the appearance of tracheophytes. PMID:22864585

  6. A distinct type of glycerol-3-phosphate acyltransferase with sn-2 preference and phosphatase activity producing 2-monoacylglycerol.

    PubMed

    Yang, Weili; Pollard, Mike; Li-Beisson, Yonghua; Beisson, Fred; Feig, Michael; Ohlrogge, John

    2010-06-29

    The first step in assembly of membrane and storage glycerolipids is acylation of glycerol-3-phosphate (G3P). All previously characterized membrane-bound, eukaryotic G3P acyltransferases (GPATs) acylate the sn-1 position to produce lysophosphatidic acid (1-acyl-LPA). Cutin is a glycerolipid with omega-oxidized fatty acids and glycerol as integral components. It occurs as an extracellular polyester on the aerial surface of all plants, provides a barrier to pathogens and resistance to stress, and maintains organ identity. We have determined that Arabidopsis acyltransferases GPAT4 and GPAT6 required for cutin biosynthesis esterify acyl groups predominantly to the sn-2 position of G3P. In addition, these acyltransferases possess a phosphatase domain that results in sn-2 monoacylglycerol (2-MAG) rather than LPA as the major product. Such bifunctional activity has not been previously described in any organism. The possible roles of 2-MAGs as intermediates in cutin synthesis are discussed. GPAT5, which is essential for the accumulation of suberin aliphatics, also exhibits a strong preference for sn-2 acylation. However, phosphatase activity is absent and 2-acyl-LPA is the major product. Clearly, plant GPATs can catalyze more reactions than the sn-1 acylation by which they are currently categorized. Close homologs of GPAT4-6 are present in all land plants, but not in animals, fungi or microorganisms (including algae). Thus, these distinctive acyltransferases may have been important for evolution of extracellular glycerolipid polymers and adaptation of plants to a terrestrial environment. These results provide insight into the biosynthetic assembly of cutin and suberin, the two most abundant glycerolipid polymers in nature. PMID:20551224

  7. Sex and depot differences in ex vivo adipose tissue fatty acid storage and glycerol-3-phosphate acyltransferase activity

    PubMed Central

    Morgan-Bathke, Maria; Chen, Liang; Oberschneider, Elisabeth; Harteneck, Debra

    2015-01-01

    Adipose tissue fatty acid storage varies according to sex, adipose tissue depot, and degree of fat gain. However, the mechanism(s) for these variations is not completely understood. We examined whether differences in adipose tissue glycerol-3-phosphate acyltransferase (GPAT) might play a role in these variations. We optimized an enzyme activity assay for total GPAT and GPAT1 activity in human adipose tissue and measured GPAT activity. Omental and subcutaneous adipose tissue was collected from obese and nonobese adults for measures of GPAT and GPAT1 activities, ex vivo palmitate storage, acyl-CoA synthetase (ACS) and diacylglycerol-acyltransferase (DGAT) activities, and CD36 protein. Total GPAT and GPAT1 activities decreased as a function of adipocyte size in both omental (r = −0.71, P = 0.003) and subcutaneous (r = −0.58, P = 0.04) fat. The relative contribution of GPAT1 to total GPAT activity increased as a function of adipocyte size, accounting for up to 60% of GPAT activity in those with the largest adipocytes. We found strong, positive correlations between ACS, GPAT, and DGAT activities for both sexes and depots (r values 0.58–0.91) and between these storage factors and palmitate storage rates into TAG (r values 0.55–0.90). We conclude that: 1) total GPAT activity decreases as a function of adipocyte size; 2) GPAT1 can account for over half of adipose GPAT activity in hypertrophic obesity; and 3) ACS, GPAT, and DGAT are coordinately regulated. PMID:25738782

  8. Pharmacological glycerol-3-phosphate acyltransferase inhibition decreases food intake and adiposity and increases insulin sensitivity in diet-induced obesity

    PubMed Central

    Kuhajda, Francis P.; Tu, Yajun; Han, Wan Fang; Medghalchi, Susan M.; El Meskini, Rajaa; Landree, Leslie E.; Peterson, Jonathan M.; Daniels, Khadija; Wong, Kody; Wydysh, Edward A.; Townsend, Craig A.; Ronnett, Gabriele V.

    2011-01-01

    Storage of excess calories as triglycerides is central to obesity and its associated disorders. Glycerol-3-phosphate acyltransferases (GPATs) catalyze the initial step in acylglyceride syntheses, including triglyceride synthesis. We utilized a novel small-molecule GPAT inhibitor, FSG67, to investigate metabolic consequences of systemic pharmacological GPAT inhibition in lean and diet-induced obese (DIO) mice. FSG67 administered intraperitoneally decreased body weight and energy intake, without producing conditioned taste aversion. Daily FSG67 (5 mg/kg, 15.3 μmol/kg) produced gradual 12% weight loss in DIO mice beyond that due to transient 9- to 10-day hypophagia (6% weight loss in pair-fed controls). Continued FSG67 maintained the weight loss despite return to baseline energy intake. Weight was lost specifically from fat mass. Indirect calorimetry showed partial protection by FSG67 against decreased rates of oxygen consumption seen with hypophagia. Despite low respiratory exchange ratio due to a high-fat diet, FSG67-treated mice showed further decreased respiratory exchange ratio, beyond pair-fed controls, indicating enhanced fat oxidation. Chronic FSG67 increased glucose tolerance and insulin sensitivity in DIO mice. Chronic FSG67 decreased gene expression for lipogenic enzymes in white adipose tissue and liver and decreased lipid accumulation in white adipose, brown adipose, and liver tissues without signs of damage. RT-PCR showed decreased gene expression for orexigenic hypothalamic neuropeptides AgRP or NPY after acute and chronic systemic FSG67. FSG67 given intracerebroventricularly (100 and 320 nmol icv) produced 24-h weight loss and feeding suppression, indicating contributions from direct central nervous system sites of action. Together, these data point to GPAT as a new potential therapeutic target for the management of obesity and its comorbidities. PMID:21490364

  9. The Glycerol-3-Phosphate Acyltransferase GPAT6 from Tomato Plays a Central Role in Fruit Cutin Biosynthesis1[OPEN

    PubMed Central

    Petit, Johann; Mauxion, Jean-Philippe; Tai, Fabienne Wong Jun; Fich, Eric A.; Joubès, Jérôme; Rothan, Christophe

    2016-01-01

    The thick cuticle covering and embedding the epidermal cells of tomato (Solanum lycopersicum) fruit acts not only as a protective barrier against pathogens and water loss but also influences quality traits such as brightness and postharvest shelf-life. In a recent study, we screened a mutant collection of the miniature tomato cultivar Micro-Tom and isolated several glossy fruit mutants in which the abundance of cutin, the polyester component of the cuticle, was strongly reduced. We employed a newly developed mapping-by-sequencing strategy to identify the causal mutation underlying the cutin deficiency in a mutant thereafter named gpat6-a (for glycerol-3-phosphate acyltransferase6). To this end, a backcross population (BC1F2) segregating for the glossy trait was phenotyped. Individuals displaying either a wild-type or a glossy fruit trait were then pooled into bulked populations and submitted to whole-genome sequencing prior to mutation frequency analysis. This revealed that the causal point mutation in the gpat6-a mutant introduces a charged amino acid adjacent to the active site of a GPAT6 enzyme. We further showed that this mutation completely abolished the GPAT activity of the recombinant protein. The gpat6-a mutant showed perturbed pollen formation but, unlike a gpat6 mutant of Arabidopsis (Arabidopsis thaliana), was not male sterile. The most striking phenotype was observed in the mutant fruit, where cuticle thickness, composition, and properties were altered. RNA sequencing analysis highlighted the main processes and pathways that were affected by the mutation at the transcriptional level, which included those associated with lipid, secondary metabolite, and cell wall biosynthesis. PMID:27208295

  10. Genetics Home Reference: GM3 synthase deficiency

    MedlinePlus

    ... GM3 synthase deficiency is characterized by recurrent seizures (epilepsy) and problems with brain development. Within the first ... diagnosis or management of GM3 synthase deficiency: American Epilepsy Society: Find a Doctor Clinic for Special Children ( ...

  11. Mycocerosic acid synthase exemplifies the architecture of reducing polyketide synthases.

    PubMed

    Herbst, Dominik A; Jakob, Roman P; Zähringer, Franziska; Maier, Timm

    2016-03-24

    Polyketide synthases (PKSs) are biosynthetic factories that produce natural products with important biological and pharmacological activities. Their exceptional product diversity is encoded in a modular architecture. Modular PKSs (modPKSs) catalyse reactions colinear to the order of modules in an assembly line, whereas iterative PKSs (iPKSs) use a single module iteratively as exemplified by fungal iPKSs (fiPKSs). However, in some cases non-colinear iterative action is also observed for modPKSs modules and is controlled by the assembly line environment. PKSs feature a structural and functional separation into a condensing and a modifying region as observed for fatty acid synthases. Despite the outstanding relevance of PKSs, the detailed organization of PKSs with complete fully reducing modifying regions remains elusive. Here we report a hybrid crystal structure of Mycobacterium smegmatis mycocerosic acid synthase based on structures of its condensing and modifying regions. Mycocerosic acid synthase is a fully reducing iPKS, closely related to modPKSs, and the prototype of mycobacterial mycocerosic acid synthase-like PKSs. It is involved in the biosynthesis of C20-C28 branched-chain fatty acids, which are important virulence factors of mycobacteria. Our structural data reveal a dimeric linker-based organization of the modifying region and visualize dynamics and conformational coupling in PKSs. On the basis of comparative small-angle X-ray scattering, the observed modifying region architecture may be common also in modPKSs. The linker-based organization provides a rationale for the characteristic variability of PKS modules as a main contributor to product diversity. The comprehensive architectural model enables functional dissection and re-engineering of PKSs. PMID:26976449

  12. Expression, purification, crystallization and preliminary X-ray analysis of an NAD-dependent glyceraldehyde-3-phosphate dehydrogenase from Helicobacter pylori

    SciTech Connect

    Elliott, Paul R.; Mohammad, Shabaz; Melrose, Helen J.; Moody, Peter C. E.

    2008-08-01

    Glyceraldehyde-3-phosphate dehydrogenase B from H. pylori has been cloned, expressed, purified and crystallized in the presence of NAD. Crystals of GAPDHB diffracted to 2.8 Å resolution and belonged to space group P6{sub 5}22, with unit-cell parameters a = b = 166.1, c = 253.1 Å. Helicobacter pylori is a dangerous human pathogen that resides in the upper gastrointestinal tract. Little is known about its metabolism and with the onset of antibiotic resistance new treatments are required. In this study, the expression, purification, crystallization and preliminary X-ray diffraction of an NAD-dependent glyceraldehyde-3-phosphate dehydrogenase from H. pylori are reported.

  13. Sucrose Synthase: Expanding Protein Function

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sucrose synthase (SUS: EC 2.4.1.13), a key enzyme in plant sucrose catabolism, is uniquely able to mobilize sucrose into multiple pathways involved in metabolic, structural, and storage functions. Our research indicates that the biological function of SUS may extend beyond its catalytic activity. Th...

  14. Glyceraldehyde-3-phosphate dehydrogenase gene over expression correlates with poor prognosis in non small cell lung cancer patients

    PubMed Central

    2013-01-01

    Background Glycolysis in presence of oxygen with high glucose consumption is known to be the metabolism of choice in many tumors. In lung cancer this phenomenon is routinely exploited in diagnostic PET imaging of fluorodeoxyglucose uptake, but not much is known about the prognostic capabilities of glycolysis level assessment in resected lung tumor samples. Methods In this retrospective study, we used real time polymerase chain reaction(RQ-PCR) to assess the expression level of the gene for Glyceraldehyde 3-phosphate dehydrogenase(GAPDH), key enzyme for glucose breakdown, in tumor samples from 82 consecutive early stages resected non small cell lung cancer(NSCLC) patients. We then compared our results in six large publicly available NSCLC microarray datasets collecting data from over 1250 total patients. Results In our study GAPDH gene over expression was found to be an adverse prognostic factor in early stages NSCLC (n = 82 HR = 1.30 p = 0.050). This result was confirmed in 5 of 6 public datasets analyzed: Shedden et al. 2008: n = 442 HR = 1.54 p < 0.0001; Lee et al. 2008: n = 138 HR = 1.31 p = 0.043; Tomida et al. 2009: n = 117 HR = 1.59 p = 0.004; Roepman et al. 2009: n = 172 (TPI1 gene) HR = 1.51 p = 0.009; Okayama et al. 2012: n = 226 HR = 3.19 p < 0.0001; Botling et al. 2013: n = 196 HR = 1.00 p = 0.97). Furthermore, in the large and clinically well annotated Shedden et al. microarray dataset, GAPDH hazard ratio did not change whether calculated for the whole dataset or for the subgroup of adjuvant naive patients only (n = 330 HR = 1.49 p < 0.0001). Conclusion GAPDH gene over expression in resected tumor samples is an adverse prognostic factor in NSCLC. Our results confirm the prognostic value of glucose metabolism assessment in NSCLC. PMID:23988223

  15. Nitric oxide-dependent NAD linkage to glyceraldehyde-3-phosphate dehydrogenase: possible involvement of a cysteine thiyl radical intermediate.

    PubMed Central

    Minetti, M; Pietraforte, D; Di Stasi, A M; Mallozzi, C

    1996-01-01

    Previous studies have demonstrated that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) undergoes NAD(H) linkage to an active site thiol when it comes into contact with .NO-related oxidants. We found that a free-radical generator 2,2'-azobis-(2-amidinopropane) hydrochloride (AAPH), which does not release either .NO or .NO-related species, was indeed able to induce the NAD(H) linkage to GAPDH. We performed spin-trapping studies with purified apo-GAPDH to identify a putative thiol intermediate produced by AAPH as well as by .NO-related oxidants. As .NO sources we used .NO gas and two .NO-donors, S-nitroso-N-acetyl-D,L-penicillamine and 3-morpholinosydno-nimine hydrochloride (SIN-1). Because SIN-1 produces .NO and a superoxide radical simultaneously, we also tested the effects of peroxynitrite. All the .NO-related oxidants were able to induce the linkage of NAD(H) to GAPDH and the formation of a protein free-radical identified as a thiyl radical (inhibited by N-ethylmaleimide). .NO gas and the .NO-donors required molecular oxygen to induce the formation of the GAPDH thiyl radical, suggesting the possible involvement of higher nitrogen oxides. Thiyl radical formation was decreased by the reconstitution of GAPDH with NAD+. Apo-GAPDH was a strong scavenger of AAPH radicals, but its scavenging ability was decreased when its cysteine residues were alkylated or when it was reconstituted with NAD+. In addition, after treatment with AAPH, a thiyl radical of GAPDH was trapped at high enzyme concentrations. We suggest that the NAD(H) linkage to GAPDH is mediated by a thiyl radical intermediate not specific to .NO or .NO-related oxidants. The cysteine residue located at the active site of GAPDH (Cys-149) is oxidized by free radicals to a thiyl radical, which reacts with the neighbouring coenzyme to form Cys-NAD(H) linkages. Studies with the NAD+ molecule radio-labelled in the nicotinamide or adenine portion revealed that both portions of the NAD+ molecule are linked to GAPDH

  16. Antibodies to inactive conformations of glyceraldehyde-3-phosphate dehydrogenase inactivate the apo- and holoforms of the enzyme.

    PubMed

    Arutiunova, E I; Pleten, A P; Nagradova, N K; Muronetz, V I

    2006-06-01

    Polyclonal antibodies produced after the immunization of a rabbit with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus were used to isolate two types of antibodies interacting with different non-native forms of the antigen. Type I antibodies were purified using Sepharose-bound apo-GAPDH that was treated with glutaraldehyde to stabilize the enzyme in the tetrameric form. Type II antibodies were isolated using immobilized denatured monomers of the enzyme. It was shown that the type I antibodies bound to the native holo- and apoforms of the enzyme with the ratio of one antibody molecule per GAPDH tetramer. While interacting with the native holoenzyme, the type I antibodies induce a time-dependent decrease in its activity by 80-90%. In the case of the apoenzyme, the decrease in the activity constitutes only 25%, this indicating that only one subunit of the tetramer is inactivated. Differential scanning calorimetry experiments showed that the formation of the complex between both forms of the enzyme and the type I antibodies resulted in a shift of the maximum of the thermal capacity curves (T(m) value) to lower temperatures. The extremely stable holoenzyme was affected to the greatest extent, the shift of the T(m) value constituting approximately 20 degrees C. We assume that the formation of the complex between the holo- or apo-GAPDH and the type I antibody results in time-dependent conformational changes in the enzyme molecule. Thus, the antibodies induce the structural rearrangements yielding the conformation that is identical to the structure of the antigen used for the selection of the antibodies (i.e., inactive). The interaction of the antibodies with the apo-GAPDH results in the inactivation of the subunit directly bound to the antibody. Virtually complete inactivation of the holoenzyme by the antibodies is likely due to the transmission of the conformational changes through the intersubunit contacts. The type II antibodies, which

  17. Glyceraldehyde 3-phosphate dehydrogenase and galectin from Dirofilaria immitis participate in heartworm disease endarteritis via plasminogen/plasmin system.

    PubMed

    González-Miguel, Javier; Larrazabal, Carmen; Loa-Mesón, Diana; Siles-Lucas, Mar; Simón, Fernando; Morchón, Rodrigo

    2016-06-15

    The interaction between parasitic protozoa and helminths, both in the blood and in tissues and the fibrinolytic system of their hosts is usually considered as a survival parasite mechanism since this system is the physiological route responsible for degrading fibrin clots. The broad-range proteolytic activity of plasmin, the final enzyme of the route, implies that its recruitment by these parasites is an important mechanism that mediates their invasion and establishment in the hosts. However, recent studies have proposed a dual role for plasmin by linking its over-production with pathological mechanisms at vascular level. Most of these studies have been conducted in Dirofilaria immitis, a blood-borne parasite that survives in the pulmonary arteries of its host for years while it produces a chronic inflammatory disease, whose main pathogenic mechanism is the appearance of proliferative endarteritis. Recently, the participation of two proteins from D. immitis, glyceraldehyde 3-phosphate dehydrogenase (DiGAPDH) and galectin (DiGAL), in the activation of the fibrinolytic system of its host has been demonstrated, which has been a priori associated with parasite survival mechanisms. The aim of the present paper was to study the role of plasmin generated by these proteins in the emergence of proliferative endarteritis. An in vitro model of canine endothelial and smooth muscle cells, as well as the two parasitic recombinant proteins were employed. The results show that DiGAPDH and DiGAL stimulate the proliferation and migration of both cell types, as well as the degradation of the extracellular matrix (ECM) via plasminogen (PLG)/plasmin system, being all of these mechanisms related to the appearance of proliferative endarteritis. Due to the high degree of evolutionary conservation of these antigens, these data support the hypothesis of the survival/pathology ambivalence in the interactions between parasites and the fibrinolytic system of their hosts and represent an

  18. Isoprene synthase genes form a monophyletic clade of acyclic terpene synthases in the TPS-B terpene synthase family.

    PubMed

    Sharkey, Thomas D; Gray, Dennis W; Pell, Heather K; Breneman, Steven R; Topper, Lauren

    2013-04-01

    Many plants emit significant amounts of isoprene, which is hypothesized to help leaves tolerate short episodes of high temperature. Isoprene emission is found in all major groups of land plants including mosses, ferns, gymnosperms, and angiosperms; however, within these groups isoprene emission is variable. The patchy distribution of isoprene emission implies an evolutionary pattern characterized by many origins or many losses. To better understand the evolution of isoprene emission, we examine the phylogenetic relationships among isoprene synthase and monoterpene synthase genes in the angiosperms. In this study we identify nine new isoprene synthases within the rosid angiosperms. We also document the capacity of a myrcene synthase in Humulus lupulus to produce isoprene. Isoprene synthases and (E)-β-ocimene synthases form a monophyletic group within the Tps-b clade of terpene synthases. No asterid genes fall within this clade. The chemistry of isoprene synthase and ocimene synthase is similar and likely affects the apparent relationships among Tps-b enzymes. The chronology of rosid evolution suggests a Cretaceous origin followed by many losses of isoprene synthase over the course of evolutionary history. The phylogenetic pattern of Tps-b genes indicates that isoprene emission from non-rosid angiosperms likely arose independently. PMID:23550753

  19. Classification of fungal chitin synthases.

    PubMed Central

    Bowen, A R; Chen-Wu, J L; Momany, M; Young, R; Szaniszlo, P J; Robbins, P W

    1992-01-01

    Comparison of the chitin synthase genes of Saccharomyces cerevisiae CHS1 and CHS2 with the Candida albicans CHS1 gene (UDP-N-acetyl-D-glucosamine:chitin 4-beta-N-acetylglucosaminyltransferase, EC 2.4.1.16) revealed two small regions of complete amino acid sequence conservation that were used to design PCR primers. Fragments homologous to chitin synthase (approximately 600 base pairs) were amplified from the genomic DNA of 14 fungal species. These fragments were sequenced, and their deduced amino acid sequences were aligned. With the exception of S. cerevisiae CHS1, the sequences fell into three distinct classes, which could represent separate functional groups. Within each class phylogenetic analysis was performed. Although not the major purpose of the investigation, this analysis tends to confirm some relationships consistent with current taxonomic groupings. Images PMID:1731323

  20. Structure of Trypanosoma cruzi glycosomal glyceraldehyde-3-phosphate dehydrogenase complexed with chalepin, a natural product inhibitor, at 1.95 A resolution.

    PubMed

    Pavão, F; Castilho, M S; Pupo, M T; Dias, R L A; Correa, A G; Fernandes, J B; da Silva, M F G F; Mafezoli, J; Vieira, P C; Oliva, G

    2002-06-01

    The structure of the glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) from Trypanosoma cruzi complexed with chalepin, a natural product from Pilocarpus spicatus, has been determined by X-ray crystallography to 1.95 A resolution. The structure is in the apo form without cofactors in the subunits of the tetrameric gGAPDH in the asymmetric unit. Unequivocal density corresponding to the inhibitor was clearly identified in one monomer. The final refined model of the complex shows extensive conformational changes when compared with the native structure. The mode of binding of chalepin to gGAPDH and its implications for inhibitor design are discussed. PMID:12044862

  1. Herbicidal inhibitors of amino acid biosynthesis and herbicide-tolerant crops.

    PubMed

    Tan, S; Evans, R; Singh, B

    2006-03-01

    Acetohydroxyacid synthase (AHAS) inhibitors interfere with branched-chain amino acid biosynthesis by inhibiting AHAS. Glyphosate affects aromatic amino acid biosynthesis by inhibiting 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Glufosinate inhibits glutamine synthetase and blocks biosynthesis of glutamine. AHAS gene variants that confer tolerance to AHAS inhibitors have been discovered in plants through selection or mutagenesis. Imidazolinone-tolerant crops have been commercialized based on these AHAS gene variants. A modified maize EPSPS gene and CP4-EPSPS gene from Agrobacterium sp. have been used to transform plants for target-based tolerance to glyphosate. A gox gene isolated from Ochrobactrum anthropi has also been employed to encode glyphosate oxidoreductase to detoxify glyphosate in plants. Glyphosate-tolerant crops with EPSPS transgene alone or both EPSPS and gox transgenes have been commercialized. Similarly, bar and pat genes isolated from Streptomyces hygroscopicus and S. viridochromogenes, respectively, have been inserted into plants to encode phosphinothricin N-acetyltransferase to detoxify glufosinate. Glufosinate-tolerant crops have been commercialized using one of these two transgenes. PMID:16547651

  2. Nitro-arginine methyl ester, a non-selective inhibitor of nitric oxide synthase reduces ibuprofen-induced gastric mucosal injury in the rat.

    PubMed

    Abraham, Premila; K, Indirani; K, Desigamani

    2005-09-01

    Ibuprofen is a commonly used non-steroidal anti-inflammatory drug. Gastrointestinal adverse drug reactions from ibuprofen usage include gastric mucosal ulcers and bleeding. The mechanism by which ibuprofen induces gastric mucosal damage is not clear. The present study is an attempt to examine the role of nitric oxide in the pathogenesis of ibuprofen-induced gastric mucosal damage. Ibuprofen administered orally at the dose of 100 mg/kg body weight for 6 days to the rats resulted in gastric mucosal injury. Serum nitrite and nitrosothiol were increased significantly as compared with the controls, which were treated with the vehicle alone. In the gastric mucosa, lipid peroxidation and protein thiols were increased, and the activity of glyceraldehyde 3-phosphate dehydrogenase, a nitric oxide sensitive enzyme was decreased significantly. Pretreatment of the rats daily with nitric oxide synthase inhibitor, nitro-arginine methyl ester (30 mg/kg body weight) 1 hr before treatment with ibuprofen reduced the gastric mucosal injury. Biochemically, it prevented the rise in serum nitrite levels and the increase in lipid peroxidation and protein thiol levels and the loss of glyceraldehyde 3-phosphate dehydrogenase activity in the gastric mucosa. The results of the present study suggest that increased nitric oxide production may be one of the mechanisms by which ibuprofen produces gastric mucosal injury and that inhibition of nitric oxide synthase reduces gastric mucosal injury. PMID:16133962

  3. A functional cellulose synthase from ascidian epidermis

    PubMed Central

    Matthysse, Ann G.; Deschet, Karine; Williams, Melanie; Marry, Mazz; White, Alan R.; Smith, William C.

    2004-01-01

    Among animals, urochordates (e.g., ascidians) are unique in their ability to biosynthesize cellulose. In ascidians cellulose is synthesized in the epidermis and incorporated into a protective coat know as the tunic. A putative cellulose synthase-like gene was first identified in the genome sequences of the ascidian Ciona intestinalis. We describe here a cellulose synthase gene from the ascidian Ciona savignyi that is expressed in the epidermis. The predicted C. savignyi cellulose synthase amino acid sequence showed conserved features found in all cellulose synthases, including plants, but was most similar to cellulose synthases from bacteria, fungi, and Dictyostelium discoidium. However, unlike other known cellulose synthases, the predicted C. savignyi polypeptide has a degenerate cellulase-like region near the carboxyl-terminal end. An expression construct carrying the C. savignyi cDNA was found to restore cellulose biosynthesis to a cellulose synthase (CelA) minus mutant of Agrobacterium tumefaciens, showing that the predicted protein has cellulose synthase activity. The lack of cellulose biosynthesis in all other groups of metazoans and the similarity of the C. savignyi cellulose synthase to enzymes from cellulose-producing organisms support the hypothesis that the urochordates acquired the cellulose biosynthetic pathway by horizontal transfer. PMID:14722352

  4. Efficient heterocyclisation by (di)terpene synthases.

    PubMed

    Mafu, S; Potter, K C; Hillwig, M L; Schulte, S; Criswell, J; Peters, R J

    2015-09-11

    While cyclic ether forming terpene synthases are known, the basis for such heterocyclisation is unclear. Here it is reported that numerous (di)terpene synthases, particularly including the ancestral ent-kaurene synthase, efficiently produce isomers of manoyl oxide from the stereochemically appropriate substrate. Accordingly, such heterocyclisation is easily accomplished by terpene synthases. Indeed, the use of single residue changes to induce production of the appropriate substrate in the upstream active site leads to efficient bifunctional enzymes producing isomers of manoyl oxide, representing novel enzymatic activity. PMID:26214384

  5. Producing biofuels using polyketide synthases

    DOEpatents

    Katz, Leonard; Fortman, Jeffrey L; Keasling, Jay D

    2013-04-16

    The present invention provides for a non-naturally occurring polyketide synthase (PKS) capable of synthesizing a carboxylic acid or a lactone, and a composition such that a carboxylic acid or lactone is included. The carboxylic acid or lactone, or derivative thereof, is useful as a biofuel. The present invention also provides for a recombinant nucleic acid or vector that encodes such a PKS, and host cells which also have such a recombinant nucleic acid or vector. The present invention also provides for a method of producing such carboxylic acids or lactones using such a PKS.

  6. Metabolomic analysis reveals hepatic metabolite perturbations in citrin/mitochondrial glycerol-3-phosphate dehydrogenase double-knockout mice, a model of human citrin deficiency.

    PubMed

    Saheki, Takeyori; Inoue, Kanako; Ono, Hiromi; Tushima, Anmi; Katsura, Natsumi; Yokogawa, Mana; Yoshidumi, Yukari; Kuhara, Tomiko; Ohse, Morimasa; Eto, Kazuhiro; Kadowaki, Takashi; Sinasac, David S; Kobayashi, Keiko

    2011-12-01

    The citrin/mitochondrial glycerol-3-phosphate dehydrogenase (mGPD) double-knockout mouse displays phenotypic attributes of both neonatal intrahepatic cholestasis and adult-onset type II citrullinemia, making it a suitable model of human citrin deficiency. In the present study, we investigated metabolic disturbances in the livers of wild-type, citrin (Ctrn) knockout, mGPD knockout, and Ctrn/mGPD double-knockout mice following oral sucrose versus saline administration using metabolomic approaches. By using gas chromatography/mass spectrometry and capillary electrophoresis/mass spectrometry, we found three general groupings of metabolite changes in the livers of the double-knockout mice following sucrose administration that were subsequently confirmed using liquid chromatography/mass spectrometry or enzymatic methods: a marked increase of hepatic glycerol 3-phosphate, a generalized decrease of hepatic tricarboxylic acid cycle intermediates, and alterations of hepatic amino acid levels related to the urea cycle or lysine catabolism including marked increases in citrulline and lysine. Furthermore, concurrent oral administration of sodium pyruvate with sucrose ameliorated the hyperammonemia induced by sucrose, as had been shown previously, as well as almost completely normalizing the hepatic metabolite perturbations found. Overall, we have identified additional metabolic disturbances in double-KO mice following oral sucrose administration, and provided further evidence for the therapeutic use of sodium pyruvate in our mouse model of citrin deficiency. PMID:21908222

  7. Over-expression of Arabidopsis thaliana SFD1/GLY1, the gene encoding plastid localized glycerol-3-phosphate dehydrogenase, increases plastidic lipid content in transgenic rice plants.

    PubMed

    Singh, Vijayata; Singh, Praveen Kumar; Siddiqui, Adnan; Singh, Subaran; Banday, Zeeshan Zahoor; Nandi, Ashis Kumar

    2016-03-01

    Lipids are the major constituents of all membranous structures in plants. Plants possess two pathways for lipid biosynthesis: the prokaryotic pathway (i.e., plastidic pathway) and the eukaryotic pathway (i.e., endoplasmic-reticulum (ER) pathway). Whereas some plants synthesize galactolipids from diacylglycerol assembled in the plastid, others, including rice, derive their galactolipids from diacylglycerols assembled by the eukaryotic pathway. Arabidopsis thaliana glycerol-3-phosphate dehydrogenase (G3pDH), coded by SUPPRESSOR OF FATTY ACID DESATURASE 1 (SFD1; alias GLY1) gene, catalyzes the formation of glycerol 3-phosphate (G3p), the backbone of many membrane lipids. Here SFD1 was introduced to rice as a transgene. Arabidopsis SFD1 localizes in rice plastids and its over-expression increases plastidic membrane lipid content in transgenic rice plants without any major impact on ER lipids. The results suggest that over-expression of plastidic G3pDH enhances biosynthesis of plastid-localized lipids in rice. Lipid composition in the transgenic plants is consistent with increased phosphatidylglycerol synthesis in the plastid and increased galactolipid synthesis from diacylglycerol produced via the ER pathway. The transgenic plants show a higher photosynthetic assimilation rate, suggesting a possible application of this finding in crop improvement. PMID:26747130

  8. Identification of arylazido-. beta. -alanyl NAD/sup +/ modified site in the rabbit muscle glyceraldehyde-3-phosphate dehydeogenase by microsequencing and fast atom bombardment mass spectrometry

    SciTech Connect

    Chen, S.; Lee, T.D.; Legesse, K.; Shively, J.E.

    1985-05-01

    Arylazido-BETA-alanyl NAD/sup +/, A3'-0-((3-(N-(4-azido-2-nitrophenyl) amino) propionyl)) NAD/sup +/, is a photoaffinity analog of NAD/sup +/. The NAD/sup +/ analog has been previously demonstrated to modify glyceraldehyde-3-phosphate dehydrogenase in a very specific manner and probably at the active site of the enzyme. The labeling experiment utilizing (/sup 3/H)-arylazido-..beta..-alanyl NAD/sup +/ revealed that the analog is associated exclusively with a tryptic peptide which has the sequence as Ile-Val-Ser-Asn-Ala-Ser-Cys-Thr-Thr-Asn. In comparison to the amino acid sequence of glyceraldehyde-3-phosphate dehydeogenase from other species this peptide is in a highly conserved region and is part of the active site of enzyme. The cysteine residue at position seven was predominantly labeled and suggested to be the site modified by arylazido-..beta..-alanyl NAD/sup +/. This cysteins residue corresponds to the Cys-149 in the pig muscle enzyme and which has been shown to be an essential residue for the enzyme activity. These results demonstrate that arylazido-..beta..-alanyl NAD/sup +/ is a useful photoaffinity probe to characterize the active site of NAD(H) dependent enzymes.

  9. Molecular Clone and Expression of a NAD+-Dependent Glycerol-3-Phosphate Dehydrogenase Isozyme Gene from the Halotolerant alga Dunaliella salina

    PubMed Central

    Cai, Ma; He, Li-Hong; Yu, Tu-Yuan

    2013-01-01

    Glycerol is an important osmotically compatible solute in Dunaliella. Glycerol-3-phosphate dehydrogenase (G3PDH) is a key enzyme in the pathway of glycerol synthesis, which converts dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate. Generally, the glycerol-DHAP cycle pathway, which is driven by G3PDH, is considered as the rate-limiting enzyme to regulate the glycerol level under osmotic shocks. Considering the peculiarity in osmoregulation, the cDNA of a NAD+-dependent G3PDH was isolated from D. salina using RACE and RT-PCR approaches in this study. Results indicated that the length of the cDNA sequence of G3PDH was 2,100 bp encoding a 699 amino acid deduced polypeptide whose computational molecular weight was 76.6 kDa. Conserved domain analysis revealed that the G3PDH protein has two independent functional domains, SerB and G3PDH domains. It was predicted that the G3PDH was a nonsecretory protein and may be located in the chloroplast of D. salina. Phylogenetic analysis demonstrated that the D. salina G3PDH had a closer relationship with the G3PDHs from the Dunaliella genus than with those from other species. In addition, the cDNA was subsequently subcloned in the pET-32a(+) vector and was transformed into E. coli strain BL21 (DE3), a expression protein with 100 kDa was identified, which was consistent with the theoretical value. PMID:23626797

  10. Plastidial Glyceraldehyde-3-Phosphate Dehydrogenase Deficiency Leads to Altered Root Development and Affects the Sugar and Amino Acid Balance in Arabidopsis1[W

    PubMed Central

    Muñoz-Bertomeu, Jesús; Cascales-Miñana, Borja; Mulet, Jose Miguel; Baroja-Fernández, Edurne; Pozueta-Romero, Javier; Kuhn, Josef M.; Segura, Juan; Ros, Roc

    2009-01-01

    Glycolysis is a central metabolic pathway that, in plants, occurs in both the cytosol and the plastids. The glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the conversion of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate with concomitant reduction of NAD+ to NADH. Both cytosolic (GAPCs) and plastidial (GAPCps) GAPDH activities have been described. However, the in vivo functions of the plastidial isoforms remain unresolved. In this work, we have identified two Arabidopsis (Arabidopsis thaliana) chloroplast/plastid-localized GAPDH isoforms (GAPCp1 and GAPCp2). gapcp double mutants display a drastic phenotype of arrested root development, dwarfism, and sterility. In spite of their low gene expression level as compared with other GAPDHs, GAPCp down-regulation leads to altered gene expression and to drastic changes in the sugar and amino acid balance of the plant. We demonstrate that GAPCps are important for the synthesis of serine in roots. Serine supplementation to the growth medium rescues root developmental arrest and restores normal levels of carbohydrates and sugar biosynthetic activities in gapcp double mutants. We provide evidence that the phosphorylated pathway of Ser biosynthesis plays an important role in supplying serine to roots. Overall, these studies provide insights into the in vivo functions of the GAPCps in plants. Our results emphasize the importance of the plastidial glycolytic pathway, and specifically of GAPCps, in plant primary metabolism. PMID:19675149

  11. Arabidopsis plants deficient in plastidial glyceraldehyde-3-phosphate dehydrogenase show alterations in abscisic acid (ABA) signal transduction: interaction between ABA and primary metabolism

    PubMed Central

    Muñoz-Bertomeu, Jesús; Bermúdez, María Angeles; Segura, Juan; Ros, Roc

    2011-01-01

    Abscisic acid (ABA) controls plant development and regulates plant responses to environmental stresses. A role for ABA in sugar regulation of plant development has also been well documented although the molecular mechanisms connecting the hormone with sugar signal transduction pathways are not well understood. In this work it is shown that Arabidopsis thaliana mutants deficient in plastidial glycolytic glyceraldehyde-3-phosphate dehydrogenase (gapcp1gapcp2) are ABA insensitive in growth, stomatal closure, and germination assays. The ABA levels of gapcp1gapcp2 were normal, suggesting that the ABA signal transduction pathway is impaired in the mutants. ABA modified gapcp1gapcp2 gene expression, but the mutant response to the hormone differed from that observed in wild-type plants. The gene expression of the transcription factor ABI4, involved in both sugar and ABA signalling, was altered in gapcp1gapcp2, suggesting that their ABA insensitivity is mediated, at least partially, through this transcriptional regulator. Serine supplementation was able partly to restore the ABA sensitivity of gapcp1gapcp2, indicating that amino acid homeostasis and/or serine metabolism may also be important determinants in the connections of ABA with primary metabolism. Overall, these studies provide new insights into the links between plant primary metabolism and ABA signalling, and demonstrate the importance of plastidial glycolytic glyceraldehyde-3-phosphate dehydrogenase in these interactions. PMID:21068209

  12. The specific role of plastidial glycolysis in photosynthetic and heterotrophic cells under scrutiny through the study of glyceraldehyde-3-phosphate dehydrogenase.

    PubMed

    Anoman, Armand Djoro; Flores-Tornero, María; Rosa-Telléz, Sara; Muñoz-Bertomeu, Jesús; Segura, Juan; Ros, Roc

    2016-01-01

    The cellular compartmentalization of metabolic processes is an important feature in plants where the same pathways could be simultaneously active in different compartments. Plant glycolysis occurs in the cytosol and plastids of green and non-green cells in which the requirements of energy and precursors may be completely different. Because of this, the relevance of plastidial glycolysis could be very different depending on the cell type. In the associated study, we investigated the function of plastidial glycolysis in photosynthetic and heterotrophic cells by specifically driving the expression of plastidial glyceraldehyde-3-phosphate dehydrogenase (GAPCp) in a glyceraldehyde-3-phosphate dehydrogenase double mutant background (gapcp1gapcp2). We showed that GAPCp is not functionally significant in photosynthetic cells, while it plays a crucial function in heterotrophic cells. We also showed that (i) GAPCp activity expression in root tips is necessary for primary root growth, (ii) its expression in heterotrophic cells of aerial parts and roots is necessary for plant growth and development, and (iii) GAPCp is an important metabolic connector of carbon and nitrogen metabolism through the phosphorylated pathway of serine biosynthesis (PPSB). We discuss here the role that this pathway could play in the control of plant growth and development. PMID:26953506

  13. Glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaebacterium Pyrococcus woesei: characterization of the enzyme, cloning and sequencing of the gene, and expression in Escherichia coli.

    PubMed Central

    Zwickl, P; Fabry, S; Bogedain, C; Haas, A; Hensel, R

    1990-01-01

    The glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaebacterium Pyrococcus woesei (optimal growth temperature, 100 to 103 degrees C) was purified to homogeneity. This enzyme was strictly phosphate dependent, utilized either NAD+ or NADP+, and was insensitive to pentalenolactone like the enzyme from the methanogenic archaebacterium Methanothermus fervidus. The enzyme exhibited a considerable thermostability, with a 44-min half-life at 100 degrees C. The amino acid sequence of the glyceraldehyde-3-phosphate dehydrogenase from P. woesei was deduced from the nucleotide sequence of the coding gene. Compared with the enzyme homologs from mesophilic archaebacteria (Methanobacterium bryantii, Methanobacterium formicicum) and an extremely thermophilic archaebacterium (Methanothermus fervidus), the primary structure of the P. woesei enzyme exhibited a strikingly high proportion of aromatic amino acid residues and a low proportion of sulfur-containing residues. The coding gene of P. woesei was expressed at a high level in Escherichia coli, thus providing an ideal basis for detailed structural and functional studies of that enzyme. Images PMID:2165475

  14. Overexpression of ACC gene from oleaginous yeast Lipomyces starkeyi enhanced the lipid accumulation in Saccharomyces cerevisiae with increased levels of glycerol 3-phosphate substrates.

    PubMed

    Wang, Jiancai; Xu, Ronghua; Wang, Ruling; Haque, Mohammad Enamul; Liu, Aizhong

    2016-06-01

    The conversion of acetyl-CoA to malonyl-CoA by acetyl-CoA carboxylase (ACC) is the rate-limiting step in fatty acid biosynthesis. In this study, a gene coding for ACC was isolated and characterized from an oleaginous yeast, Lipomyces starkeyi. Real-time quantitative PCR (qPCR) analysis of L. starkeyi acetyl-CoA carboxylase gene (LsACC1) showed that the expression levels were upregulated with the fast accumulation of lipids. The LsACC1 was co-overexpressed with the glycerol 3-phosphate dehydrogenase gene (GPD1), which regulates lipids biosynthesis by supplying another substrates glycerol 3-phosphate for storage lipid assembly, in the non-oleaginous yeast Saccharomyces cerevisiae. Further, the S. cerevisiae acetyl-CoA carboxylase (ScACC1) was transferred with GPD1 and its function was analyzed in comparison with LsACC1. The results showed that overexpressed LsACC1 and GPD1 resulted in a 63% increase in S. cerevisiae. This study gives new data in understanding of the molecular mechanisms underlying the regulation of fatty acids and lipid biosynthesis in yeasts. PMID:26865376

  15. Polyester synthases: natural catalysts for plastics.

    PubMed Central

    Rehm, Bernd H A

    2003-01-01

    Polyhydroxyalkanoates (PHAs) are biopolyesters composed of hydroxy fatty acids, which represent a complex class of storage polyesters. They are synthesized by a wide range of different Gram-positive and Gram-negative bacteria, as well as by some Archaea, and are deposited as insoluble cytoplasmic inclusions. Polyester synthases are the key enzymes of polyester biosynthesis and catalyse the conversion of (R)-hydroxyacyl-CoA thioesters to polyesters with the concomitant release of CoA. These soluble enzymes turn into amphipathic enzymes upon covalent catalysis of polyester-chain formation. A self-assembly process is initiated resulting in the formation of insoluble cytoplasmic inclusions with a phospholipid monolayer and covalently attached polyester synthases at the surface. Surface-attached polyester synthases show a marked increase in enzyme activity. These polyester synthases have only recently been biochemically characterized. An overview of these recent findings is provided. At present, 59 polyester synthase structural genes from 45 different bacteria have been cloned and the nucleotide sequences have been obtained. The multiple alignment of the primary structures of these polyester synthases show an overall identity of 8-96% with only eight strictly conserved amino acid residues. Polyester synthases can been assigned to four classes based on their substrate specificity and subunit composition. The current knowledge on the organization of the polyester synthase genes, and other genes encoding proteins related to PHA metabolism, is compiled. In addition, the primary structures of the 59 PHA synthases are aligned and analysed with respect to highly conserved amino acids, and biochemical features of polyester synthases are described. The proposed catalytic mechanism based on similarities to alpha/beta-hydrolases and mutational analysis is discussed. Different threading algorithms suggest that polyester synthases belong to the alpha/beta-hydrolase superfamily, with

  16. Molecular evolution and sequence divergence of plant chalcone synthase and chalcone synthase-Like genes.

    PubMed

    Han, Yingying; Zhao, Wenwen; Wang, Zhicui; Zhu, Jingying; Liu, Qisong

    2014-06-01

    Plant chalcone synthase (CHS) and CHS-Like (CHSL) proteins are polyketide synthases. In this study, we evaluated the molecular evolution of this gene family using representative types of CHSL genes, including stilbene synthase (STS), 2-pyrone synthase (2-PS), bibenzyl synthase (BBS), acridone synthase (ACS), biphenyl synthase (BIS), benzalacetone synthase, coumaroyl triacetic acid synthase (CTAS), and benzophenone synthase (BPS), along with their CHS homologs from the same species of both angiosperms and gymnosperms. A cDNA-based phylogeny indicated that CHSLs had diverse evolutionary patterns. STS, ACS, and 2-PS clustered with CHSs from the same species (late diverged pattern), while CTAS, BBS, BPS, and BIS were distant from their CHS homologs (early diverged pattern). The amino-acid phylogeny suggested that CHS and CHSL proteins formed clades according to enzyme function. The CHSs and CHSLs from Polygonaceae and Arachis had unique evolutionary histories. Synonymous mutation rates were lower in late diverged CHSLs than in early diverged ones, indicating that gene duplications occurred more recently in late diverged CHSLs than in early diverged ones. Relative rate tests proved that late diverged CHSLs had unequal rates to CHSs from the same species when using fatty acid synthase, which evolved from the common ancestor with the CHS superfamily, as the outgroup, while the early diverged lineages had equal rates. This indicated that late diverged CHSLs experienced more frequent mutation than early diverged CHSLs after gene duplication, allowing obtaining new functions in relatively short period of time. PMID:24849013

  17. Crystal structure of riboflavin synthase

    SciTech Connect

    Liao, D.-I.; Wawrzak, Z.; Calabrese, J.C.; Viitanen, P.V.; Jordan, D.B.

    2010-03-05

    Riboflavin synthase catalyzes the dismutation of two molecules of 6,7-dimethyl-8-(1'-D-ribityl)-lumazine to yield riboflavin and 4-ribitylamino-5-amino-2,6-dihydroxypyrimidine. The homotrimer of 23 kDa subunits has no cofactor requirements for catalysis. The enzyme is nonexistent in humans and is an attractive target for antimicrobial agents of organisms whose pathogenicity depends on their ability to biosynthesize riboflavin. The first three-dimensional structure of the enzyme was determined at 2.0 {angstrom} resolution using the multiwavelength anomalous diffraction (MAD) method on the Escherichia coli protein containing selenomethionine residues. The homotrimer consists of an asymmetric assembly of monomers, each of which comprises two similar {beta} barrels and a C-terminal {alpha} helix. The similar {beta} barrels within the monomer confirm a prediction of pseudo two-fold symmetry that is inferred from the sequence similarity between the two halves of the protein. The {beta} barrels closely resemble folds found in phthalate dioxygenase reductase and other flavoproteins. The three active sites of the trimer are proposed to lie between pairs of monomers in which residues conserved among species reside, including two Asp-His-Ser triads and dyads of Cys-Ser and His-Thr. The proposed active sites are located where FMN (an analog of riboflavin) is modeled from an overlay of the {beta} barrels of phthalate dioxygenase reductase and riboflavin synthase. In the trimer, one active site is formed, and the other two active sites are wide open and exposed to solvent. The nature of the trimer configuration suggests that only one active site can be formed and be catalytically competent at a time.

  18. The 2',4'-dihydroxychalcone could be explored to develop new inhibitors against the glycerol-3-phosphate dehydrogenase from Leishmania species.

    PubMed

    Passalacqua, Thais G; Torres, Fábio A E; Nogueira, Camila T; de Almeida, Leticia; Del Cistia, Mayara L; dos Santos, Mariana B; Regasini, Luis O; Graminha, Márcia A S; Marchetto, Reinaldo; Zottis, Aderson

    2015-09-01

    The enzyme glycerol-3-phosphate dehydrogenase (G3PDH) from Leishmania species is considered as an attractive target to design new antileishmanial drugs and a previous in silico study reported on the importance of chalcones to achieve its inhibition. Here, we report the identification of a synthetic chalcone in our in vitro assays with promastigote cells from Leishmania amazonensis, its biological activity in animal models, and docking followed by molecular dynamics simulation to investigate the molecular interactions and structural patterns that are crucial to achieve the inhibition complex between this compound and G3PDH. A molecular fragment of this natural product derivative can provide new inhibitors with increased potency and selectivity. PMID:26169126

  19. Cloning and characterization of murine 1-acyl-sn-glycerol 3-phosphate acyltransferases and their regulation by PPARalpha in murine heart.

    PubMed

    Lu, Biao; Jiang, Yan J; Zhou, Yaling; Xu, Fred Y; Hatch, Grant M; Choy, Patrick C

    2005-01-15

    AGPAT (1-acyl-sn-glycerol 3-phosphate acyltransferase) exists in at least five isoforms in humans, termed as AGPAT1, AGPAT2, AGPAT3, AGPAT4 and AGPAT5. Although they catalyse the same biochemical reaction, their relative function, tissue expression and regulation are poorly understood. Linkage studies in humans have revealed that AGPAT2 contributes to glycerolipid synthesis and plays an important role in regulating lipid metabolism. We report the molecular cloning, tissue distribution, and enzyme characterization of mAGPATs (murine AGPATs) and regulation of cardiac mAGPATs by PPARalpha (peroxisome-proliferator-activated receptor alpha). mAGPATs demonstrated differential tissue expression profiles: mAGPAT1 and mAGPAT3 were ubiquitously expressed in most tissues, whereas mAGPAT2, mAGPAT4 and mAGPAT5 were expressed in a tissue-specific manner. mAGPAT2 expressed in in vitro transcription and translation reactions and in transfected COS-1 cells exhibited specificity for 1-acyl-sn-glycerol 3-phosphate. When amino acid sequences of five mAGPATs were compared, three highly conserved motifs were identified, including one novel motif/pattern KX2LX6GX12R. Cardiac mAGPAT activities were 25% lower (P<0.05) in PPARalpha null mice compared with wild-type. In addition, cardiac mAGPAT activities were 50% lower (P<0.05) in PPARalpha null mice fed clofibrate compared with clofibrate fed wild-type animals. This modulation of AGPAT activity was accompanied by significant enhancement/reduction of the mRNA levels of mAGPAT3/mAGPAT2 respectively. Finally, mRNA expression of cardiac mAGPAT3 appeared to be regulated by PPARalpha activation. We conclude that cardiac mAGPAT activity may be regulated by both the composition of mAGPAT isoforms and the levels of each isoform. PMID:15367102

  20. Glycerol-3-phosphate acyltranferase-2 behaves as a cancer testis gene and promotes growth and tumorigenicity of the breast cancer MDA-MB-231 cell line.

    PubMed

    Pellon-Maison, Magali; Montanaro, Mauro A; Lacunza, Ezequiel; Garcia-Fabiani, Maria B; Soler-Gerino, Mercedes C; Cattaneo, Elizabeth R; Quiroga, Ivana Y; Abba, Martin C; Coleman, Rosalind A; Gonzalez-Baro, Maria R

    2014-01-01

    The de novo synthesis of glycerolipids in mammalian cells begins with the acylation of glycerol-3-phosphate, catalyzed by glycerol-3-phosphate acyltransferase (GPAT). GPAT2 is a mitochondrial isoform primarily expressed in testis under physiological conditions. Because it is aberrantly expressed in multiple myeloma, it has been proposed as a novel cancer testis gene. Using a bioinformatics approach, we found that GPAT2 is highly expressed in melanoma, lung, prostate and breast cancer, and we validated GPAT2 expression at the protein level in breast cancer by immunohistochemistry. In this case GPAT2 expression correlated with a higher histological grade. 5-Aza-2' deoxycytidine treatment of human cells lines induced GPAT2 expression suggesting epigenetic regulation of gene expression. In order to evaluate the contribution of GPAT2 to the tumor phenotype, we silenced its expression in MDA-MB-231 cells. GPAT2 knockdown diminished cell proliferation, anchorage independent growth, migration and tumorigenicity, and increased staurosporine-induced apoptosis. In contrast, GPAT2 over-expression increased cell proliferation rate and resistance to staurosporine-induced apoptosis. To understand the functional role of GPAT2, we performed a co-expression analysis in mouse and human testis and found a significant association with semantic terms involved in cell cycle, DNA integrity maintenance, piRNA biogenesis and epigenetic regulation. Overall, these results indicate the GPAT2 would be directly associated with the control of cell proliferation. In conclusion, we confirm GPAT2 as a cancer testis gene and that its expression contributes to the tumor phenotype of MDA-MB-231 cells. PMID:24967918

  1. Inactivation of glyceraldehyde-3-phosphate dehydrogenase by a reactive metabolite of acetaminophen and mass spectral characterization of an arylated active site peptide.

    PubMed

    Dietze, E C; Schäfer, A; Omichinski, J G; Nelson, S D

    1997-10-01

    Acetaminophen (4'-hydroxyacetanilide, APAP) is a widely used analgesic and antipyretic drug that can cause hepatic necrosis under some circumstances via cytochrome P450-mediated oxidation to a reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI). Although the mechanism of hepatocellular injury caused by APAP is not fully understood, it is known that NAPQI forms covalent adducts with several hepatocellular proteins. Reported here is the identification of one of these proteins as glyceraldehyde-3-phosphate dehydrogenase [GAPDH, D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12]. Two hours after the administration of hepatotoxic doses of [14C]APAP to mice, at a time prior to overt cell damage, hepatocellular GAPDH activity was significantly decreased concurrent with the formation of a 14C-labeled GAPDH adduct. A nonhepatotoxic regioisomer of APAP, 3'-hydroxyacetanilide (AMAP), was found to decrease GAPDH activity to a lesser extent than APAP, and radiolabel from [14C]AMAP bound to a lesser extent to GAPDH at a time when its overall binding to hepatocellular proteins was almost equivalent to that of APAP. In order to determine the nature of the covalent adduct between GAPDH and APAP, its major reactive and toxic metabolite, NAPQI, was incubated with purified porcine muscle GAPDH. Microsequencing analysis and fast atom bombardment mass spectrometry (FAB-MS) with collision-induced dissociation (CID) were used to characterize one of the adducts as APAP bound to the cysteinyl sulfhydryl group of Cys-149 in the active site peptide of GAPDH. PMID:9348431

  2. The Class II Phosphatidylinositol 3-Phosphate Kinase PIK3C2A Promotes Shigella flexneri Dissemination through Formation of Vacuole-Like Protrusions

    PubMed Central

    Dragoi, Ana-Maria

    2015-01-01

    Intracellular pathogens such as Shigella flexneri and Listeria monocytogenes achieve dissemination in the intestinal epithelium by displaying actin-based motility in the cytosol of infected cells. As they reach the cell periphery, motile bacteria form plasma membrane protrusions that resolve into vacuoles in adjacent cells, through a poorly understood mechanism. Here, we report on the role of the class II phosphatidylinositol 3-phosphate kinase PIK3C2A in S. flexneri dissemination. Time-lapse microscopy revealed that PIK3C2A was required for the resolution of protrusions into vacuoles through the formation of an intermediate membrane-bound compartment that we refer to as a vacuole-like protrusion (VLP). Genetic rescue of PIK3C2A depletion with RNA interference (RNAi)-resistant cDNA constructs demonstrated that VLP formation required the activity of PIK3C2A in primary infected cells. PIK3C2A expression was required for production of phosphatidylinositol 3-phosphate [PtdIns(3)P] at the plasma membrane surrounding protrusions. PtdIns(3)P production was not observed in the protrusions formed by L. monocytogenes, whose dissemination did not rely on PIK3C2A. PIK3C2A-mediated PtdIns(3)P production in S. flexneri protrusions was regulated by host cell tyrosine kinase signaling and relied on the integrity of the S. flexneri type 3 secretion system (T3SS). We suggest a model of S. flexneri dissemination in which the formation of VLPs is mediated by the PIK3C2A-dependent production of the signaling lipid PtdIns(3)P in the protrusion membrane, which relies on the T3SS-dependent activation of tyrosine kinase signaling in protrusions. PMID:25667265

  3. Site-Directed Mutagenesis from Arg195 to His of a Microalgal Putatively Chloroplastidial Glycerol-3-Phosphate Acyltransferase Causes an Increase in Phospholipid Levels in Yeast

    PubMed Central

    Ouyang, Long-Ling; Li, Hui; Yan, Xiao-Jun; Xu, Ji-Lin; Zhou, Zhi-Gang

    2016-01-01

    To analyze the contribution of glycerol-3-phosphate acyltransferase (GPAT) to the first acylation of glycerol-3-phosphate (G-3-P), the present study focused on a functional analysis of the GPAT gene from Lobosphaera incisa (designated as LiGPAT). A full-length cDNA of LiGPAT consisting of a 1,305-bp ORF, a 1,652-bp 5′-UTR, and a 354-bp 3′-UTR, was cloned. The ORF encoded a 434-amino acid peptide, of which 63 residues at the N-terminus defined a chloroplast transit peptide. Multiple sequence alignment and phylogeny analysis of GPAT homologs provided the convincible bioinformatics evidence that LiGPAT was localized to chloroplasts. Considering the conservation of His among the G-3-P binding sites from chloroplastidial GPATs and the substitution of His by Arg at position 195 in the LiGPAT mature protein (designated mLiGPAT), we established the heterologous expression of either mLiGPAT or its mutant (Arg195His) (sdmLiGPAT) in the GPAT-deficient yeast mutant gat1Δ. Lipid profile analyses of these transgenic yeasts not only validated the acylation function of LiGPAT but also indicated that the site-directed mutagenesis from Arg195 to His led to an increase in the phospholipid level in yeast. Semi-quantitative analysis of mLiGPAT and sdmLiGPAT, together with the structural superimposition of their G-3-P binding sites, indicated that the increased enzymatic activity was caused by the enlarged accessible surface of the phosphate group binding pocket when Arg195 was mutated to His. Thus, the potential of genetic manipulation of GPAT to increase the glycerolipid level in L. incisa and other microalgae would be of great interest. PMID:27014309

  4. Topology of 1-Acyl-sn-glycerol-3-phosphate Acyltransferases SLC1 and ALE1 and Related Membrane-bound O-Acyltransferases (MBOATs) of Saccharomyces cerevisiae*

    PubMed Central

    Pagac, Martin; de la Mora, Hector Vazquez; Duperrex, Cécile; Roubaty, Carole; Vionnet, Christine; Conzelmann, Andreas

    2011-01-01

    In yeast, phosphatidic acid, the biosynthetic precursor for all glycerophospholipids and triacylglycerols, is made de novo by the 1-acyl-sn-glycerol-3-phosphate acyltransferases Ale1p and Slc1p. Ale1p belongs to the membrane-bound O-acyltransferase (MBOAT) family, which contains many enzymes acylating lipids but also others that acylate secretory proteins residing in the lumen of the ER. A histidine present in a very short loop between two predicted transmembrane domains is the only residue that is conserved throughout the MBOAT gene family. The yeast MBOAT proteins of known function comprise Ale1p, the ergosterol acyltransferases Are1p and Are2p, and Gup1p, the last of which acylates lysophosphatidylinositol moieties of GPI anchors on ER lumenal GPI proteins. C-terminal topology reporters added to truncated versions of Gup1p yield a topology predicting a lumenal location of its uniquely conserved histidine 447 residue. The same approach shows that Ale1p and Are2p also have the uniquely conserved histidine residing in the ER lumen. Because these data raised the possibility that phosphatidic acid could be made in the lumen of the ER, we further investigated the topology of the second yeast 1-acyl-sn-glycerol-3-phosphate acyltransferase, Slc1p. The location of C-terminal topology reporters, microsomal assays probing the protease sensitivity of inserted tags, and the accessibility of natural or artificially inserted cysteines to membrane-impermeant alkylating agents all indicate that the most conserved motif containing the presumed active site histidine of Slc1p is oriented toward the ER lumen, whereas other conserved motifs are cytosolic. The implications of these findings are discussed. PMID:21849510

  5. Inducible nitric oxide synthase and inflammation.

    PubMed

    Salvemini, D; Marino, M H

    1998-01-01

    Nitric oxide (NO), derived from L-arginine (L-Arg) by the enzyme nitric oxide synthase (NOS), is involved in acute and chronic inflammatory events. In view of the complexity associated with the inflammatory response, the dissection of possible mechanisms by which NO modulates this response will be profitable in designing novel and more efficacious NOS inhibitors. In this review we describe the consequences associated with the induction of inducible nitric oxide synthase (iNOS) and its therapeutic implications. PMID:15991919

  6. Unique animal prenyltransferase with monoterpene synthase activity

    NASA Astrophysics Data System (ADS)

    Gilg, Anna B.; Tittiger, Claus; Blomquist, Gary J.

    2009-06-01

    Monoterpenes are structurally diverse natural compounds that play an essential role in the chemical ecology of a wide array of organisms. A key enzyme in monoterpene biosynthesis is geranyl diphosphate synthase (GPPS). GPPS is an isoprenyl diphosphate synthase that catalyzes a single electrophilic condensation reaction between dimethylallyl diphosphate (C5) and isopentenyl diphosphate (C5) to produce geranyl diphosphate (GDP; C10). GDP is the universal precursor to all monoterpenes. Subsequently, monoterpene synthases are responsible for the transformation of GDP to a variety of acyclic, monocyclic, and bicyclic monoterpene products. In pheromone-producing male Ips pini bark beetles (Coleoptera: Scolytidae), the acyclic monoterpene myrcene is required for the production of the major aggregation pheromone component, ipsdienol. Here, we report monoterpene synthase activity associated with GPPS of I. pini. Enzyme assays were performed on recombinant GPPS to determine the presence of monoterpene synthase activity, and the reaction products were analyzed by coupled gas chromatography-mass spectrometry. The functionally expressed recombinant enzyme produced both GDP and myrcene, making GPPS of I. pini a bifunctional enzyme. This unique insect isoprenyl diphosphate synthase possesses the functional plasticity that is characteristic of terpene biosynthetic enzymes of plants, contributing toward the current understanding of product specificity of the isoprenoid pathway.

  7. Collaborative trial validation studies of real-time PCR-based GMO screening methods for detection of the bar gene and the ctp2-cp4epsps construct.

    PubMed

    Grohmann, Lutz; Brünen-Nieweler, Claudia; Nemeth, Anne; Waiblinger, Hans-Ulrich

    2009-10-14

    Polymerase Chain Reaction (PCR)-based screening methods targeting genetic elements commonly used in genetically modified (GM) plants are important tools for the detection of GM materials in food, feed, and seed samples. To expand and harmonize the screening capability of enforcement laboratories, the German Federal Office of Consumer Protection and Food Safety conducted collaborative trials for interlaboratory validation of real-time PCR methods for detection of the phosphinothricin acetyltransferase (bar) gene from Streptomyces hygroscopicus and a construct containing the 5-enolpyruvylshikimate-3-phosphate synthase gene from Agrobacterium tumefaciens sp. strain CP4 (ctp2-cp4epsps), respectively. To assess the limit of detection, precision, and accuracy of the methods, laboratories had to analyze two sets of 18 coded genomic DNA samples of events LLRice62 and MS8 with the bar method and NK603 and GT73 with the ctp2-cp4epsps method at analyte levels of 0, 0.02, and 0.1% GM content, respectively. In addition, standard DNAs were provided to the laboratories to generate calibration curves for copy number quantification of the bar and ctp2-cp4epsps target sequences present in the test samples. The study design and the results obtained are discussed with respect to the difficult issue of developing general guidelines and concepts for the collaborative trial validation of qualitative PCR screening methods. PMID:19807158

  8. A red and far-red light receptor mutation confers resistance to the herbicide glyphosate.

    PubMed

    Sharkhuu, Altanbadralt; Narasimhan, Meena L; Merzaban, Jasmeen S; Bressan, Ray A; Weller, Steve; Gehring, Chris

    2014-06-01

    Glyphosate is a widely applied broad-spectrum systemic herbicide that inhibits competitively the penultimate enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) from the shikimate pathway, thereby causing deleterious effects. A glyphosate-resistant Arabidopsis mutant (gre1) was isolated and genetic analyses indicated that a dysfunctional red (R) and far-red (FR) light receptor, phytochrome B (phyB), caused this phenotype. This finding is consistent with increased glyphosate sensitivity and glyphosate-induced shikimate accumulation in low R:FR light, and the induction of genes encoding enzymes of the shikimate pathway in high R:FR light. Expression of the shikimate pathway genes exhibited diurnal oscillation and this oscillation was altered in the phyB mutant. Furthermore, transcript analysis suggested that this diurnal oscillation was not only dependent on phyB but was also due to circadian regulatory mechanisms. Our data offer an explanation of the well documented observation that glyphosate treatment at various times throughout the day, with their specific composition of light quality and intensity, results in different efficiencies of the herbicide. PMID:24654847

  9. A red and far-red light receptor mutation confers resistance to the herbicide glyphosate

    PubMed Central

    Sharkhuu, Altanbadralt; Narasimhan, Meena L; Merzaban, Jasmeen S; Bressan, Ray A; Weller, Steve; Gehring, Chris

    2014-01-01

    Glyphosate is a widely applied broad-spectrum systemic herbicide that inhibits competitively the penultimate enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) from the shikimate pathway, thereby causing deleterious effects. A glyphosate-resistant Arabidopsis mutant (gre1) was isolated and genetic analyses indicated that a dysfunctional red (R) and far-red (FR) light receptor, phytochrome B (phyB), caused this phenotype. This finding is consistent with increased glyphosate sensitivity and glyphosate-induced shikimate accumulation in low R:FR light, and the induction of genes encoding enzymes of the shikimate pathway in high R:FR light. Expression of the shikimate pathway genes exhibited diurnal oscillation and this oscillation was altered in the phyB mutant. Furthermore, transcript analysis suggested that this diurnal oscillation was not only dependent on phyB but was also due to circadian regulatory mechanisms. Our data offer an explanation of the well documented observation that glyphosate treatment at various times throughout the day, with their specific composition of light quality and intensity, results in different efficiencies of the herbicide. PMID:24654847

  10. Population genetics structure of glyphosate-resistant Johnsongrass (Sorghum halepense L. Pers) does not support a single origin of the resistance

    PubMed Central

    Fernández, Luis; de Haro, Luis Alejandro; Distefano, Ana J; Carolina Martínez, Maria; Lía, Verónica; Papa, Juan C; Olea, Ignacio; Tosto, Daniela; Esteban Hopp, Horacio

    2013-01-01

    Single sequence repeats (SSR) developed for Sorghum bicolor were used to characterize the genetic distance of 46 different Sorghum halepense (Johnsongrass) accessions from Argentina some of which have evolved toward glyphosate resistance. Since Johnsongrass is an allotetraploid and only one subgenome is homologous to cultivated sorghum, some SSR loci amplified up to two alleles while others (presumably more conserved loci) amplified up to four alleles. Twelve SSR providing information of 24 loci representative of Johnsongrass genome were selected for genetic distance characterization. All of them were highly polymorphic, which was evidenced by the number of different alleles found in the samples studied, in some of them up to 20. UPGMA and Mantel analysis showed that Johnsongrass glyphosate-resistant accessions that belong to different geographic regions do not share similar genetic backgrounds. In contrast, they show closer similarity to their neighboring susceptible counterparts. Discriminant Analysis of Principal Components using the clusters identified by K-means support the lack of a clear pattern of association among samples and resistance status or province of origin. Consequently, these results do not support a single genetic origin of glyphosate resistance. Nucleotide sequencing of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoding gene from glyphosate-resistant and susceptible accessions collected from different geographic origins showed that none presented expected mutations in aminoacid positions 101 and 106 which are diagnostic of target-site resistance mechanism. PMID:24223277

  11. Involvement of facultative apomixis in inheritance of EPSPS gene amplification in glyphosate-resistant Amaranthus palmeri.

    PubMed

    Ribeiro, Daniela N; Pan, Zhiqiang; Duke, Stephen O; Nandula, Vijay K; Baldwin, Brian S; Shaw, David R; Dayan, Franck E

    2014-01-01

    The inheritance of glyphosate resistance in two Amaranthus palmeri populations (R1 and R2) was examined in reciprocal crosses (RC) and second reciprocal crosses (2RC) between glyphosate-resistant (R) and -susceptible (S) parents of this dioecious species. R populations and Female-R × Male-S crosses contain higher 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene copy numbers than the S population. EPSPS expression, EPSPS enzyme activity, EPSPS protein quantity, and level of resistance to glyphosate correlated positively with genomic EPSPS relative copy number. Transfer of resistance was more influenced by the female than the male parent in spite of the fact that the multiple copies of EPSPS are amplified in the nuclear genome. This led us to hypothesize that this perplexing pattern of inheritance may result from apomictic seed production in A. palmeri. We confirmed that reproductively isolated R and S female plants produced seeds, indicating that A. palmeri can produce seeds both sexually and apomictically (facultative apomixis). This apomictic trait accounts for the low copy number inheritance in the Female-S × Male-R offsprings. Apomixis may also enhance the stability of the glyphosate resistance trait in the R populations in the absence of reproductive partners. PMID:24142112

  12. A built-in strategy for containment of transgenic plants: creation of selectively terminable transgenic rice.

    PubMed

    Lin, Chaoyang; Fang, Jun; Xu, Xiaoli; Zhao, Te; Cheng, Jiaan; Tu, Juming; Ye, Gongyin; Shen, Zhicheng

    2008-01-01

    Plant transgenic technology has been widely utilized for engineering crops for trait improvements and for production of high value proteins such as pharmaceuticals. However, the unintended spreading of commercial transgenic crops by pollination and seed dispersal is a major concern for environmental and food safety. Simple and reliable containment strategies for transgenes are highly desirable. Here we report a novel method for creating selectively terminable transgenic rice. In this method, the gene(s) of interest is tagged with a RNA interference cassette, which specifically suppresses the expression of the bentazon detoxification enzyme CYP81A6 and thus renders transgenic rice to be sensitive to bentazon, a herbicide used for rice weed control. We generated transgenic rice plants by this method using a new glyphosate resistant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene from Pesudomonas putida as the gene of interest, and demonstrated that these transgenic rice plants were highly sensitive to bentazon but tolerant to glyphosate, which is exactly the opposite of conventional rice. Field trial of these transgenic rice plants further confirmed that they can be selectively killed at 100% by one spray of bentazon at a regular dose used for conventional rice weed control. Furthermore, we found that the terminable transgenic rice created in this study shows no difference in growth, development and yield compared to its non-transgenic control. Therefore, this method of creating transgenic rice constitutes a novel strategy of transgene containment, which appears simple, reliable and inexpensive for implementation. PMID:18350155

  13. A Built-In Strategy for Containment of Transgenic Plants: Creation of Selectively Terminable Transgenic Rice

    PubMed Central

    Zhao, Te; Cheng, Jiaan; Tu, Juming; Ye, Gongyin; Shen, Zhicheng

    2008-01-01

    Plant transgenic technology has been widely utilized for engineering crops for trait improvements and for production of high value proteins such as pharmaceuticals. However, the unintended spreading of commercial transgenic crops by pollination and seed dispersal is a major concern for environmental and food safety. Simple and reliable containment strategies for transgenes are highly desirable. Here we report a novel method for creating selectively terminable transgenic rice. In this method, the gene(s) of interest is tagged with a RNA interference cassette, which specifically suppresses the expression of the bentazon detoxification enzyme CYP81A6 and thus renders transgenic rice to be sensitive to bentazon, a herbicide used for rice weed control. We generated transgenic rice plants by this method using a new glyphosate resistant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene from Pesudomonas putida as the gene of interest, and demonstrated that these transgenic rice plants were highly sensitive to bentazon but tolerant to glyphosate, which is exactly the opposite of conventional rice. Field trial of these transgenic rice plants further confirmed that they can be selectively killed at 100% by one spray of bentazon at a regular dose used for conventional rice weed control. Furthermore, we found that the terminable transgenic rice created in this study shows no difference in growth, development and yield compared to its non-transgenic control. Therefore, this method of creating transgenic rice constitutes a novel strategy of transgene containment, which appears simple, reliable and inexpensive for implementation. PMID:18350155

  14. Quantification of genetically modified soya using strong anion exchange chromatography and time-of-flight mass spectrometry.

    PubMed

    Chang, Po-Chih; Reddy, P Muralidhar; Ho, Yen-Peng

    2014-09-01

    Stable-isotope dimethyl labeling was applied to the quantification of genetically modified (GM) soya. The herbicide-resistant gene-related protein 5-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) was labeled using a dimethyl labeling reagent, formaldehyde-H2 or -D2. The identification and quantification of CP4 EPSPS was performed using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The CP4 EPSPS protein was separated from high abundance proteins using strong anion exchange chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Then, the tryptic peptides from the samples and reference were labeled with formaldehyde-H2 and formaldehyde-D2, respectively. The two labeled pools were mixed and analyzed using MALDI-MS. The data showed a good correlation between the peak ratio of the H- and D-labeled peptides and the GM soya percentages at 0.5, 1, 3, and 5 %, with R (2) of 0.99. The labeling reagents are readily available. The labeling experiments and the detection procedures are simple. The approach is useful for the quantification of GM soya at a level as low as 0.5 %. PMID:24969465

  15. Gene amplification confers glyphosate resistance in Amaranthus palmeri

    PubMed Central

    Gaines, Todd A.; Zhang, Wenli; Wang, Dafu; Bukun, Bekir; Chisholm, Stephen T.; Shaner, Dale L.; Nissen, Scott J.; Patzoldt, William L.; Tranel, Patrick J.; Culpepper, A. Stanley; Grey, Timothy L.; Webster, Theodore M.; Vencill, William K.; Sammons, R. Douglas; Jiang, Jiming; Preston, Christopher; Leach, Jan E.; Westra, Philip

    2009-01-01

    The herbicide glyphosate became widely used in the United States and other parts of the world after the commercialization of glyphosate-resistant crops. These crops have constitutive overexpression of a glyphosate-insensitive form of the herbicide target site gene, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Increased use of glyphosate over multiple years imposes selective genetic pressure on weed populations. We investigated recently discovered glyphosate-resistant Amaranthus palmeri populations from Georgia, in comparison with normally sensitive populations. EPSPS enzyme activity from resistant and susceptible plants was equally inhibited by glyphosate, which led us to use quantitative PCR to measure relative copy numbers of the EPSPS gene. Genomes of resistant plants contained from 5-fold to more than 160-fold more copies of the EPSPS gene than did genomes of susceptible plants. Quantitative RT-PCR on cDNA revealed that EPSPS expression was positively correlated with genomic EPSPS relative copy number. Immunoblot analyses showed that increased EPSPS protein level also correlated with EPSPS genomic copy number. EPSPS gene amplification was heritable, correlated with resistance in pseudo-F2 populations, and is proposed to be the molecular basis of glyphosate resistance. FISH revealed that EPSPS genes were present on every chromosome and, therefore, gene amplification was likely not caused by unequal chromosome crossing over. This occurrence of gene amplification as an herbicide resistance mechanism in a naturally occurring weed population is particularly significant because it could threaten the sustainable use of glyphosate-resistant crop technology. PMID:20018685

  16. Oligonucleotide-Mediated Genome Editing Provides Precision and Function to Engineered Nucleases and Antibiotics in Plants.

    PubMed

    Sauer, Noel J; Narváez-Vásquez, Javier; Mozoruk, Jerry; Miller, Ryan B; Warburg, Zachary J; Woodward, Melody J; Mihiret, Yohannes A; Lincoln, Tracey A; Segami, Rosa E; Sanders, Steven L; Walker, Keith A; Beetham, Peter R; Schöpke, Christian R; Gocal, Greg F W

    2016-04-01

    Here, we report a form of oligonucleotide-directed mutagenesis for precision genome editing in plants that uses single-stranded oligonucleotides (ssODNs) to precisely and efficiently generate genome edits at DNA strand lesions made by DNA double strand break reagents. Employing a transgene model in Arabidopsis (Arabidopsis thaliana), we obtained a high frequency of precise targeted genome edits when ssODNs were introduced into protoplasts that were pretreated with the glycopeptide antibiotic phleomycin, a nonspecific DNA double strand breaker. Simultaneous delivery of ssODN and a site-specific DNA double strand breaker, either transcription activator-like effector nucleases (TALENs) or clustered, regularly interspaced, short palindromic repeats (CRISPR/Cas9), resulted in a much greater targeted genome-editing frequency compared with treatment with DNA double strand-breaking reagents alone. Using this site-specific approach, we applied the combination of ssODN and CRISPR/Cas9 to develop an herbicide tolerance trait in flax (Linum usitatissimum) by precisely editing the 5'-ENOLPYRUVYLSHIKIMATE-3-PHOSPHATE SYNTHASE (EPSPS) genes. EPSPS edits occurred at sufficient frequency that we could regenerate whole plants from edited protoplasts without employing selection. These plants were subsequently determined to be tolerant to the herbicide glyphosate in greenhouse spray tests. Progeny (C1) of these plants showed the expected Mendelian segregation of EPSPS edits. Our findings show the enormous potential of using a genome-editing platform for precise, reliable trait development in crop plants. PMID:26864017

  17. A Built-In Strategy to Mitigate Transgene Spreading from Genetically Modified Corn

    PubMed Central

    Li, Jing; Yu, Hui; Zhang, Fengzhen; Lin, Chaoyang; Gao, Jianhua; Fang, Jun; Ding, Xiahui; Shen, Zhicheng; Xu, Xiaoli

    2013-01-01

    Transgene spreading is a major concern in cultivating genetically modified (GM) corn. Cross-pollination may cause the spread of transgenes from GM cornfields to conventional fields. Occasionally, seed lot contamination, volunteers, mixing during sowing, harvest, and trade can also lead to transgene escape. Obviously, new biological confinement technologies are highly desired to mitigate transgene spreading in addition to physical separation and isolation methods. In this study, we report the development of a built-in containment method to mitigate transgene spreading in corn. In this method, an RNAi cassette for suppressing the expression of the nicosulfuron detoxifying enzyme CYP81A9 and an expression cassette for the glyphosate tolerant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene G10 were constructed and transformed into corn via Agrobacterium-mediated transformation. The GM corn plants that were generated were found to be sensitive to nicosulfuron but resistant to glyphosate, which is exactly the opposite of conventional corn. Field tests demonstrated that GM corn plants with silenced CYP81A9 could be killed by applying nicosulfuron at 40 g/ha, which is the recommended dose for weed control in cornfields. This study suggests that this built-in containment method for controlling the spread of corn transgenes is effective and easy to implement. PMID:24324711

  18. Piezoelectric Sensor for Determination of Genetically Modified Soybean Roundup Ready® in Samples not Amplified by PCR

    PubMed Central

    Stobiecka, Magdalena; Cieśla, Jarosław M.; Janowska, Beata; Tudek, Barbara; Radecka, Hanna

    2007-01-01

    The chemically modified piezoelectrodes were utilized to develop relatively cheap and easy to use biosensor for determination of genetically modified Roundup Ready soybean (RR soybean). The biosensor relies on the immobilization onto gold piezoelectrodes of the 21-mer single stranded oligonucleotide (probes) related to 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene, which is an active component of an insert integrated into RR soybean genome. The hybridization reaction between the probe and the target complementary sequence in solution was monitored. The system was optimized using synthetic oligonucleotides, which were applied for EPSPS gene detection in DNA samples extracted from animal feed containing 30% RR soybean amplified by the PCR and nonamplified by PCR. The detection limit for genomic DNA was in the range of 4.7·105 numbers of genom copies contained EPSPS gene in the QCM cell. The properties such as sensitivity and selectivity of piezoelectric senor presented here indicated that it could be applied for the direct determination of genetically modified RR soybean in the samples non-amplified by PCR.

  19. Metabonomic strategy for the investigation of the mode of action of the phytotoxin (5S,8R,13S,16R)-(-)-pyrenophorol using 1H nuclear magnetic resonance fingerprinting.

    PubMed

    Aliferis, Konstantinos A; Chrysayi-Tokousbalides, Maria

    2006-03-01

    The biochemical mode of action of (5S,8R,13S,16R)-(-)-pyrenophorol isolated from a Drechslera avenae pathotype was investigated by using metabolic fingerprinting. (1)H NMR spectra of crude leaf extracts from untreated Avena sterilis seedlings and A. sterilis seedlings treated with pyrenophorol were compared with those obtained from treatments with the herbicides diuron, glyphosate, mesotrione, norflurazon, oxadiazon, and paraquat. Multivariate analysis was carried out to group treatments according to the mode of action of the phytotoxic substances applied. Analysis results revealed that none of the herbicide treatments fitted the pyrenophorol model and indicate that the effect of the phytotoxin on A. sterilis differs than those caused by glyphosate, mesotrione, norflurazon, oxadiazon, paraquat, and diuron, which inhibit 5-enolpyruvylshikimate-3-phosphate synthase, 4-hydroxyphenyl-pyruvate-dioxygenase, phytoene desaturase, protoporphyrinogen oxidase, photosystem I, and photosystem II, respectively. The method applied, combined with appropriate data preprocessing and analysis, was found to be rapid for the screening of phytotoxic substances for metabolic effects. PMID:16506820

  20. Compositional equivalence of DAS-444Ø6-6 (AAD-12 + 2mEPSPS + PAT) herbicide-tolerant soybean and nontransgenic soybean.

    PubMed

    Lepping, Miles D; Herman, Rod A; Potts, Brian L

    2013-11-20

    Soybeans from transgenic event DAS-444Ø6-6 are the first to express three proteins that provide tolerance to broad-spectrum herbicides. DAS-444Ø6-6 soybean expresses the aryloxyalkanoate dioxygenase-12 (AAD-12) enzyme from the soil bacterium Delftia acidovorans , which provides tolerance to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D); the double-mutant 5-enolpyruvylshikimate-3-phosphate synthase (2mEPSPS) enzyme encoded by a modified version of the epsps gene from maize ( Zea mays ), which provides tolerance to the herbicide glyphosate; and the phosphinothricin acetyltransferase (PAT) enzyme from Streptomyces viridochromogenes , which provides tolerance to the herbicide glufosinate. The purpose of this study was to determine if the nutrient and antinutrient composition of forage and grain from DAS-444Ø6-6 soybean are similar to those of nontransgenic soybean. Forage was analyzed for proximates, fiber, and minerals; grain analyses further included vitamins, amino acid and fatty acid profiles, and antinutrients and bioactive components (lectin, phytic acid, raffinose, stachyose, trypsin inhibitor, and isoflavones). Results indicate that DAS-444Ø6-6 soybean is compositionally equivalent to nontransgenic soybean. Findings are consistent with similar studies for other input traits, as endogenous plant metabolic pathways that influence composition are expected to be less affected by transgenesis compared with traditional plant-breeding methods. PMID:24191699

  1. Strategies to improve low copy transgenic events in Agrobacterium-mediated transformation of maize.

    PubMed

    Sivamani, Elumalai; Li, Xianggan; Nalapalli, Samson; Barron, Yoshimi; Prairie, Anna; Bradley, David; Doyle, Michele; Que, Qiudeng

    2015-12-01

    Transgenic plants containing low copy transgene insertion free of vector backbone are highly desired for many biotechnological applications. We have investigated two different strategies for increasing the percentage of low copy events in Agrobacterium-mediated transformation experiments in maize. One of the strategies is to use a binary vector with two separate T-DNAs, one T-DNA containing an intact E.coli manA gene encoding phosphomannose isomerase (PMI) as selectable marker gene cassette and another T-DNA containing an RNAi cassette of PMI sequences. By using this strategy, low copy transgenic events containing the transgenes were increased from 43 to 60 % in maize. An alternate strategy is using selectable marker gene cassettes containing regulatory or coding sequences derived from essential plant genes such as 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) or MADS box transcription factor. In this paper we demonstrate that higher percentage of low copy transgenic events can be obtained in Agrobacterium-mediated maize transformation experiments using both strategies. We propose that the above two strategies can be used independently or in combination to increase transgenic events that contain low copy transgene insertion in Agrobacterium-mediated transformation experiments. PMID:26338266

  2. EPSPS variability, gene expression, and enzymatic activity in glyphosate-resistant biotypes of Digitaria insularis.

    PubMed

    Galeano, E; Barroso, A A M; Vasconcelos, T S; López-Rubio, A; Albrecht, A J P; Victoria Filho, R; Carrer, H

    2016-01-01

    Weed resistance to herbicides is a natural phenomenon that exerts selection on individuals in a population. In Brazil, glyphosate resistance was recently detected in Digitaria insularis. The objective of this study was to elucidate mechanisms of weed resistance in this plant, including genetic variability, allelism, amino acid substitutions, gene expression, and enzymatic activity levels. Most of these have not previously been studied in this species. D. insularis DNA sequences were used to analyze genetic variability. cDNA from resistant and susceptible plants was used to identify mutations, alleles, and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) expression, using real-time quantitative reverse transcription-polymerase chain reaction. In addition, EPSPS activity was measured. We found a decrease in genetic variability between populations related to glyphosate application. Substitutions from proline to threonine and tyrosine to cysteine led to a decrease in EPSPS affinity for the glyphosate. In addition, the EPSPS enzymatic activity was slightly higher in resistant plants, whereas EPSPS gene expression was almost identical in both biotypes, suggesting feedback regulation at different levels. To conclude, our results suggest new molecular mechanisms used by D. insularis to increase glyphosate resistance. PMID:27525929

  3. Gene replacements and insertions in rice by intron targeting using CRISPR-Cas9.

    PubMed

    Li, Jun; Meng, Xiangbing; Zong, Yuan; Chen, Kunling; Zhang, Huawei; Liu, Jinxing; Li, Jiayang; Gao, Caixia

    2016-01-01

    Sequence-specific nucleases have been exploited to create targeted gene knockouts in various plants(1), but replacing a fragment and even obtaining gene insertions at specific loci in plant genomes remain a serious challenge. Here, we report efficient intron-mediated site-specific gene replacement and insertion approaches that generate mutations using the non-homologous end joining (NHEJ) pathway using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system. Using a pair of single guide RNAs (sgRNAs) targeting adjacent introns and a donor DNA template including the same pair of sgRNA sites, we achieved gene replacements in the rice endogenous gene 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) at a frequency of 2.0%. We also obtained targeted gene insertions at a frequency of 2.2% using a sgRNA targeting one intron and a donor DNA template including the same sgRNA site. Rice plants harbouring the OsEPSPS gene with the intended substitutions were glyphosate-resistant. Furthermore, the site-specific gene replacements and insertions were faithfully transmitted to the next generation. These newly developed approaches can be generally used to replace targeted gene fragments and to insert exogenous DNA sequences into specific genomic sites in rice and other plants. PMID:27618611

  4. Ceramide synthases in biomedical research.

    PubMed

    Cingolani, Francesca; Futerman, Anthony H; Casas, Josefina

    2016-05-01

    Sphingolipid metabolism consists of multiple metabolic pathways that converge upon ceramide, one of the key molecules among sphingolipids (SLs). In mammals, ceramide synthesis occurs via N-acylation of sphingoid backbones, dihydrosphingosine (dhSo) or sphingosine (So). The reaction is catalyzed by ceramide synthases (CerS), a family of enzymes with six different isoforms, with each one showing specificity towards a restricted group of acyl-CoAs, thus producing ceramides (Cer) and dihydroceramides (dhCer) with different fatty acid chain lengths. A large body of evidence documents the role of both So and dhSo as bioactive molecules, as well as the involvement of dhCer and Cer in physiological and pathological processes. In particular, the fatty acid composition of Cer has different effects in cell biology and in the onset and progression of different diseases. Therefore, modulation of CerS activity represents an attractive target in biomedical research and in finding new treatment modalities. In this review, we discuss functional, structural and biochemical features of CerS and examine CerS inhibitors that are currently available. PMID:26248326

  5. MicroRNA-24 can control triacylglycerol synthesis in goat mammary epithelial cells by targeting the fatty acid synthase gene.

    PubMed

    Wang, H; Luo, J; Chen, Z; Cao, W T; Xu, H F; Gou, D M; Zhu, J J

    2015-12-01

    In nonruminants it has been demonstrated that microRNA-24 (miR-24) is involved in preadipocyte differentiation, hepatic lipid, and plasma triacylglycerol synthesis. However, its role in ruminant mammary gland remains unclear. In this study we measured miR-24 expression in goat mammary gland tissue at 4 different stages of lactation and observed that it had highest expression at peak lactation when compared with the dry period. Overexpression or downregulation of miR-24 in goat mammary epithelial cells (GMEC) strongly affected fatty acid profiles; in particular, miR-24 enhanced unsaturated fatty acid concentration. Additional effects of miR-24 included changes in triacylglycerol content and the expression of fatty acid synthase, sterol regulatory element binding transcription protein 1, stearoyl-CoA desaturase, glycerol-3-phosphate acyltransferase mitochondrial, and acetyl-CoA carboxylase. Luciferase reporter assay confirmed that fatty acid synthase is a target of miR-24. Taken together, these results not only highlight the physiological importance of miR-24 in fatty acid metabolism in GMEC, but also laid the foundation for further research on regulatory mechanisms among miR-24 and other microRNA expressed in GMEC. PMID:26476938

  6. Endothelial nitric oxide synthase (NOS) deficiency affects energy metabolism pattern in murine oxidative skeletal muscle.

    PubMed Central

    Momken, Iman; Fortin, Dominique; Serrurier, Bernard; Bigard, Xavier; Ventura-Clapier, Renée; Veksler, Vladimir

    2002-01-01

    Oxidative capacity of muscles correlates with capillary density and with microcirculation, which in turn depend on various regulatory factors, including NO generated by endothelial nitric oxide synthase (eNOS). To determine the role of eNOS in patterns of regulation of energy metabolism in various muscles, we studied mitochondrial respiration in situ in saponin-permeabilized fibres as well as the energy metabolism enzyme profile in the cardiac, soleus (oxidative) and gastrocnemius (glycolytic) muscles isolated from mice lacking eNOS (eNOS(-/-)). In soleus muscle, the absence of eNOS induced a marked decrease in both basal mitochondrial respiration without ADP (-32%; P <0.05) and maximal respiration in the presence of ADP (-29%; P <0.05). Furthermore, the eNOS(-/-) soleus muscle showed a decrease in total creatine kinase (-29%; P <0.05), citrate synthase (-31%; P <0.01), adenylate kinase (-27%; P <0.05), glyceraldehyde-3-phosphate dehydrogenase (-43%; P <0.01) and pyruvate kinase (-26%; P <0.05) activities. The percentage of myosin heavy chains I (slow isoform) was significantly increased from 24.3+/-1.5% in control to 30.1+/-1.1% in eNOS(-/-) soleus muscle ( P <0.05) at the expense of a slight non-significant decrease in the three other (fast) isoforms. Besides, eNOS(-/-) soleus showed a 28% loss of weight. Interestingly, we did not find differences in any parameters in cardiac and gastrocnemius muscles compared with respective controls. These results show that eNOS knockout has an important effect on muscle oxidative capacity as well on the activities of energy metabolism enzymes in oxidative (soleus) muscle. The absence of such effects in cardiac and glycolytic (gastrocnemius) muscle suggests a specific role for eNOS-produced NO in oxidative skeletal muscle. PMID:12123418

  7. The tomato terpene synthase gene family.

    PubMed

    Falara, Vasiliki; Akhtar, Tariq A; Nguyen, Thuong T H; Spyropoulou, Eleni A; Bleeker, Petra M; Schauvinhold, Ines; Matsuba, Yuki; Bonini, Megan E; Schilmiller, Anthony L; Last, Robert L; Schuurink, Robert C; Pichersky, Eran

    2011-10-01

    Compounds of the terpenoid class play numerous roles in the interactions of plants with their environment, such as attracting pollinators and defending the plant against pests. We show here that the genome of cultivated tomato (Solanum lycopersicum) contains 44 terpene synthase (TPS) genes, including 29 that are functional or potentially functional. Of these 29 TPS genes, 26 were expressed in at least some organs or tissues of the plant. The enzymatic functions of eight of the TPS proteins were previously reported, and here we report the specific in vitro catalytic activity of 10 additional tomato terpene synthases. Many of the tomato TPS genes are found in clusters, notably on chromosomes 1, 2, 6, 8, and 10. All TPS family clades previously identified in angiosperms are also present in tomato. The largest clade of functional TPS genes found in tomato, with 12 members, is the TPS-a clade, and it appears to encode only sesquiterpene synthases, one of which is localized to the mitochondria, while the rest are likely cytosolic. A few additional sesquiterpene synthases are encoded by TPS-b clade genes. Some of the tomato sesquiterpene synthases use z,z-farnesyl diphosphate in vitro as well, or more efficiently than, the e,e-farnesyl diphosphate substrate. Genes encoding monoterpene synthases are also prevalent, and they fall into three clades: TPS-b, TPS-g, and TPS-e/f. With the exception of two enzymes involved in the synthesis of ent-kaurene, the precursor of gibberellins, no other tomato TPS genes could be demonstrated to encode diterpene synthases so far. PMID:21813655

  8. Terpene synthases are widely distributed in bacteria

    PubMed Central

    Yamada, Yuuki; Kuzuyama, Tomohisa; Komatsu, Mamoru; Shin-ya, Kazuo; Omura, Satoshi; Cane, David E.; Ikeda, Haruo

    2015-01-01

    Odoriferous terpene metabolites of bacterial origin have been known for many years. In genome-sequenced Streptomycetaceae microorganisms, the vast majority produces the degraded sesquiterpene alcohol geosmin. Two minor groups of bacteria do not produce geosmin, with one of these groups instead producing other sesquiterpene alcohols, whereas members of the remaining group do not produce any detectable terpenoid metabolites. Because bacterial terpene synthases typically show no significant overall sequence similarity to any other known fungal or plant terpene synthases and usually exhibit relatively low levels of mutual sequence similarity with other bacterial synthases, simple correlation of protein sequence data with the structure of the cyclized terpene product has been precluded. We have previously described a powerful search method based on the use of hidden Markov models (HMMs) and protein families database (Pfam) search that has allowed the discovery of monoterpene synthases of bacterial origin. Using an enhanced set of HMM parameters generated using a training set of 140 previously identified bacterial terpene synthase sequences, a Pfam search of 8,759,463 predicted bacterial proteins from public databases and in-house draft genome data has now revealed 262 presumptive terpene synthases. The biochemical function of a considerable number of these presumptive terpene synthase genes could be determined by expression in a specially engineered heterologous Streptomyces host and spectroscopic identification of the resulting terpene products. In addition to a wide variety of terpenes that had been previously reported from fungal or plant sources, we have isolated and determined the complete structures of 13 previously unidentified cyclic sesquiterpenes and diterpenes. PMID:25535391

  9. Properties of phosphorylated thymidylate synthase.

    PubMed

    Frączyk, Tomasz; Ruman, Tomasz; Wilk, Piotr; Palmowski, Paweł; Rogowska-Wrzesinska, Adelina; Cieśla, Joanna; Zieliński, Zbigniew; Nizioł, Joanna; Jarmuła, Adam; Maj, Piotr; Gołos, Barbara; Wińska, Patrycja; Ostafil, Sylwia; Wałajtys-Rode, Elżbieta; Shugar, David; Rode, Wojciech

    2015-12-01

    Thymidylate synthase (TS) may undergo phosphorylation endogenously in mammalian cells, and as a recombinant protein expressed in bacterial cells, as indicated by the reaction of purified enzyme protein with Pro-Q® Diamond Phosphoprotein Gel Stain (PGS). With recombinant human, mouse, rat, Trichinella spiralis and Caenorhabditis elegans TSs, expressed in Escherichia coli, the phosphorylated, compared to non-phosphorylated recombinant enzyme forms, showed a decrease in Vmax(app), bound their cognate mRNA (only rat enzyme studied), and repressed translation of their own and several heterologous mRNAs (human, rat and mouse enzymes studied). However, attempts to determine the modification site(s), whether endogenously expressed in mammalian cells, or recombinant proteins, did not lead to unequivocal results. Comparative ESI-MS/analysis of IEF fractions of TS preparations from parental and FdUrd-resistant mouse leukemia L1210 cells, differing in sensitivity to inactivation by FdUMP, demonstrated phosphorylation of Ser(10) and Ser(16) in the resistant enzyme only, although PGS staining pointed to the modification of both L1210 TS proteins. The TS proteins phosphorylated in bacterial cells were shown by (31)P NMR to be modified only on histidine residues, like potassium phosphoramidate (KPA)-phosphorylated TS proteins. NanoLC-MS/MS, enabling the use of CID and ETD peptide fragmentation methods, identified several phosphohistidine residues, but certain phosphoserine and phosphothreonine residues were also implicated. Molecular dynamics studies, based on the mouse TS crystal structure, allowed one to assess potential of several phosphorylated histidine residues to affect catalytic activity, the effect being phosphorylation site dependent. PMID:26315778

  10. Aromatic Polyketide Synthases (Purification, Characterization, and Antibody Development to Benzalacetone Synthase from Raspberry Fruits).

    PubMed Central

    Borejsza-Wysocki, W.; Hrazdina, G.

    1996-01-01

    p-Hydroxyphenylbutan-2-one, the characteristic aroma compound of raspberries (Rubus idaeus L.), is synthesized from p-coumaryl-coenzyme A and malonyl-coenzyme A in a two-step reaction sequence that is catalyzed by benzalacetone synthase and benzalacetone reductase (W. Borejsza-Wysocki and G. Hrazdina [1994] Phytochemistry 35: 623-628). Benzalacetone synthase condenses one malonate with p-coumarate to form the pathway intermediate p-hydroxyphenylbut-3-ene-2-one (p-hydroxybenzalacetone) in a reaction that is similar to those catalyzed by chalcone and stilbene synthases. We have obtained an enzyme preparation from ripe raspberries that was preferentially enriched in benzalacetone synthase (approximately 170-fold) over chalcone synthase (approximately 14-fold) activity. This preparation was used to characterize benzalacetone synthase and to develop polyclonal antibodies in rabbits. Benzalacetone synthase showed similarity in its molecular properties to chalcone synthase but differed distinctly in its substrate specificity, response to 2-mercaptoethanol and ethylene glycol, and induction in cell-suspension cultures. The product of the enzyme, p-hydroxybenzalacetone, inhibited mycelial growth of the raspberry pathogen Phytophthora fragariae var rubi at 250 [mu]M. We do not know whether the dual activity in the benzalacetone synthase preparation is the result of a bifunctional enzyme or is caused by contamination with chalcone synthase that was also present. The rapid induction of the enzyme in cell-suspension cultures upon addition of yeast extract and the toxicity of its product, p-hydroxybenzalacetone, to phytopathogenic fungi also suggest that the pathway may be part of a plant defense response. PMID:12226219

  11. Apicoplast-Localized Lysophosphatidic Acid Precursor Assembly Is Required for Bulk Phospholipid Synthesis in Toxoplasma gondii and Relies on an Algal/Plant-Like Glycerol 3-Phosphate Acyltransferase

    PubMed Central

    Callahan, Damien L.; Dubois, David; van Dooren, Giel G.; Shears, Melanie J.; Cesbron-Delauw, Marie-France; Maréchal, Eric; McConville, Malcolm J.; McFadden, Geoffrey I.; Yamaryo-Botté, Yoshiki; Botté, Cyrille Y.

    2016-01-01

    Most apicomplexan parasites possess a non-photosynthetic plastid (the apicoplast), which harbors enzymes for a number of metabolic pathways, including a prokaryotic type II fatty acid synthesis (FASII) pathway. In Toxoplasma gondii, the causative agent of toxoplasmosis, the FASII pathway is essential for parasite growth and infectivity. However, little is known about the fate of fatty acids synthesized by FASII. In this study, we have investigated the function of a plant-like glycerol 3-phosphate acyltransferase (TgATS1) that localizes to the T. gondii apicoplast. Knock-down of TgATS1 resulted in significantly reduced incorporation of FASII-synthesized fatty acids into phosphatidic acid and downstream phospholipids and a severe defect in intracellular parasite replication and survival. Lipidomic analysis demonstrated that lipid precursors are made in, and exported from, the apicoplast for de novo biosynthesis of bulk phospholipids. This study reveals that the apicoplast-located FASII and ATS1, which are primarily used to generate plastid galactolipids in plants and algae, instead generate bulk phospholipids for membrane biogenesis in T. gondii. PMID:27490259

  12. Plastidial Glycolytic Glyceraldehyde-3-Phosphate Dehydrogenase Is an Important Determinant in the Carbon and Nitrogen Metabolism of Heterotrophic Cells in Arabidopsis.

    PubMed

    Anoman, Armand D; Muñoz-Bertomeu, Jesús; Rosa-Téllez, Sara; Flores-Tornero, María; Serrano, Ramón; Bueso, Eduardo; Fernie, Alisdair R; Segura, Juan; Ros, Roc

    2015-11-01

    This study functionally characterizes the Arabidopsis (Arabidopsis thaliana) plastidial glycolytic isoforms of glyceraldehyde-3-phosphate dehydrogenase (GAPCp) in photosynthetic and heterotrophic cells. We expressed the enzyme in gapcp double mutants (gapcp1gapcp2) under the control of photosynthetic (Rubisco small subunit RBCS2B [RBCS]) or heterotrophic (phosphate transporter PHT1.2 [PHT]) cell-specific promoters. Expression of GAPCp1 under the control of RBCS in gapcp1gapcp2 had no significant effect on the metabolite profile or growth in the aerial part (AP). GAPCp1 expression under the control of the PHT promoter clearly affected Arabidopsis development by increasing the number of lateral roots and having a major effect on AP growth and metabolite profile. Our results indicate that GAPCp1 is not functionally important in photosynthetic cells but plays a fundamental role in roots and in heterotrophic cells of the AP. Specifically, GAPCp activity may be required in root meristems and the root cap for normal primary root growth. Transcriptomic and metabolomic analyses indicate that the lack of GAPCp activity affects nitrogen and carbon metabolism as well as mineral nutrition and that glycerate and glutamine are the main metabolites responding to GAPCp activity. Thus, GAPCp could be an important metabolic connector of glycolysis with other pathways, such as the phosphorylated pathway of serine biosynthesis, the ammonium assimilation pathway, or the metabolism of γ-aminobutyrate, which in turn affect plant development. PMID:26134167

  13. A de novo NADPH generation pathway for improving lysine production of Corynebacterium glutamicum by rational design of the coenzyme specificity of glyceraldehyde 3-phosphate dehydrogenase.

    PubMed

    Bommareddy, Rajesh Reddy; Chen, Zhen; Rappert, Sugima; Zeng, An-Ping

    2014-09-01

    Engineering the cofactor availability is a common strategy of metabolic engineering to improve the production of many industrially important compounds. In this work, a de novo NADPH generation pathway is proposed by altering the coenzyme specificity of a native NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) to NADP, which consequently has the potential to produce additional NADPH in the glycolytic pathway. Specifically, the coenzyme specificity of GAPDH of Corynebacterium glutamicum is systematically manipulated by rational protein design and the effect of the manipulation for cellular metabolism and lysine production is evaluated. By a combinatorial modification of four key residues within the coenzyme binding sites, different GAPDH mutants with varied coenzyme specificity were constructed. While increasing the catalytic efficiency of GAPDH towards NADP enhanced lysine production in all of the tested mutants, the most significant improvement of lysine production (~60%) was achieved with the mutant showing similar preference towards both NAD and NADP. Metabolic flux analysis with (13)C isotope studies confirmed that there was no significant change of flux towards the pentose phosphate pathway and the increased lysine yield was mainly attributed to the NADPH generated by the mutated GAPDH. The present study highlights the importance of protein engineering as a key strategy in de novo pathway design and overproduction of desired products. PMID:24953302

  14. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of glyceraldehyde-3-phosphate dehydrogenase from Streptococcus agalactiae NEM316.

    PubMed

    Nagarajan, Revathi; Ponnuraj, Karthe

    2014-07-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an essential enzyme involved in glycolysis. Despite lacking the secretory signal sequence, this cytosolic enzyme has been found localized at the surface of several bacteria and fungi. As a surface protein, GAPDH exhibits various adhesive functions, thereby facilitating colonization and invasion of host tissues. Streptococcus agalactiae, also known as group B streptococcus (GBS), binds onto the host using its surface adhesins and causes sepsis and pneumonia in neonates. GAPDH is one of the surface adhesins of GBS binding to human plasminogen and is a virulent factor associated with host colonization. Although the surface-associated GAPDH has been shown to bind to a variety of host extracellular matrix (ECM) molecules in various bacteria, the molecular mechanism underlying their interaction is not fully understood. To investigate this, structural studies on GAPDH of S. agalactiae were initiated. The gapC gene of S. agalactiae NEM316 encoding GAPDH protein was cloned into pET-28a vector, overexpressed in Escherichia coli BL21(DE3) cells and purified to homogeneity. The purified protein was crystallized using the hanging-drop vapour-diffusion method. The GAPDH crystals obtained in two different crystallization conditions diffracted to 2.8 and 2.6 Å resolution, belonging to two different space groups P2₁ and P2₁2₁2₁, respectively. The structure was solved by molecular replacement and structure refinement is now in progress. PMID:25005093

  15. Cytosolic Glyceraldehyde-3-Phosphate Dehydrogenases Interact with Phospholipase Dδ to Transduce Hydrogen Peroxide Signals in the Arabidopsis Response to Stress[C][W

    PubMed Central

    Guo, Liang; Devaiah, Shivakumar P.; Narasimhan, Rama; Pan, Xiangqing; Zhang, Yanyan; Zhang, Wenhua; Wang, Xuemin

    2012-01-01

    Reactive oxygen species (ROS) are produced in plants under various stress conditions and serve as important mediators in plant responses to stresses. Here, we show that the cytosolic glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenases (GAPCs) interact with the plasma membrane–associated phospholipase D (PLDδ) to transduce the ROS hydrogen peroxide (H2O2) signal in Arabidopsis thaliana. Genetic ablation of PLDδ impeded stomatal response to abscisic acid (ABA) and H2O2, placing PLDδ downstream of H2O2 in mediating ABA-induced stomatal closure. To determine the molecular link between H2O2 and PLDδ, GAPC1 and GAPC2 were identified to bind to PLDδ, and the interaction was demonstrated by coprecipitation using proteins expressed in Escherichia coli and yeast, surface plasmon resonance, and bimolecular fluorescence complementation. H2O2 promoted the GAPC–PLDδ interaction and PLDδ activity. Knockout of GAPCs decreased ABA- and H2O2-induced activation of PLD and stomatal sensitivity to ABA. The loss of GAPCs or PLDδ rendered plants less responsive to water deficits than the wild type. The results indicate that the H2O2-promoted interaction of GAPC and PLDδ may provide a direct connection between membrane lipid–based signaling, energy metabolism and growth control in the plant response to ROS and water stress. PMID:22589465

  16. A H2 very high frequency capacitively coupled plasma inactivates glyceraldehyde 3-phosphate dehydrogenase(GapDH) more efficiently than UV photons and heat combined

    NASA Astrophysics Data System (ADS)

    Stapelmann, Katharina; Lackmann, Jan-Wilm; Buerger, Ines; Bandow, Julia Elisabeth; Awakowicz, Peter

    2014-02-01

    Plasma sterilization is a promising alternative to commonly used sterilization techniques, because the conventional methods suffer from certain limitations, e.g. incompatibility with heat-sensitive materials, or use of toxic agents. However, plasma-based sterilization mechanisms are not fully understood yet. A low-pressure very high frequency capacitively coupled plasma is used to investigate the impact of a hydrogen discharge on the protein glyceraldehyde 3-phosphate dehydrogenase (GapDH). GapDH is an enzyme of glycolysis. As a part of the central metabolism, it occurs in nearly all organisms from bacteria to humans. The plasma is investigated with absolutely calibrated optical emission spectroscopy in order to identify and to quantify plasma components that can contribute to enzyme inactivation. The contribution of UV photons and heat to GapDH inactivation is investigated separately, and neither seems to be a major factor. In order to investigate the mechanisms of GapDH inactivation by the hydrogen discharge, samples are investigated for etching, induction of amino acid backbone breaks, and chemical modifications. While neither etching nor strand breaks are observed, chemical modifications occur at different amino acid residues of GapDH. Deamidations of asparagines as well as methionine and cysteine oxidations are detected after VHF-CCP treatment. In particular, oxidation of the cysteine in the active centre is known to lead to GapDH inactivation.

  17. Cloning and heterologous overexpression of three gap genes encoding different glyceraldehyde-3-phosphate dehydrogenases from the plant pathogenic bacterium Pseudomonas syringae pv. tomato strain DC3000.

    PubMed

    Elkhalfi, Bouchra; Araya-Garay, José Miguel; Rodríguez-Castro, Jorge; Rey-Méndez, Manuel; Soukri, Abdelaziz; Serrano Delgado, Aurelio

    2013-06-01

    The gammaproteobacterium Pseudomonas syringae pv. tomato DC3000 is the causal agent of bacterial speck, a common disease of tomato. The mode of infection of this pathogen is not well understood, but according to molecular biological, genomic and proteomic data it produces a number of proteins that may promote infection and draw nutrients from the plant. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a major enzyme of carbon metabolism that was reported to be a surface antigen and virulence factor in other pathogenic microorganisms, but its possible role in the infection process of P. syringae has so far not been studied. Whole-genome sequence analyses revealed the occurrence in this phytopathogenic bacterium of three paralogous gap genes encoding distinct GAPDHs, namely two class I enzymes having different molecular mass subunits and one class III bifunctional D-erythrose-4-phosphate dehydrogenase/GAPDH enzyme. By using genome bioinformatics data, as well as alignments of both DNA and deduced protein sequences, the three gap genes of P. syringae were one-step cloned with a His-Tag in pET21a vector using a PCR-based strategy, and its expression optimized in Escherichia coli BL21 to achieve high yield of the heterologous proteins. In accordance with their distinct molecular phylogenies, these bacterial gap genes encode functional GAPDHs of diverse molecular masses and nicotinamide-coenzyme specificities, suggesting specific metabolic and/or cellular roles. PMID:23507306

  18. Evolutionary engineering of a glycerol-3-phosphate dehydrogenase-negative, acetate-reducing Saccharomyces cerevisiae strain enables anaerobic growth at high glucose concentrations

    PubMed Central

    Guadalupe-Medina, Víctor; Metz, Benjamin; Oud, Bart; van Der Graaf, Charlotte M; Mans, Robert; Pronk, Jack T; van Maris, Antonius J A

    2014-01-01

    Glycerol production by Saccharomyces cerevisiae, which is required for redox-cofactor balancing in anaerobic cultures, causes yield reduction in industrial bioethanol production. Recently, glycerol formation in anaerobic S. cerevisiae cultures was eliminated by expressing Escherichia coli (acetylating) acetaldehyde dehydrogenase (encoded by mhpF) and simultaneously deleting the GPD1 and GPD2 genes encoding glycerol-3-phosphate dehydrogenase, thus coupling NADH reoxidation to reduction of acetate to ethanol. Gpd– strains are, however, sensitive to high sugar concentrations, which complicates industrial implementation of this metabolic engineering concept. In this study, laboratory evolution was used to improve osmotolerance of a Gpd– mhpF-expressing S. cerevisiae strain. Serial batch cultivation at increasing osmotic pressure enabled isolation of an evolved strain that grew anaerobically at 1 M glucose, at a specific growth rate of 0.12 h−1. The evolved strain produced glycerol at low concentrations (0.64 ± 0.33 g l−1). However, these glycerol concentrations were below 10% of those observed with a Gpd+ reference strain. Consequently, the ethanol yield on sugar increased from 79% of the theoretical maximum in the reference strain to 92% for the evolved strains. Genetic analysis indicated that osmotolerance under aerobic conditions required a single dominant chromosomal mutation, and one further mutation in the plasmid-borne mhpF gene for anaerobic growth. PMID:24004455

  19. Apicoplast-Localized Lysophosphatidic Acid Precursor Assembly Is Required for Bulk Phospholipid Synthesis in Toxoplasma gondii and Relies on an Algal/Plant-Like Glycerol 3-Phosphate Acyltransferase.

    PubMed

    Amiar, Souad; MacRae, James I; Callahan, Damien L; Dubois, David; van Dooren, Giel G; Shears, Melanie J; Cesbron-Delauw, Marie-France; Maréchal, Eric; McConville, Malcolm J; McFadden, Geoffrey I; Yamaryo-Botté, Yoshiki; Botté, Cyrille Y

    2016-08-01

    Most apicomplexan parasites possess a non-photosynthetic plastid (the apicoplast), which harbors enzymes for a number of metabolic pathways, including a prokaryotic type II fatty acid synthesis (FASII) pathway. In Toxoplasma gondii, the causative agent of toxoplasmosis, the FASII pathway is essential for parasite growth and infectivity. However, little is known about the fate of fatty acids synthesized by FASII. In this study, we have investigated the function of a plant-like glycerol 3-phosphate acyltransferase (TgATS1) that localizes to the T. gondii apicoplast. Knock-down of TgATS1 resulted in significantly reduced incorporation of FASII-synthesized fatty acids into phosphatidic acid and downstream phospholipids and a severe defect in intracellular parasite replication and survival. Lipidomic analysis demonstrated that lipid precursors are made in, and exported from, the apicoplast for de novo biosynthesis of bulk phospholipids. This study reveals that the apicoplast-located FASII and ATS1, which are primarily used to generate plastid galactolipids in plants and algae, instead generate bulk phospholipids for membrane biogenesis in T. gondii. PMID:27490259

  20. ATP-driven transhydrogenation and ionization of water in a reconstituted glyceraldehyde-3-phosphate dehydrogenases (phosphorylating and non-phosphorylating) model system.

    PubMed

    Serrano, A; Mateos, M I; Losada, M

    1993-12-30

    In an unbuffered medium, an intense acidification occurs during the oxidation of D-glyceraldehyde-3-phosphate (G3P) to 3-phospho-D-glycerate (PGA) catalyzed by NADP(+)-specific non-phosphorylating G3P dehydrogenase, an enzyme that photosynthetic eukaryotic cells contain exclusively in their cytosol. The true enzymatic character of this proton release is the consequence of the following redox/acid-base reaction: G3P + NADP+ + H2O-->PGA + NADPH + 2H+. When the well-established ATP-dependent reduction of PGA to G3P, catalyzed by PGA kinase and NAD(+)-specific phosphorylating G3P dehydrogenase, was coupled through the intermediate G3P to the above reverse oxidation reaction, a transient alkalinization of the medium followed by its acidification accompanied transhydrogenation from NADH to NADP+. The significance of the observed endergonic transhydrogenation and ionization of water at the expense of the chemical energy of ATP in this reconstituted enzyme system as well as its relevance for the export of reducing power (H-) across the chloroplast membrane and the maintenance of the pH gradient that exists between the stroma and the cytosol are discussed. PMID:8280152

  1. The N-terminal segment of glyceraldehyde-3-phosphate dehydrogenase of Haemonchus contortus interacts with complements C1q and C3.

    PubMed

    Vedamurthy, G V; Sahoo, S; Devi, I K; Murugavel, S; Joshi, P

    2015-11-01

    Haemonchus contortus, an economically important blood-sucking parasite of sheep and goats, survives the harsh host gut environment by secreting a number of proteins referred as excretory/secretory (ES) products. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme, is one of the components of H. contortus ES products. The parasite enzyme binds to complement C3 and inhibits its activity. In this study, the C3-binding activity of the parasite GAPDH was mapped to the N-terminal part of the enzyme by generating defined recombinant fragments of the protein. The N-terminal fragment also trapped complement C1q but not C5 and inhibited complement-mediated lysis of sensitized sheep erythrocytes. Competitive binding assay indicates different binding regions for C1q and C3 proteins. GAPDH stimulated proliferation of goat peripheral blood mononuclear cells in vitro and reacted with the sera from H. contortus-infected animals. However, the fragments of GAPDH did not stimulate cell proliferation nor reacted with the infected animal sera. Furthermore, denatured GAPDH failed to react with the infected animal sera in dot blot suggesting conformation-dependent epitope. These results demonstrate an elegant strategy of the parasite to completely shut down complement activation and identify GAPDH as a promising target for future therapeutic intervention. PMID:26332726

  2. Possible role of NAD-dependent glyceraldehyde-3-phosphate dehydrogenase in growth promotion of Arabidopsis seedlings by low levels of selenium.

    PubMed

    Takeda, Toru; Fukui, Yuki

    2015-01-01

    We explored functional significance of selenium (Se) in Arabidopsis physiology. Se at very low concentrations in cultivation exerted a considerable positive effect on Arabidopsis growth with no indication of oxidative stress, whereas Se at higher concentrations significantly suppressed the growth and brought serious oxidative damage. Respiration, ATP levels, and the activity of NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (NAD-GAPDH) were enhanced in Arabidopsis grown in the medium containing 1.0 μM Se. Addition of an inhibitor of glutathione (GSH) synthesis to the medium abolished both of the Se-dependent growth promotion and NAD-GAPDH up-regulation. Assay of NAD-GAPDH purified from seedlings subjected to Se interventions raised the possibility of a direct connection between the activity of this enzyme and Arabidopsis growth. These results reveal that trace amounts of Se accelerate Arabidopsis growth, and suggest that this pro-growth effect of Se arises enhancing mitochondrial performance in a GSH-dependent manner, in which NAD-GAPDH may serve as a key regulator. PMID:25988618

  3. Defective Hyphal Induction of a Candida albicans Phosphatidylinositol 3-Phosphate 5-Kinase Null Mutant on Solid Media Does Not Lead to Decreased Virulence

    PubMed Central

    Augsten, Martin; Hübner, Claudia; Nguyen, Monika; Künkel, Waldemar; Härtl, Albert; Eck, Raimund

    2002-01-01

    A phosphatidylinositol 3-phosphate [PI(3)P] 5-kinase gene (CaFAB1) of the most important human pathogenic yeast, Candida albicans, was cloned and sequenced. An open reading frame was detected which encodes a 2,369-amino-acid protein with a calculated molecular mass of 268 kDa and a relative isoelectric point of 6.76. This protein exhibits 38% overall amino acid sequence identity with Saccharomyces cerevisiae Fab1p. We localized the CaFAB1 gene on chromosome R. To determine the influence of the PI(3)P 5-kinase CaFab1p on processes involved in C. albicans morphogenesis and pathogenicity, we sequentially disrupted both copies of the gene. Homozygous deletion of C. albicans CaFAB1 resulted in a mutant strain which exhibited defects in morphogenesis. A Cafab1 null mutant had enlarged vacuoles, an acidification defect, and increased generation times and was unable to form hyphae on different solid media. The sensitivities to hyperosmotic and high-temperature stresses, adherence, and virulence compared to those of wild-type strain SC5314 were not affected. PMID:12117957

  4. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of glyceraldehyde-3-phosphate dehydrogenase from Streptococcus agalactiae NEM316

    PubMed Central

    Nagarajan, Revathi; Ponnuraj, Karthe

    2014-01-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an essential enzyme involved in glycolysis. Despite lacking the secretory signal sequence, this cytosolic enzyme has been found localized at the surface of several bacteria and fungi. As a surface protein, GAPDH exhibits various adhesive functions, thereby facilitating colonization and invasion of host tissues. Streptococcus agalactiae, also known as group B streptococcus (GBS), binds onto the host using its surface adhesins and causes sepsis and pneumonia in neonates. GAPDH is one of the surface adhesins of GBS binding to human plasminogen and is a virulent factor associated with host colonization. Although the surface-associated GAPDH has been shown to bind to a variety of host extracellular matrix (ECM) molecules in various bacteria, the molecular mechanism underlying their interaction is not fully understood. To investigate this, structural studies on GAPDH of S. agalactiae were initiated. The gapC gene of S. agalactiae NEM316 encoding GAPDH protein was cloned into pET-28a vector, overexpressed in Escherichia coli BL21(DE3) cells and purified to homogeneity. The purified protein was crystallized using the hanging-drop vapour-diffusion method. The GAPDH crystals obtained in two different crystallization conditions diffracted to 2.8 and 2.6 Å resolution, belonging to two different space groups P21 and P212121, respectively. The structure was solved by molecular replacement and structure refinement is now in progress. PMID:25005093

  5. Two glycerol 3-phosphate dehydrogenase isogenes from Candida versatilis SN-18 play an important role in glycerol biosynthesis under osmotic stress.

    PubMed

    Mizushima, Daiki; Iwata, Hisashi; Ishimaki, Yuki; Ogihara, Jun; Kato, Jun; Kasumi, Takafumi

    2016-05-01

    Two isogenes of glycerol 3-phosphate dehydrogenase (GPD) from Candida versatilis SN-18 were cloned and sequenced. These intronless genes (Cagpd1 and Cagpd2) were both predicted to encode a 378 amino acid polypeptide, and the deduced amino acid sequences mutually showed 76% identity. Interestingly, Cagpd1 and Cagpd2 were located tandemly in a locus of genomic DNA within a 262 bp interval. To our knowledge, this represents a novel instance of isogenic genes relating to glucose metabolism. The stress response element (STRE) was found respectively at -93 to -89 bp upstream of the 5'end of Cagpd1 and -707 to -703 bp upstream of Cagpd2, indicating that these genes are involved in osmotic stress response. In heterologous expression using a gpd1Δgpd2Δ double deletion mutant of Saccharomyces cerevisiae, Cagpd1 and Cagpd2 transformants complemented the function of GPD, with Cagpd2 being much more effective than Cagpd1 in promoting growth and glycerol synthesis. Phylogenetic analysis of the amino acid sequences suggested that Cagpd1p and Cagpd2p are NADP(+)-dependent GPDs (EC 1.1.1.94). However, crude enzyme extract from Cagpd1 and Cagpd2 transformants showed GPD activity with only NAD(+) as cofactor. Hence, both Cagpd1p and Cagpd2p are likely NAD(+)-dependent GPDs (EC 1.1.1.8), similar to GPDs from S. cerevisiae and Candida magnoliae. PMID:26906228

  6. Three Homologous Genes Encoding sn-Glycerol-3-Phosphate Acyltransferase 4 Exhibit Different Expression Patterns and Functional Divergence in Brassica napus1[C][W][OA

    PubMed Central

    Chen, Xue; Truksa, Martin; Snyder, Crystal L.; El-Mezawy, Aliaa; Shah, Saleh; Weselake, Randall J.

    2011-01-01

    Brassica napus is an allotetraploid (AACC) formed from the fusion of two diploid progenitors, Brassica rapa (AA) and Brassica oleracea (CC). Polyploidy and genome-wide rearrangement during the evolution process have resulted in genes that are present as multiple homologs in the B. napus genome. In this study, three B. napus homologous genes encoding endoplasmic reticulum-bound sn-glycerol-3-phosphate acyltransferase 4 (GPAT4) were identified and characterized. Although the three GPAT4 homologs share a high sequence similarity, they exhibit different expression patterns and altered epigenetic features. Heterologous expression in yeast further revealed that the three BnGPAT4 homologs encoded functional GPAT enzymes but with different levels of polypeptide accumulation. Complementation of the Arabidopsis (Arabidopsis thaliana) gpat4 gpat8 double mutant line with individual BnGPAT4 homologs suggested their physiological roles in cuticle formation. Analysis of gpat4 RNA interference lines of B. napus revealed that the BnGPAT4 deficiency resulted in reduced cutin content and altered stomatal structures in leaves. Our results revealed that the BnGPAT4 homologs have evolved into functionally divergent forms and play important roles in cutin synthesis and stomatal development. PMID:21173024

  7. Cloning of a coconut endosperm cDNA encoding a 1-acyl-sn-glycerol-3-phosphate acyltransferase that accepts medium-chain-length substrates.

    PubMed Central

    Knutzon, D S; Lardizabal, K D; Nelsen, J S; Bleibaum, J L; Davies, H M; Metz, J G

    1995-01-01

    Immature coconut (Cocos nucifera) endosperm contains a 1-acyl-sn-glycerol-3-phosphate acyltransferase (LPAAT) activity that shows a preference for medium-chain-length fatty acyl-coenzyme A substrates (H.M. Davies, D.J. Hawkins, J.S. Nelsen [1995] Phytochemistry 39:989-996). Beginning with solubilized membrane preparations, we have used chromatographic separations to identify a polypeptide with an apparent molecular mass of 29 kD, whose presence in various column fractions correlates with the acyltransferase activity detected in those same fractions. Amino acid sequence data obtained from several peptides generated from this protein were used to isolate a full-length clone from a coconut endosperm cDNA library. Clone pCGN5503 contains a 1325-bp cDNA insert with an open reading frame encoding a 308-amino acid protein with a calculated molecular mass of 34.8 kD. Comparison of the deduced amino acid sequence of pCGN5503 to sequences in the data banks revealed significant homology to other putative LPAAT sequences. Expression of the coconut cDNA in Escherichia coli conferred upon those cells a novel LPAAT activity whose substrate activity profile matched that of the coconut enzyme. PMID:8552723

  8. Distribution of Callose Synthase, Cellulose Synthase, and Sucrose Synthase in Tobacco Pollen Tube Is Controlled in Dissimilar Ways by Actin Filaments and Microtubules1[W

    PubMed Central

    Cai, Giampiero; Faleri, Claudia; Del Casino, Cecilia; Emons, Anne Mie C.; Cresti, Mauro

    2011-01-01

    Callose and cellulose are fundamental components of the cell wall of pollen tubes and are probably synthesized by distinct enzymes, callose synthase and cellulose synthase, respectively. We examined the distribution of callose synthase and cellulose synthase in tobacco (Nicotiana tabacum) pollen tubes in relation to the dynamics of actin filaments, microtubules, and the endomembrane system using specific antibodies to highly conserved peptide sequences. The role of the cytoskeleton and membrane flow was investigated using specific inhibitors (latrunculin B, 2,3-butanedione monoxime, taxol, oryzalin, and brefeldin A). Both enzymes are associated with the plasma membrane, but cellulose synthase is present along the entire length of pollen tubes (with a higher concentration at the apex) while callose synthase is located in the apex and in distal regions. In longer pollen tubes, callose synthase accumulates consistently around callose plugs, indicating its involvement in plug synthesis. Actin filaments and endomembrane dynamics are critical for the distribution of callose synthase and cellulose synthase, showing that enzymes are transported through Golgi bodies and/or vesicles moving along actin filaments. Conversely, microtubules appear to be critical in the positioning of callose synthase in distal regions and around callose plugs. In contrast, cellulose synthases are only partially coaligned with cortical microtubules and unrelated to callose plugs. Callose synthase also comigrates with tubulin by Blue Native-polyacrylamide gel electrophoresis. Membrane sucrose synthase, which expectedly provides UDP-glucose to callose synthase and cellulose synthase, binds to actin filaments depending on sucrose concentration; its distribution is dependent on the actin cytoskeleton and the endomembrane system but not on microtubules. PMID:21205616

  9. An investigation into eukaryotic pseudouridine synthases.

    PubMed

    King, Ross D; Lu, Chuan

    2014-08-01

    A common post-transcriptional modification of RNA is the conversion of uridine to its isomer pseudouridine. We investigated the biological significance of eukaryotic pseudouridine synthases using the yeast Saccharomyces cerevisiae. We conducted a comprehensive statistical analysis on growth data from automated perturbation (gene deletion) experiments, and used bi-logistic curve analysis to characterise the yeast phenotypes. The deletant strains displayed different alteration in growth properties, including in some cases enhanced growth and/or biphasic growth curves not seen in wild-type strains under matched conditions. These results demonstrate that disrupting pseudouridine synthases can have a significant qualitative effect on growth. We further investigated the significance of post-transcriptional pseudouridine modification through investigation of the scientific literature. We found that (1) In Toxoplasma gondii, a pseudouridine synthase gene is critical in cellular differentiation between the two asexual forms: Tachyzoites and bradyzoites; (2) Mutation of pseudouridine synthase genes has also been implicated in human diseases (mitochondrial myopathy and sideroblastic anemia (MLASA); dyskeratosis congenita). Taken together, these results are consistent with pseudouridine synthases having a Gene Ontology function of "biological regulation". PMID:25152040

  10. Chitin synthase homologs in three ectomycorrhizal truffles.

    PubMed

    Lanfranco, L; Garnero, L; Delpero, M; Bonfante, P

    1995-12-01

    Degenerate PCR primers were used to amplify a conserved gene portion coding chitin synthase from genomic DNA of six species of ectomycorrhizal truffles. DNA was extracted from both hypogeous fruitbodies and in vitro growing mycelium of Tuber borchii. A single fragment of about 600 bp was amplified for each species. The amplification products from Tuber magnatum, T. borchii and T. ferrugineum were cloned and sequenced, revealing a high degree of identity (91.5%) at the nucleotide level. On the basis of the deduced amino acid sequences these clones were assigned to class II chitin synthase. Southern blot experiments performed on genomic DNA showed that the amplification products derive from a single copy gene. Phylogenetic analysis of the nucleotide sequences of class II chitin synthase genes confirmed the current taxonomic position of the genus Tuber, and suggested a close relationship between T. magnatum and T. uncinatum. PMID:8593947

  11. Homology study of two polyhydroxyalkanoate (PHA) synthases from Pseudomonas aureofaciens.

    PubMed

    Umeda, F; Nishikawa, T; Miyasaka, H; Maeda, I; Kawase, M; Yagi, K

    2001-11-01

    Recently, we have cloned and analyzed two polyhydroxyalkanoate (PHA) synthase genes (phaC1 and phaC2 in the pha cluster) from Pseudomonas aureofaciens. In this report, the deduced amino acid (AA) sequences of PHA synthase 1 and PHA synthase 2 from P. aureofaciens are compared with those from three other bacterial strains (Pseudomonas sp. 61-3, P. oleovorans and P. aeruginosa) containing the homologous pha cluster. The level of homology of either PHA synthase 1 or PHA synthase 2 was high with each enzyme from these three bacterial strains. Furthermore, multialignment of PHA synthase AA sequences implied that both enzymes of PHA synthase 1 and PHA synthase 2 were highly conserved in the four strains including P. aureofaciens. PMID:11916262

  12. Secretion of the housekeeping protein glyceraldehyde-3-phosphate dehydrogenase by the LEE-encoded type III secretion system in enteropathogenic Escherichia coli.

    PubMed

    Aguilera, Laura; Ferreira, Elaine; Giménez, Rosa; Fernández, Francisco José; Taulés, Marta; Aguilar, Juan; Vega, M Cristina; Badia, Josefa; Baldomà, Laura

    2012-06-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional housekeeping protein secreted by pathogens and involved in adhesion and/or virulence. Previously we reported that enterohemorrhagic (EHEC) and enteropathogenic (EPEC) Escherichia coli secrete GAPDH into the culture medium. This bacterial protein binds human plasminogen and fibrinogen and remains associated with Caco-2 cells upon infection. In these pathogens, GAPDH secretion is not linked to outer membrane vesicles and depends on growth conditions, although the secretion mechanism is still unknown. EPEC is an attaching and effacing pathogen able to secrete and translocate multiple effector proteins into infected cells through a type III secretion system (T3SS). The secretion process is often dependent on a bacterial chaperone. The chaperone CesT displays broad substrate specificity and plays a central role in the recruitment of multiple type III effectors to the T3SS apparatus. Here we provide genetic evidences on GAPDH secretion through T3SS by EPEC grown in DMEM. Secretion of GAPDH is increased in ΔsepD mutants and abolished in mutants defective in the type III ATPase EscN. Complementation with escN gene restores GAPDH secretion. In addition, we prove by means of pull down experiments, overlay immunoblotting and biolayer interferometry a novel interaction between GAPDH and the chaperone CesT. This interaction, which is strong and slow dissociating, may stabilize a population of GAPDH molecules in a secretion competent-state and target them to the type III secretion apparatus. This is the first description of CesT interaction with a housekeeping protein and its export through T3SS. PMID:22433988

  13. Glycerol-3-phosphate acyltransferase-4-deficient mice are protected from diet-induced insulin resistance by the enhanced association of mTOR and rictor.

    PubMed

    Zhang, Chongben; Cooper, Daniel E; Grevengoed, Trisha J; Li, Lei O; Klett, Eric L; Eaton, James M; Harris, Thurl E; Coleman, Rosalind A

    2014-08-01

    Glycerol-3-phosphate acyltransferase (GPAT) activity is highly induced in obese individuals with insulin resistance, suggesting a correlation between GPAT function, triacylglycerol accumulation, and insulin resistance. We asked whether microsomal GPAT4, an isoform regulated by insulin, might contribute to the development of hepatic insulin resistance. Compared with control mice fed a high fat diet, Gpat4(-/-) mice were more glucose tolerant and were protected from insulin resistance. Overexpression of GPAT4 in mouse hepatocytes impaired insulin-suppressed gluconeogenesis and insulin-stimulated glycogen synthesis. Impaired glucose homeostasis was coupled to inhibited insulin-stimulated phosphorylation of Akt(Ser⁴⁷³) and Akt(Thr³⁰⁸). GPAT4 overexpression inhibited rictor's association with the mammalian target of rapamycin (mTOR), and mTOR complex 2 (mTORC2) activity. Compared with overexpressed GPAT3 in mouse hepatocytes, GPAT4 overexpression increased phosphatidic acid (PA), especially di16:0-PA. Conversely, in Gpat4(-/-) hepatocytes, both mTOR/rictor association and mTORC2 activity increased, and the content of PA in Gpat4(-/-) hepatocytes was lower than in controls, with the greatest decrease in 16:0-PA species. Compared with controls, liver and skeletal muscle from Gpat4(-/-)-deficient mice fed a high-fat diet were more insulin sensitive and had a lower hepatic content of di16:0-PA. Taken together, these data demonstrate that a GPAT4-derived lipid signal, likely di16:0-PA, impairs insulin signaling in mouse liver and contributes to hepatic insulin resistance. PMID:24939733

  14. DNA vaccine encoding the moonlighting protein Onchocerca volvulus glyceraldehyde-3-phosphate dehydrogenase (Ov-GAPDH) leads to partial protection in a mouse model of human filariasis.

    PubMed

    Steisslinger, Vera; Korten, Simone; Brattig, Norbert W; Erttmann, Klaus D

    2015-10-26

    River blindness, caused by the filarial parasite Onchocerca volvulus, is a major socio-economic and public health problem in Sub-Saharan Africa. In January 2015, The Onchocerciasis Vaccine for Africa (TOVA) Initiative has been launched with the aim of providing new tools to complement mass drug administration (MDA) of ivermectin, thereby promoting elimination of onchocerciasis in Africa. In this context we here present Onchocerca volvulus glyceraldehyde-3-phosphate dehydrogenase (Ov-GAPDH) as a possible DNA vaccine candidate. We report that in a laboratory model for filariasis, immunization with Ov-GAPDH led to a significant reduction of adult worm load and microfilaraemia in BALB/c mice after challenge infection with the filarial parasite Litomosoides sigmodontis. Mice were either vaccinated with Ov-GAPDH.DNA plasmid (Ov-pGAPDH.DNA) alone or in combination with recombinantly expressed Ov-GAPDH protein (Ov-rGAPDH). During the following challenge infection of immunized and control mice with L. sigmodontis, those formulations which included the DNA plasmid, led to a significant reduction of adult worm loads (up to 57% median reduction) and microfilaraemia (up to 94% reduction) in immunized animals. In a further experiment, immunization with a mixture of four overlapping, synthetic Ov-GAPDH peptides (Ov-GAPDHpept), with alum as adjuvant, did not significantly reduce worm loads. Our results indicate that DNA vaccination with Ov-GAPDH has protective potential against filarial challenge infection in the mouse model. This suggests a transfer of the approach into the cattle Onchocerca ochengi model, where it is possible to investigate the effects of this vaccination in the context of a natural host-parasite relationship. PMID:26320419

  15. Nrbf2 Protein Suppresses Autophagy by Modulating Atg14L Protein-containing Beclin 1-Vps34 Complex Architecture and Reducing Intracellular Phosphatidylinositol-3 Phosphate Levels*

    PubMed Central

    Zhong, Yu; Morris, Deanna H.; Jin, Lin; Patel, Mittul S.; Karunakaran, Senthil K.; Fu, You-Jun; Matuszak, Emily A.; Weiss, Heidi L.; Chait, Brian T.; Wang, Qing Jun

    2014-01-01

    Autophagy is a tightly regulated lysosomal degradation pathway for maintaining cellular homeostasis and responding to stresses. Beclin 1 and its interacting proteins, including the class III phosphatidylinositol-3 kinase Vps34, play crucial roles in autophagy regulation in mammals. We identified nuclear receptor binding factor 2 (Nrbf2) as a Beclin 1-interacting protein from Becn1−/−;Becn1-EGFP/+ mouse liver and brain. We also found that Nrbf2-Beclin 1 interaction required the N terminus of Nrbf2. We next used the human retinal pigment epithelial cell line RPE-1 as a model system and showed that transiently knocking down Nrbf2 by siRNA increased autophagic flux under both nutrient-rich and starvation conditions. To investigate the mechanism by which Nrbf2 regulates autophagy, we demonstrated that Nrbf2 interacted and colocalized with Atg14L, suggesting that Nrbf2 is a component of the Atg14L-containing Beclin 1-Vps34 complex. Moreover, ectopically expressed Nrbf2 formed cytosolic puncta that were positive for isolation membrane markers. These results suggest that Nrbf2 is involved in autophagosome biogenesis. Furthermore, we showed that Nrbf2 deficiency led to increased intracellular phosphatidylinositol-3 phosphate levels and diminished Atg14L-Vps34/Vps15 interactions, suggesting that Nrbf2-mediated Atg14L-Vps34/Vps15 interactions likely inhibit Vps34 activity. Therefore, we propose that Nrbf2 may interact with the Atg14L-containing Beclin 1-Vps34 protein complex to modulate protein-protein interactions within the complex, leading to suppression of Vps34 activity, autophagosome biogenesis, and autophagic flux. This work reveals a novel aspect of the intricate mechanism for the Beclin 1-Vps34 protein-protein interaction network to achieve precise control of autophagy. PMID:25086043

  16. A Land-Plant-Specific Glycerol-3-Phosphate Acyltransferase Family in Arabidopsis: Substrate Specificity, sn-2 Preference, and Evolution1[W][OA

    PubMed Central

    Yang, Weili; Simpson, Jeffrey P.; Li-Beisson, Yonghua; Beisson, Fred; Pollard, Mike; Ohlrogge, John B.

    2012-01-01

    Arabidopsis (Arabidopsis thaliana) has eight glycerol-3-phosphate acyltransferase (GPAT) genes that are members of a plant-specific family with three distinct clades. Several of these GPATs are required for the synthesis of cutin or suberin. Unlike GPATs with sn-1 regiospecificity involved in membrane or storage lipid synthesis, GPAT4 and -6 are unique bifunctional enzymes with both sn-2 acyltransferase and phosphatase activity resulting in 2-monoacylglycerol products. We present enzymology, pathway organization, and evolutionary analysis of this GPAT family. Within the cutin-associated clade, GPAT8 is demonstrated as a bifunctional sn-2 acyltransferase/phosphatase. GPAT4, -6, and -8 strongly prefer C16:0 and C18:1 ω-oxidized acyl-coenzyme As (CoAs) over unmodified or longer acyl chain substrates. In contrast, suberin-associated GPAT5 can accommodate a broad chain length range of ω-oxidized and unsubstituted acyl-CoAs. These substrate specificities (1) strongly support polyester biosynthetic pathways in which acyl transfer to glycerol occurs after oxidation of the acyl group, (2) implicate GPAT specificities as one major determinant of cutin and suberin composition, and (3) argue against a role of sn-2-GPATs (Enzyme Commission 2.3.1.198) in membrane/storage lipid synthesis. Evidence is presented that GPAT7 is induced by wounding, produces suberin-like monomers when overexpressed, and likely functions in suberin biosynthesis. Within the third clade, we demonstrate that GPAT1 possesses sn-2 acyltransferase but not phosphatase activity and can utilize dicarboxylic acyl-CoA substrates. Thus, sn-2 acyltransferase activity extends to all subbranches of the Arabidopsis GPAT family. Phylogenetic analyses of this family indicate that GPAT4/6/8 arose early in land-plant evolution (bryophytes), whereas the phosphatase-minus GPAT1 to -3 and GPAT5/7 clades diverged later with the appearance of tracheophytes. PMID:22864585

  17. A BAR-Domain Protein SH3P2, Which Binds to Phosphatidylinositol 3-Phosphate and ATG8, Regulates Autophagosome Formation in Arabidopsis[C][W

    PubMed Central

    Zhuang, Xiaohong; Wang, Hao; Lam, Sheung Kwan; Gao, Caiji; Wang, Xiangfeng; Cai, Yi; Jiang, Liwen

    2013-01-01

    Autophagy is a well-defined catabolic mechanism whereby cytoplasmic materials are engulfed into a structure termed the autophagosome. In plants, little is known about the underlying mechanism of autophagosome formation. In this study, we report that SH3 DOMAIN-CONTAINING PROTEIN2 (SH3P2), a Bin-Amphiphysin-Rvs domain–containing protein, translocates to the phagophore assembly site/preautophagosome structure (PAS) upon autophagy induction and actively participates in the membrane deformation process. Using the SH3P2–green fluorescent protein fusion as a reporter, we found that the PAS develops from a cup-shaped isolation membranes or endoplasmic reticulum–derived omegasome-like structures. Using an inducible RNA interference (RNAi) approach, we show that RNAi knockdown of SH3P2 is developmentally lethal and significantly suppresses autophagosome formation. An in vitro membrane/lipid binding assay demonstrates that SH3P2 is a membrane-associated protein that binds to phosphatidylinositol 3-phosphate. SH3P2 may facilitate membrane expansion or maturation in coordination with the phosphatidylinositol 3-kinase (PI3K) complex during autophagy, as SH3P2 promotes PI3K foci formation, while PI3K inhibitor treatment inhibits SH3P2 from translocating to autophagosomes. Further interaction analysis shows that SH3P2 associates with the PI3K complex and interacts with ATG8s in Arabidopsis thaliana, whereby SH3P2 may mediate autophagy. Thus, our study has identified SH3P2 as a novel regulator of autophagy and provided a conserved model for autophagosome biogenesis in Arabidopsis. PMID:24249832

  18. Covalent immobilization of lipase, glycerol kinase, glycerol-3-phosphate oxidase & horseradish peroxidase onto plasticized polyvinyl chloride (PVC) strip & its application in serum triglyceride determination

    PubMed Central

    Chauhan, Nidhi; Narang, Jagriti; Pundir, Chandra Shekhar

    2014-01-01

    Background & objectives: Reusable biostrip consisting enzymes immobilized onto alkylamine glass beads affixed on plasticized PVC strip for determination of triglyceride (TG) suffers from high cost of beads and their detachments during washings for reuse, leading to loss of activity. The purpose of this study was to develop a cheaper and stable biostrip for investigation of TG levels in serum. Methods: A reusable enzyme-strip was prepared for TG determination by co-immobilizing lipase, glycerol kinase (GK), glycerol-3-phosphate oxidase (GPO) and peroxidase (HRP) directly onto plasticized polyvinyl chloride (PVC) strip through glutaraldehyde coupling. The method was evaluated by studying its recovery, precision and reusability. Results: The enzyme-strip showed optimum activity at pH 7.0, 35°C and a linear relationship between its activity and triolein concentration in the range 0.1 to 15 mM. The strip was used for determination of serum TG. The detection limit of the method was 0.1 mM. Analytical recovery of added triolein was 96 per cent. Within and between batch coefficients of variation (CV) were 2.2 and 3.7 per cent, respectively. A good correlation (r=0.99) was found between TG values by standard enzymic colrimetric method employing free enzymes and the present method. The strip lost 50 per cent of its initial activity after its 200 uses during the span of 100 days, when stored at 4°C. Interpretation & conclusions: The nitrating acidic treatment of plasticized PVC strip led to glutaraldehyde coupling of four enzymes used for enzymic colourimetric determination of serum TG. The strip provided 200 reuses of enzymes with only 50 per cent loss of its initial activity. The method could be used for preparation of other enzyme strips also. PMID:24927348

  19. Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) Protein-Protein Interaction Inhibitor Reveals a Non-catalytic Role for GAPDH Oligomerization in Cell Death.

    PubMed

    Qvit, Nir; Joshi, Amit U; Cunningham, Anna D; Ferreira, Julio C B; Mochly-Rosen, Daria

    2016-06-24

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an important glycolytic enzyme, has a non-catalytic (thus a non-canonical) role in inducing mitochondrial elimination under oxidative stress. We recently demonstrated that phosphorylation of GAPDH by δ protein kinase C (δPKC) inhibits this GAPDH-dependent mitochondrial elimination. δPKC phosphorylation of GAPDH correlates with increased cell injury following oxidative stress, suggesting that inhibiting GAPDH phosphorylation should decrease cell injury. Using rational design, we identified pseudo-GAPDH (ψGAPDH) peptide, an inhibitor of δPKC-mediated GAPDH phosphorylation that does not inhibit the phosphorylation of other δPKC substrates. Unexpectedly, ψGAPDH decreased mitochondrial elimination and increased cardiac damage in an animal model of heart attack. Either treatment with ψGAPDH or direct phosphorylation of GAPDH by δPKC decreased GAPDH tetramerization, which corresponded to reduced GAPDH glycolytic activity in vitro and ex vivo Taken together, our study identified the potential mechanism by which oxidative stress inhibits the protective GAPDH-mediated elimination of damaged mitochondria. Our study also identified a pharmacological tool, ψGAPDH peptide, with interesting properties. ψGAPDH peptide is an inhibitor of the interaction between δPKC and GAPDH and of the resulting phosphorylation of GAPDH by δPKC. ψGAPDH peptide is also an inhibitor of GAPDH oligomerization and thus an inhibitor of GAPDH glycolytic activity. Finally, we found that ψGAPDH peptide is an inhibitor of the elimination of damaged mitochondria. We discuss how this unique property of increasing cell damage following oxidative stress suggests a potential use for ψGAPDH peptide-based therapy. PMID:27129213

  20. Pleurocidin Peptide Enhances Grouper Anti-Vibrio harveyi Immunity Elicited by Poly(lactide-co-glycolide)-Encapsulated Recombinant Glyceraldehyde-3-phosphate Dehydrogenase

    PubMed Central

    Chuang, Shu-Chun; Huang, Wan-Ling; Kau, Sau-Wei; Yang, Yun-Pei; Yang, Chung-Da

    2014-01-01

    Outer membrane proteins, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), are considered immunodominant antigens for eliciting protective immunity against Vibrio harveyi, the main etiological agent of vibriosis in fish. Cationic antimicrobial peptides (AMPs), such as pleurocidin (PLE), play important roles in activating and recruiting immune cells, thereby contributing to subsequent innate and adaptive immune responses. In the present study, we aimed to use PLE peptide as a potent adjuvant to improve the immunogenicity of V. harveyi recombinant GAPDH (rGAPDH). In order to prepare a controlled-release vaccine, PLE peptide and rGAPDH protein were simultaneously encapsulated into polymeric microparticles made from the biodegradable poly(lactide-co-glycolide) (PLG) polymer. The resulting PLG-encapsulated PLE plus rGAPDH (PLG-PLE/rGAPDH) microparticles, 3.21–6.27 μm in diameter, showed 72%–83% entrapment efficiency and durably released both PLE and rGAPDH for a long 30-day period. Following peritoneal immunization in grouper (Epinephelus coioides), PLG-PLE/rGAPDH microparticles resulted in significantly higher (p < 0.05, nested design) long-lasting GAPDH-specific immunity (serum titers and lymphocyte proliferation) than PLG-encapsulated rGAPDH (PLG-rGAPDH) microparticles. After an experimental challenge of V. harveyi, PLG-PLE/rGAPDH microparticles conferred a high survival rate (85%), which was significantly higher (p < 0.05, chi-square test) than that induced by PLG-rGAPDH microparticles (67%). In conclusion, PLE peptide exhibits an efficacious adjuvant effect to elicit not only improved immunity, but also enhanced protection against V. harveyi in grouper induced by rGAPDH protein encapsulated in PLG microparticles. PMID:26344624

  1. Pleurocidin Peptide Enhances Grouper Anti-Vibrio harveyi Immunity Elicited by Poly(lactide-co-glycolide)-Encapsulated Recombinant Glyceraldehyde-3-phosphate Dehydrogenase.

    PubMed

    Chuang, Shu-Chun; Huang, Wan-Ling; Kau, Sau-Wei; Yang, Yun-Pei; Yang, Chung-Da

    2014-01-01

    Outer membrane proteins, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), are considered immunodominant antigens for eliciting protective immunity against Vibrio harveyi, the main etiological agent of vibriosis in fish. Cationic antimicrobial peptides (AMPs), such as pleurocidin (PLE), play important roles in activating and recruiting immune cells, thereby contributing to subsequent innate and adaptive immune responses. In the present study, we aimed to use PLE peptide as a potent adjuvant to improve the immunogenicity of V. harveyi recombinant GAPDH (rGAPDH). In order to prepare a controlled-release vaccine, PLE peptide and rGAPDH protein were simultaneously encapsulated into polymeric microparticles made from the biodegradable poly(lactide-co-glycolide) (PLG) polymer. The resulting PLG-encapsulated PLE plus rGAPDH (PLG-PLE/rGAPDH) microparticles, 3.21-6.27 μm in diameter, showed 72%-83% entrapment efficiency and durably released both PLE and rGAPDH for a long 30-day period. Following peritoneal immunization in grouper (Epinephelus coioides), PLG-PLE/rGAPDH microparticles resulted in significantly higher (p < 0.05, nested design) long-lasting GAPDH-specific immunity (serum titers and lymphocyte proliferation) than PLG-encapsulated rGAPDH (PLG-rGAPDH) microparticles. After an experimental challenge of V. harveyi, PLG-PLE/rGAPDH microparticles conferred a high survival rate (85%), which was significantly higher (p < 0.05, chi-square test) than that induced by PLG-rGAPDH microparticles (67%). In conclusion, PLE peptide exhibits an efficacious adjuvant effect to elicit not only improved immunity, but also enhanced protection against V. harveyi in grouper induced by rGAPDH protein encapsulated in PLG microparticles. PMID:26344624

  2. Genetic Variability of Yersinia pestis Isolates as Predicted by PCR-Based IS100 Genotyping and Analysis of Structural Genes Encoding Glycerol-3-Phosphate Dehydrogenase (glpD)

    PubMed Central

    Motin, Vladimir L.; Georgescu, Anca M.; Elliott, Jeffrey M.; Hu, Ping; Worsham, Patricia L.; Ott, Linda L.; Slezak, Tomas R.; Sokhansanj, Bahrad A.; Regala, Warren M.; Brubaker, Robert R.; Garcia, Emilio

    2002-01-01

    A PCR-based genotyping system that detects divergence of IS100 locations within the Yersinia pestis genome was used to characterize a large collection of isolates of different biovars and geographical origins. Using sequences derived from the glycerol-negative biovar orientalis strain CO92, a set of 27 locus-specific primers was designed to amplify fragments between the end of IS100 and its neighboring gene. Geographically diverse members of the orientalis biovar formed a homogeneous group with identical genotype with the exception of strains isolated in Indochina. In contrast, strains belonging to the glycerol-positive biovar antiqua showed a variety of fingerprinting profiles. Moreover, strains of the biovar medievalis (also glycerol positive) clustered together with the antiqua isolates originated from Southeast Asia, suggesting their close phylogenetic relationships. Interestingly, a Manchurian biovar antiqua strain Nicholisk 51 displayed a genotyping pattern typical of biovar orientalis isolates. Analysis of the glycerol pathway in Y. pestis suggested that a 93-bp deletion within the glpD gene encoding aerobic glycerol-3-phosphate dehydrogenase might account for the glycerol-negative phenotype of the orientalis biovar. The glpD gene of strain Nicholisk 51 did not possess this deletion, although it contained two nucleotide substitutions characteristic of the glpD version found exclusively in biovar orientalis strains. To account for this close relationship between biovar orientalis strains and the antiqua Nicholisk 51 isolate, we postulate that the latter represents a variant of this biovar with restored ability to ferment glycerol. The fact that such a genetic lesion might be repaired as part of the natural evolutionary process suggests the existence of genetic exchange between different Yersinia strains in nature. The relevance of this observation on the emergence of epidemic Y. pestis strains is discussed. PMID:11807062

  3. Potentiation of thermal inactivation of glyceraldehyde-3-phosphate dehydrogenase by photodynamic treatment. A possible model for the synergistic interaction between photodynamic therapy and hyperthermia.

    PubMed Central

    Prinsze, C; Dubbelman, T M; Van Steveninck, J

    1991-01-01

    Thermal inactivation of glyceraldehyde-3-phosphate dehydrogenase appeared to be caused by a conformational mechanism, without involvement of covalent reactions. On the other hand, photodynamic inactivation of the enzyme (induced by illumination in the presence of Photofrin II) was caused by photo-oxidation of the essential thiol group in the active centre. A short photodynamic treatment of the enzyme, leading to only a limited inactivation, caused a pronounced potentiation of subsequent thermal inactivation, as measured over the temperature range 40-50 degrees C. Analysis of the experimental results according to the Arrhenius equation revealed that both the activation energy of thermal inactivation and the frequency factor (the proportionality constant) were significantly decreased by the preceding photodynamic treatment. The experimental results indicate a mechanism in which limited photodynamic treatment induced a conformational change of the protein molecule. This conformational change did not contribute to photodynamic enzyme inhibition, but was responsible for the decreased frequency factor and activation energy of subsequent thermal inactivation of the enzyme. The opposing effects of decreased activation energy and decreased frequency factor resulted in potentiation of thermal inactivation of the enzyme over the temperature range 40-50 degrees C. With other proteins, different results were obtained. With amylase the combined photodynamic and thermal effects were not synergistic, but additive, and photodynamic treatment had no effect on the frequency factor and the activation energy of thermal inactivation. With respect to myoglobin denaturation, the photodynamic and thermal effects were antagonistic over the whole practically applicable temperature range. Limited photodynamic treatment protected the protein against heat-induced precipitation, concomitantly increasing both the frequency factor and the activation energy of the process. These results offer a

  4. Oxygen transfer as a tool for fine-tuning recombinant protein production by Pichia pastoris under glyceraldehyde-3-phosphate dehydrogenase promoter.

    PubMed

    Güneş, Hande; Çalık, Pınar

    2016-07-01

    Effects of oxygen transfer on recombinant protein production by Pichia pastoris under glyceraldehyde-3-phosphate dehydrogenase promoter were investigated. Recombinant glucose isomerase was chosen as the model protein. Two groups of oxygen transfer strategies were applied, one of which was based on constant oxygen transfer rate where aeration rate was Q O/V = 3 and 10 vvm, and agitation rate was N = 900 min(-1); while the other one was based on constant dissolved oxygen concentrations, C DO = 5, 10, 15, 20 and 40 % in the fermentation broth, by using predetermined exponential glucose feeding with μ o = 0.15 h(-1). The highest cell concentration was obtained as 44 g L(-1) at t = 9 h of the glucose fed-batch phase at C DO = 20 % operation while the highest volumetric and specific enzyme activities were obtained as 4440 U L(-1) and 126 U g(-1) cell, respectively at C DO = 15 % operation. Investigation of specific enzyme activities revealed that keeping C DO at 15 % was more advantageous with an expense of relatively higher by-product formation and lower specific cell growth rate. For this strategy, the highest oxygen transfer coefficient and oxygen uptake rate were K L a = 0.045 s(-1) and OUR = 8.91 mmol m(-3) s(-1), respectively. PMID:27003824

  5. Misfolded forms of glyceraldehyde-3-phosphate dehydrogenase interact with GroEL and inhibit chaperonin-assisted folding of the wild-type enzyme.

    PubMed

    Polyakova, Oxana V; Roitel, Olivier; Asryants, Regina A; Poliakov, Alexei A; Branlant, Guy; Muronetz, Vladimir I

    2005-04-01

    We studied the interaction of chaperonin GroEL with different misfolded forms of tetrameric phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH): (1) GAPDH from rabbit muscles with all SH-groups modified by 5,5'-dithiobis(2-nitrobenzoate); (2) O-R-type dimers of mutant GAPDH from Bacillus stearothermophilus with amino acid substitutions Y283V, D282G, and Y283V/W84F, and (3) O-P-type dimers of mutant GAPDH from B. stearothermophilus with amino acid substitutions Y46G/S48G and Y46G/R52G. It was shown that chemically modified GAPDH and the O-R-type mutant dimers bound to GroEL with 1:1 stoichiometry and dissociation constants K(d) of 0.4 and 0.9 muM, respectively. A striking feature of the resulting complexes with GroEL was their stability in the presence of Mg-ATP. Chemically modified GAPDH and the O-R-type mutant dimers inhibited GroEL-assisted refolding of urea-denatured wild-type GAPDH from B. stearothermophilus but did not affect its spontaneous reactivation. In contrast to the O-R-dimers, the O-P-type mutant dimers neither bound nor affected GroEL-assisted refolding of the wild-type GAPDH. Thus, we suggest that interaction of GroEL with certain types of misfolded proteins can result in the formation of stable complexes and the impairment of chaperonin activity. PMID:15741339

  6. Misfolded forms of glyceraldehyde-3-phosphate dehydrogenase interact with GroEL and inhibit chaperonin-assisted folding of the wild-type enzyme

    PubMed Central

    Polyakova, Oxana V.; Roitel, Olivier; Asryants, Regina A.; Poliakov, Alexei A.; Branlant, Guy; Muronetz, Vladimir I.

    2005-01-01

    We studied the interaction of chaperonin GroEL with different misfolded forms of tetrameric phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH): (1) GAPDH from rabbit muscles with all SH-groups modified by 5,5′-dithiobis(2-nitrobenzoate); (2) O-R-type dimers of mutant GAPDH from Bacillus stearothermophilus with amino acid substitutions Y283V, D282G, and Y283V/W84F, and (3) O-P-type dimers of mutant GAPDH from B. stearothermophilus with amino acid substitutions Y46G/S48G and Y46G/R52G. It was shown that chemically modified GAPDH and the O-R-type mutant dimers bound to GroEL with 1:1 stoichiometry and dissociation constants Kd of 0.4 and 0.9 μM, respectively. A striking feature of the resulting complexes with GroEL was their stability in the presence of Mg-ATP. Chemically modified GAPDH and the O-R-type mutant dimers inhibited GroEL-assisted refolding of urea-denatured wild-type GAPDH from B. stearothermophilus but did not affect its spontaneous reactivation. In contrast to the O-R-dimers, the O-P-type mutant dimers neither bound nor affected GroEL-assisted refolding of the wild-type GAPDH. Thus, we suggest that interaction of GroEL with certain types of misfolded proteins can result in the formation of stable complexes and the impairment of chaperonin activity. PMID:15741339

  7. Glycerol-3-Phosphate Levels Are Associated with Basal Resistance to the Hemibiotrophic Fungus Colletotrichum higginsianum in Arabidopsis1[W][OA

    PubMed Central

    Chanda, Bidisha; Venugopal, Srivathsa C.; Kulshrestha, Saurabh; Navarre, Duroy A.; Downie, Bruce; Vaillancourt, Lisa; Kachroo, Aardra; Kachroo, Pradeep

    2008-01-01

    Glycerol-3-phosphate (G3P) is an important component of carbohydrate and lipid metabolic processes. In this article, we provide evidence that G3P levels in plants are associated with defense to a hemibiotrophic fungal pathogen Colletotrichum higginsianum. Inoculation of Arabidopsis (Arabidopsis thaliana) with C. higginsianum was correlated with an increase in G3P levels and a concomitant decrease in glycerol levels in the host. Plants impaired in utilization of plastidial G3P (act1) accumulated elevated levels of pathogen-induced G3P and displayed enhanced resistance. Furthermore, overexpression of the host GLY1 gene, which encodes a G3P dehydrogenase (G3Pdh), conferred enhanced resistance. In contrast, the gly1 mutant accumulated reduced levels of G3P after pathogen inoculation and showed enhanced susceptibility to C. higginsianum. Unlike gly1, a mutation in a cytosolic isoform of G3Pdh did not alter basal resistance to C. higginsianum. Furthermore, act1 gly1 double-mutant plants were as susceptible as the gly1 plants. Increased resistance or susceptibility of act1 and gly1 plants to C. higginsianum, respectively, was not due to effects of these mutations on salicylic acid- or ethylene-mediated defense pathways. The act1 mutation restored a wild-type-like response in camalexin-deficient pad3 plants, which were hypersusceptible to C. higginsianum. These data suggest that G3P-associated resistance to C. higginsianum occurs independently or downstream of the camalexin pathway. Together, these results suggest a novel and specific link between G3P metabolism and plant defense. PMID:18567828

  8. Mutation in Glycerol-3-Phosphate Dehydrogenase 1–Like Gene (GPD1-L) Decreases Cardiac Na+ Current and Causes Inherited Arrhythmias

    PubMed Central

    London, Barry; Michalec, Michael; Mehdi, Haider; Zhu, Xiaodong; Kerchner, Laurie; Sanyal, Shamarendra; Viswanathan, Prakash C.; Pfahnl, Arnold E.; Shang, Lijuan L.; Madhusudanan, Mohan; Baty, Catherine J.; Lagana, Stephen; Aleong, Ryan; Gutmann, Rebecca; Ackerman, Michael J.; McNamara, Dennis M.; Weiss, Raul; Dudley, Samuel C.

    2011-01-01

    Background Brugada syndrome is a rare, autosomal-dominant, male-predominant form of idiopathic ventricular fibrillation characterized by a right bundle-branch block and ST elevation in the right precordial leads of the surface ECG. Mutations in the cardiac Na+ channel SCN5A on chromosome 3p21 cause ≈20% of the cases of Brugada syndrome; most mutations decrease inward Na+ current, some by preventing trafficking of the channels to the surface membrane. We previously used positional cloning to identify a new locus on chromosome 3p24 in a large family with Brugada syndrome and excluded SCN5A as a candidate gene. Methods and Results We used direct sequencing to identify a mutation (A280V) in a conserved amino acid of the glycerol-3-phosphate dehydrogenase 1–like (GPD1-L) gene. The mutation was present in all affected individuals and absent in >500 control subjects. GPD1-L RNA and protein are abundant in the heart. Compared with wild-type GPD1-L, coexpression of A280V GPD1-L with SCN5A in HEK cells reduced inward Na+ currents by ≈50% (P<0.005). Wild-type GPD1-L localized near the cell surface to a greater extent than A280V GPD1-L. Coexpression of A280V GPD1-L with SCN5A reduced SCN5A cell surface expression by 31±5% (P=0.01). Conclusions GPD1-L is a novel gene that may affect trafficking of the cardiac Na+ channel to the cell surface. A GPD1-L mutation decreases SCN5A surface membrane expression, reduces inward Na+ current, and causes Brugada syndrome. PMID:17967977

  9. Identification of novel sesterterpene/triterpene synthase from Bacillus clausii.

    PubMed

    Sato, Tsutomu; Yamaga, Hiroaki; Kashima, Shoji; Murata, Yusuke; Shinada, Tetsuro; Nakano, Chiaki; Hoshino, Tsutomu

    2013-05-10

    Basic enzyme: The tetraprenyl-β-curcumene synthase homologue from the alkalophilic Bacillus clausii catalyses conversions of a geranylfarnesyl diphosphate and a hexaprenyl diphosphate into novel head-to-tail acyclic sesterterpene and triterpene. Tetraprenyl-β-curcumene synthase homologues represent a new family of terpene synthases that form not only sesquarterpene but also sesterterpene and triterpene. PMID:23554321

  10. Lessons from 455 Fusarium polyketide synthases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In fungi, polyketide synthases (PKSs) synthesize a structurally diverse array of secondary metabolites (SMs) with a range of biological activities. The most studied SMs are toxic to animals and/or plants, alter plant growth, have beneficial pharmaceutical activities, and/or are brightly colored pigm...

  11. Producing dicarboxylic acids using polyketide synthases

    SciTech Connect

    Katz, Leonard; Fortman, Jeffrey L; Keasling, Jay D

    2013-10-29

    The present invention provides for a polyketide synthase (PKS) capable of synthesizing a dicarboxylic acid (diacid). Such diacids include diketide-diacids and triketide-diacids. The invention includes recombinant nucleic acid encoding the PKS, and host cells comprising the PKS. The invention also includes methods for producing the diacids.

  12. Producing dicarboxylic acids using polyketide synthases

    SciTech Connect

    Katz, Leonard; Fortman, Jeffrey L.; Keasling, Jay D.

    2015-05-26

    The present invention provides for a polyketide synthase (PKS) capable of synthesizing a dicarboxylic acid (diacid). Such diacids include diketide-diacids and triketide-diacids. The invention includes recombinant nucleic acid encoding the PKS, and host cells comprising the PKS. The invention also includes methods for producing the diacids.

  13. BIOGENESIS FACTOR REQUIRED FOR ATP SYNTHASE 3 Facilitates Assembly of the Chloroplast ATP Synthase Complex.

    PubMed

    Zhang, Lin; Duan, Zhikun; Zhang, Jiao; Peng, Lianwei

    2016-06-01

    Thylakoid membrane-localized chloroplast ATP synthases use the proton motive force generated by photosynthetic electron transport to produce ATP from ADP. Although it is well known that the chloroplast ATP synthase is composed of more than 20 proteins with α3β3γ1ε1δ1I1II1III14IV1 stoichiometry, its biogenesis process is currently unclear. To unravel the molecular mechanisms underlying the biogenesis of chloroplast ATP synthase, we performed extensive screening for isolating ATP synthase mutants in Arabidopsis (Arabidopsis thaliana). In the recently identified bfa3 (biogenesis factors required for ATP synthase 3) mutant, the levels of chloroplast ATP synthase subunits were reduced to approximately 25% of wild-type levels. In vivo labeling analysis showed that assembly of the CF1 component of chloroplast ATP synthase was less efficient in bfa3 than in the wild type, indicating that BFA3 is required for CF1 assembly. BFA3 encodes a chloroplast stromal protein that is conserved in higher plants, green algae, and a few species of other eukaryotic algae, and specifically interacts with the CF1β subunit. The BFA3 binding site was mapped to a region in the catalytic site of CF1β. Several residues highly conserved in eukaryotic CF1β are crucial for the BFA3-CF1β interaction, suggesting a coevolutionary relationship between BFA3 and CF1β. BFA3 appears to function as a molecular chaperone that transiently associates with unassembled CF1β at its catalytic site and facilitates subsequent association with CF1α during assembly of the CF1 subcomplex of chloroplast ATP synthase. PMID:27208269

  14. Trichinella pseudospiralis vs. T. spiralis thymidylate synthase gene structure and T. pseudospiralis thymidylate synthase retrogene sequence

    PubMed Central

    2014-01-01

    Background Thymidylate synthase is a housekeeping gene, designated ancient due to its role in DNA synthesis and ubiquitous phyletic distribution. The genomic sequences were characterized coding for thymidylate synthase in two species of the genus Trichinella, an encapsulating T. spiralis and a non-encapsulating T. pseudospiralis. Methods Based on the sequence of parasitic nematode Trichinella spiralis thymidylate synthase cDNA, PCR techniques were employed. Results Each of the respective gene structures encompassed 6 exons and 5 introns located in conserved sites. Comparison with the corresponding gene structures of other eukaryotic species revealed lack of common introns that would be shared among selected fungi, nematodes, mammals and plants. The two deduced amino acid sequences were 96% identical. In addition to the thymidylate synthase gene, the intron-less retrocopy, i.e. a processed pseudogene, with sequence identical to the T. spiralis gene coding region, was found to be present within the T. pseudospiralis genome. This pseudogene, instead of the gene, was confirmed by RT-PCR to be expressed in the parasite muscle larvae. Conclusions Intron load, as well as distribution of exon and intron phases in thymidylate synthase genes from various sources, point against the theory of gene assembly by the primordial exon shuffling and support the theory of evolutionary late intron insertion into spliceosomal genes. Thymidylate synthase pseudogene expressed in T. pseudospiralis muscle larvae is designated a retrogene. PMID:24716800

  15. Geranyl diphosphate synthase large subunit, and methods of use

    DOEpatents

    Croteau, Rodney B.; Burke, Charles C.; Wildung, Mark R.

    2001-10-16

    A cDNA encoding geranyl diphosphate synthase large subunit from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase large subunit). In another aspect, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase large subunit. In yet another aspect, the present invention provides isolated, recombinant geranyl diphosphate synthase protein comprising an isolated, recombinant geranyl diphosphate synthase large subunit protein and an isolated, recombinant geranyl diphosphate synthase small subunit protein. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase.

  16. The cellulose synthase companion proteins act non-redundantly with CELLULOSE SYNTHASE INTERACTING1/POM2 and CELLULOSE SYNTHASE 6

    PubMed Central

    Endler, Anne; Schneider, Rene; Kesten, Christopher; Lampugnani, Edwin R.; Persson, Staffan

    2016-01-01

    ABSTRACT Cellulose is a cell wall constituent that is essential for plant growth and development, and an important raw material for a range of industrial applications. Cellulose is synthesized at the plasma membrane by massive cellulose synthase (CesA) complexes that track along cortical microtubules in elongating cells of Arabidopsis through the activity of the protein CELLULOSE SYNTHASE INTERACTING1 (CSI1). In a recent study we identified another family of proteins that also are associated with the CesA complex and microtubules, and that we named COMPANIONS OF CELLULOSE SYNTHASE (CC). The CC proteins protect the cellulose synthesising capacity of Arabidopsis seedlings during exposure to adverse environmental conditions by enhancing microtubule dynamics. In this paper we provide cell biology and genetic evidence that the CSI1 and the CC proteins fulfil distinct functions during cellulose synthesis. We also show that the CC proteins are necessary to aid cellulose synthesis when components of the CesA complex are impaired. These data indicate that the CC proteins have a broad role in aiding cellulose synthesis during environmental changes and when core complex components are non-functional. PMID:26829351

  17. (R)-citramalate synthase in methanogenic archaea.

    PubMed

    Howell, D M; Xu, H; White, R H

    1999-01-01

    The Methanococcus jannaschii gene MJ1392 was cloned, and its protein product was hyperexpressed in Escherichia coli. The resulting protein was purified and shown to catalyze the condensation of pyruvate and acetyl coenzyme A, with the formation of (R)-citramalate. Thus, this gene (cimA) encodes an (R)-citramalate synthase (CimA). This is the first identification of this enzyme, which is likely involved in the biosynthesis of isoleucine. PMID:9864346

  18. Chrysanthemyl Diphosphate Synthase Operates in Planta as a Bifunctional Enzyme with Chrysanthemol Synthase Activity*

    PubMed Central

    Yang, Ting; Gao, Liping; Hu, Hao; Stoopen, Geert; Wang, Caiyun; Jongsma, Maarten A.

    2014-01-01

    Chrysanthemyl diphosphate synthase (CDS) is the first pathway-specific enzyme in the biosynthesis of pyrethrins, the most widely used plant-derived pesticide. CDS catalyzes c1′-2-3 cyclopropanation reactions of two molecules of dimethylallyl diphosphate (DMAPP) to yield chrysanthemyl diphosphate (CPP). Three proteins are known to catalyze this cyclopropanation reaction of terpene precursors. Two of them, phytoene and squalene synthase, are bifunctional enzymes with both prenyltransferase and terpene synthase activity. CDS, the other member, has been reported to perform only the prenyltransferase step. Here we show that the NDXXD catalytic motif of CDS, under the lower substrate conditions prevalent in plants, also catalyzes the next step, converting CPP into chrysanthemol by hydrolyzing the diphosphate moiety. The enzymatic hydrolysis reaction followed conventional Michaelis-Menten kinetics, with a Km value for CPP of 196 μm. For the chrysanthemol synthase activity, DMAPP competed with CPP as substrate. The DMAPP concentration required for half-maximal activity to produce chrysanthemol was ∼100 μm, and significant substrate inhibition was observed at elevated DMAPP concentrations. The N-terminal peptide of CDS was identified as a plastid-targeting peptide. Transgenic tobacco plants overexpressing CDS emitted chrysanthemol at a rate of 0.12–0.16 μg h−1 g−1 fresh weight. We propose that CDS should be renamed a chrysanthemol synthase utilizing DMAPP as substrate. PMID:25378387

  19. Chrysanthemyl diphosphate synthase operates in planta as a bifunctional enzyme with chrysanthemol synthase activity.

    PubMed

    Yang, Ting; Gao, Liping; Hu, Hao; Stoopen, Geert; Wang, Caiyun; Jongsma, Maarten A

    2014-12-26

    Chrysanthemyl diphosphate synthase (CDS) is the first pathway-specific enzyme in the biosynthesis of pyrethrins, the most widely used plant-derived pesticide. CDS catalyzes c1'-2-3 cyclopropanation reactions of two molecules of dimethylallyl diphosphate (DMAPP) to yield chrysanthemyl diphosphate (CPP). Three proteins are known to catalyze this cyclopropanation reaction of terpene precursors. Two of them, phytoene and squalene synthase, are bifunctional enzymes with both prenyltransferase and terpene synthase activity. CDS, the other member, has been reported to perform only the prenyltransferase step. Here we show that the NDXXD catalytic motif of CDS, under the lower substrate conditions prevalent in plants, also catalyzes the next step, converting CPP into chrysanthemol by hydrolyzing the diphosphate moiety. The enzymatic hydrolysis reaction followed conventional Michaelis-Menten kinetics, with a Km value for CPP of 196 μm. For the chrysanthemol synthase activity, DMAPP competed with CPP as substrate. The DMAPP concentration required for half-maximal activity to produce chrysanthemol was ∼100 μm, and significant substrate inhibition was observed at elevated DMAPP concentrations. The N-terminal peptide of CDS was identified as a plastid-targeting peptide. Transgenic tobacco plants overexpressing CDS emitted chrysanthemol at a rate of 0.12-0.16 μg h(-1) g(-1) fresh weight. We propose that CDS should be renamed a chrysanthemol synthase utilizing DMAPP as substrate. PMID:25378387

  20. CTP synthase forms cytoophidia in the cytoplasm and nucleus

    SciTech Connect

    Gou, Ke-Mian; Chang, Chia-Chun; Shen, Qing-Ji; Sung, Li-Ying; Liu, Ji-Long

    2014-04-15

    CTP synthase is an essential metabolic enzyme responsible for the de novo synthesis of CTP. Multiple studies have recently showed that CTP synthase protein molecules form filamentous structures termed cytoophidia or CTP synthase filaments in the cytoplasm of eukaryotic cells, as well as in bacteria. Here we report that CTP synthase can form cytoophidia not only in the cytoplasm, but also in the nucleus of eukaryotic cells. Both glutamine deprivation and glutamine analog treatment promote formation of cytoplasmic cytoophidia (C-cytoophidia) and nuclear cytoophidia (N-cytoophidia). N-cytoophidia are generally shorter and thinner than their cytoplasmic counterparts. In mammalian cells, both CTP synthase 1 and CTP synthase 2 can form cytoophidia. Using live imaging, we have observed that both C-cytoophidia and N-cytoophidia undergo multiple rounds of fusion upon glutamine analog treatment. Our study reveals the coexistence of cytoophidia in the cytoplasm and nucleus, therefore providing a good opportunity to investigate the intracellular compartmentation of CTP synthase. - Highlights: • CTP synthase forms cytoophidia not only in the cytoplasm but also in the nucleus. • Glutamine deprivation and Glutamine analogs promotes cytoophidium formation. • N-cytoophidia exhibit distinct morphology when compared to C-cytoophidia. • Both CTP synthase 1 and CTP synthase 2 form cytoophidia in mammalian cells. • Fusions of cytoophidia occur in the cytoplasm and nucleus.

  1. Oligosaccharide Binding in Escherichia coli Glycogen Synthase

    SciTech Connect

    Sheng, Fang; Yep, Alejandra; Feng, Lei; Preiss, Jack; Geiger, James H.

    2010-11-17

    Glycogen/starch synthase elongates glucan chains and is the key enzyme in the synthesis of glycogen in bacteria and starch in plants. Cocrystallization of Escherichia coli wild-type glycogen synthase (GS) with substrate ADPGlc and the glucan acceptor mimic HEPPSO produced a closed form of GS and suggests that domain-domain closure accompanies glycogen synthesis. Cocrystallization of the inactive GS mutant E377A with substrate ADPGlc and oligosaccharide results in the first oligosaccharide-bound glycogen synthase structure. Four bound oligosaccharides are observed, one in the interdomain cleft (G6a) and three on the N-terminal domain surface (G6b, G6c, and G6d). Extending from the center of the enzyme to the interdomain cleft opening, G6a mostly interacts with the highly conserved N-terminal domain residues lining the cleft of GS. The surface-bound oligosaccharides G6c and G6d have less interaction with enzyme and exhibit a more curled, helixlike structural arrangement. The observation that oligosaccharides bind only to the N-terminal domain of GS suggests that glycogen in vivo probably binds to only one side of the enzyme to ensure unencumbered interdomain movement, which is required for efficient, continuous glucan-chain synthesis.

  2. Thermostable malate synthase of Streptomyces thermovulgaris.

    PubMed

    Goh, L L; Koh, R; Loke, P; Sim, T S

    2003-10-01

    The gene, encoding malate synthase (MS), aceB, was cloned from the thermophilic bacterium Streptomyces thermovulgaris by homology-based PCR. The 1,626-bp cloned fragment encodes a protein consisting of 541 amino acids. S. thermovulgaris malate synthase (stMS) gene was over-expressed in Escherichia coli using a glutathione-S transferase (GST) fusion vector (pGEX-6P-1), purified by affinity chromatography, and subsequently cleaved from its GST fusion partner. The purified stMS was characterized and compared to a mesophilic malate synthase (scMS) from Streptomyces coelicolor. stMS exhibited higher temperature optima (40-60 degrees C) than those of scMS (28-37 degrees C). It was more thermostable and very resistant to the chemical denaturant urea. Amino acid sequence comparison of stMS with four mesophilic streptomycete MSs indicated that they share 70.9-91.4% amino acid identities, with stMS possessing slightly more charged residues (approximately 31%) than its mesophilic counterparts (approximately 28-29%). Seven charged residues (E85, R187, R209, H239, H364, R382 and K520) that were unique to stMS may be selectively and strategically placed to support its peculiar characteristics. PMID:13680388

  3. Structure of a modular polyketide synthase

    PubMed Central

    Dutta, Somnath; Whicher, Jonathan R.; Hansen, Douglas A.; Hale, Wendi A.; Chemler, Joseph A.; Congdon, Grady R.; Narayan, Alison R.; Håkansson, Kristina; Sherman, David H.; Smith, Janet L.

    2014-01-01

    Polyketide natural products constitute a broad class of compounds with diverse structural features and biological activities. Their biosynthetic machinery, represented by type I polyketide synthases, has an architecture in which successive modules catalyze two-carbon linear extensions and keto group processing reactions on intermediates covalently tethered to carrier domains. We employed electron cryo-microscopy to visualize a full-length module and determine sub-nanometer resolution 3D reconstructions that revealed an unexpectedly different architecture compared to the homologous dimeric mammalian fatty acid synthase. A single reaction chamber provides access to all catalytic sites for the intra-module carrier domain. In contrast, the carrier from the preceding module uses a separate entrance outside the reaction chamber to deliver the upstream polyketide intermediate for subsequent extension and modification. This study reveals for the first time the structural basis for both intra-module and inter-module substrate transfer in polyketide synthases, and establishes a new model for molecular dissection of these multifunctional enzyme systems. PMID:24965652

  4. Genomic organization of plant terpene synthases and molecular evolutionary implications.

    PubMed Central

    Trapp, S C; Croteau, R B

    2001-01-01

    Terpenoids are the largest, most diverse class of plant natural products and they play numerous functional roles in primary metabolism and in ecological interactions. The first committed step in the formation of the various terpenoid classes is the transformation of the prenyl diphosphate precursors, geranyl diphosphate, farnesyl diphosphate, and geranylgeranyl diphosphate, to the parent structures of each type catalyzed by the respective monoterpene (C(10)), sesquiterpene (C(15)), and diterpene synthases (C(20)). Over 30 cDNAs encoding plant terpenoid synthases involved in primary and secondary metabolism have been cloned and characterized. Here we describe the isolation and analysis of six genomic clones encoding terpene synthases of conifers, [(-)-pinene (C(10)), (-)-limonene (C(10)), (E)-alpha-bisabolene (C(15)), delta-selinene (C(15)), and abietadiene synthase (C(20)) from Abies grandis and taxadiene synthase (C(20)) from Taxus brevifolia], all of which are involved in natural products biosynthesis. Genome organization (intron number, size, placement and phase, and exon size) of these gymnosperm terpene synthases was compared to eight previously characterized angiosperm terpene synthase genes and to six putative terpene synthase genomic sequences from Arabidopsis thaliana. Three distinct classes of terpene synthase genes were discerned, from which assumed patterns of sequential intron loss and the loss of an unusual internal sequence element suggest that the ancestral terpenoid synthase gene resembled a contemporary conifer diterpene synthase gene in containing at least 12 introns and 13 exons of conserved size. A model presented for the evolutionary history of plant terpene synthases suggests that this superfamily of genes responsible for natural products biosynthesis derived from terpene synthase genes involved in primary metabolism by duplication and divergence in structural and functional specialization. This novel molecular evolutionary approach focused

  5. Rational Design of Nanoparticle Platforms for "Cutting-the-Fat": Covalent Immobilization of Lipase, Glycerol Kinase, and Glycerol-3-Phosphate Oxidase on Metal Nanoparticles.

    PubMed

    Aggarwal, V; Pundir, C S

    2016-01-01

    The aggregates of nanoparticles (NPs) are considered better supports for the immobilization of enzymes, as these promote enzyme kinetics, due to their unusual but favorable properties such as larger surface area to volume ratio, high catalytic efficiency of certain immobilized enzymes, non-toxicity of some of the nanoparticle matrices, high stability, strong adsorption of the enzyme of interest by a number of different approaches, and faster electron transportability. Co-immobilization of multiple enzymes required for a multistep reaction cascade on a single support is more efficient than separately immobilizing the corresponding enzymes and mixing them physically, since products of one enzyme could serve as reactants for another. These products can diffuse much more easily between enzymes on the same particle than diffusion from one particle to the next, in the reaction medium. Thus, co-immobilization of enzymes onto NP aggregates is expected to produce faster kinetics than their individual immobilizations on separate matrices. Lipase, glycerol kinase, and glycerol-3-phosphate oxidase are required for lipid analysis in a cascade reaction, and we describe the co-immobilization of these three enzymes on nanocomposites of zinc oxide nanoparticles (ZnONPs)-chitosan (CHIT) and gold nanoparticles-polypyrrole-polyindole carboxylic acid (AuPPy-Pin5COOH) which are electrodeposited on Pt and Au electrodes, respectively. The kinetic properties and analytes used for amperometric determination of TG are fully described for others to practice in a trained laboratory. Cyclic voltammetry, scanning electron microscopy, Fourier transform infra-red spectra, and electrochemical impedance spectra confirmed their covalent co-immobilization onto electrode surfaces through glutaraldehyde coupling on CHIT-ZnONPs and amide bonding on AuPPy/Pin5COOH. The combined activities of co-immobilized enzymes was tested amperometrically, and these composite nanobiocatalysts showed optimum activity

  6. Geranyl diphosphate synthase molecules, and nucleic acid molecules encoding same

    SciTech Connect

    Croteau, Rodney Bruce; Burke, Charles Cullen

    2008-06-24

    In one aspect, the present invention provides isolated nucleic acid molecules that each encode a geranyl diphosphate synthase protein, wherein each isolated nucleic acid molecule hybridizes to a nucleic acid molecule consisting of the sequence set forth in SEQ ID NO:1 under conditions of 5.times.SSC at 45.degree. C. for one hour. The present invention also provides isolated geranyl diphosphate synthase proteins, and methods for altering the level of expression of geranyl diphosphate synthase protein in a host cell.

  7. Regulators of floral fragrance production and their target genes in petunia are not exclusively active in the epidermal cells of petals.

    PubMed

    Van Moerkercke, Alex; Galván-Ampudia, Carlos S; Verdonk, Julian C; Haring, Michel A; Schuurink, Robert C

    2012-05-01

    In which cells of the flower volatile biosynthesis takes place is unclear. In rose and snapdragon, some enzymes of the volatile phenylpropanoid/benzenoid pathway have been shown to be present in the epidermal cells of petals. It is therefore generally believed that the production of these compounds occurs in these cells. However, whether the entire pathway is active in these cells and whether it is exclusively active in these cells remains to be proven. Cell-specific transcription factors activating these genes will determine in which cells they are expressed. In petunia, the transcription factor EMISSION OF BENZENOIDS II (EOBII) activates the ODORANT1 (ODO1) promoter and the promoter of the biosynthetic gene isoeugenol synthase (IGS). The regulator ODO1 in turn activates the promoter of the shikimate gene 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Here the identification of a new target gene of ODO1, encoding an ABC transporter localized on the plasma membrane, PhABCG1, which is co-expressed with ODO1, is described. PhABCG1 expression is up-regulated in petals overexpressing ODO1 through activation of the PhABCG1 promoter. Interestingly, the ODO1, PhABCG1, and IGS promoters were active in petunia protoplasts originating from both epidermal and mesophyll cell layers of the petal, suggesting that the volatile phenylpropanoid/benzenoid pathway in petunia is active in these different cell types. Since volatile release occurs from epidermal cells, trafficking of (volatile) compounds between cell layers must be involved, but the exact function of PhABCG1 remains to be resolved. PMID:22345641

  8. Metabonomics classifies pathways affected by bioactive compounds. Artificial neural network classification of NMR spectra of plant extracts.

    PubMed

    Ott, Karl-Heinz; Araníbar, Nelly; Singh, Bijay; Stockton, Gerald W

    2003-03-01

    The biochemical mode-of-action (MOA) for herbicides and other bioactive compounds can be rapidly and simultaneously classified by automated pattern recognition of the metabonome that is embodied in the 1H NMR spectrum of a crude plant extract. The ca. 300 herbicides that are used in agriculture today affect less than 30 different biochemical pathways. In this report, 19 of the most interesting MOAs were automatically classified. Corn (Zea mays) plants were treated with various herbicides such as imazethapyr, glyphosate, sethoxydim, and diuron, which represent various biochemical modes-of-action such as inhibition of specific enzymes (acetohydroxy acid synthase [AHAS], protoporphyrin IX oxidase [PROTOX], 5-enolpyruvylshikimate-3-phosphate synthase [EPSPS], acetyl CoA carboxylase [ACC-ase], etc.), or protein complexes (photosystems I and II), or major biological process such as oxidative phosphorylation, auxin transport, microtubule growth, and mitosis. Crude isolates from the treated plants were subjected to 1H NMR spectroscopy, and the spectra were classified by artificial neural network analysis to discriminate the herbicide modes-of-action. We demonstrate the use and refinement of the method, and present cross-validated assignments for the metabolite NMR profiles of over 400 plant isolates. The MOA screen also recognizes when a new mode-of-action is present, which is considered extremely important for the herbicide discovery process, and can be used to study deviations in the metabolism of compounds from a chemical synthesis program. The combination of NMR metabolite profiling and neural network classification is expected to be similarly relevant to other metabonomic profiling applications, such as in drug discovery. PMID:12590124

  9. β-Glucan Synthase Gene Overexpression and β-Glucans Overproduction in Pleurotus ostreatus Using Promoter Swapping

    PubMed Central

    Liu, Dongren; Qi, Yuancheng; Gao, Yuqian; Shen, Jinwen; Qiu, Liyou

    2013-01-01

    Mushroom β-glucans are potent immunological stimulators in medicine, but their productivities are very low. In this study, we successfully improved its production by promoter engineering in Pleurotus ostreatus. The promoter for β-1,3-glucan synthase gene (GLS) was replaced by the promoter of glyceraldehyde-3-phosphate dehydrogenase gene of Aspergillus nidulans. The homologous recombination fragment for swapping GLS promoter comprised five segments, which were fused by two rounds of combined touchdown PCR and overlap extension PCR (TD-OE PCR), and was introduced into P. ostreatus through PEG/CaCl2-mediated protoplast transformation. The transformants exhibited one to three fold higher transcription of GLS gene and produced 32% to 131% higher yield of β-glucans than the wild type. The polysaccharide yields had a significant positive correlation to the GLS gene expression. The infrared spectra of the polysaccharides all displayed the typical absorption peaks of β-glucans. This is the first report of successful swapping of promoters in filamentous fungi. PMID:23637884

  10. Divinyl ether synthase gene and protein, and uses thereof

    DOEpatents

    Howe, Gregg A.; Itoh, Aya

    2011-09-13

    The present invention relates to divinyl ether synthase genes, proteins, and methods of their use. The present invention encompasses both native and recombinant wild-type forms of the synthase, as well as mutants and variant forms, some of which possess altered characteristics relative to the wild-type synthase. The present invention also relates to methods of using divinyl ether synthase genes and proteins, including in their expression in transgenic organisms and in the production of divinyl ether fatty acids, and to methods of suing divinyl ether fatty acids, including in the protection of plants from pathogens.

  11. Divinyl ether synthase gene, and protein and uses thereof

    DOEpatents

    Howe, Gregg A.; Itoh, Aya

    2006-12-26

    The present invention relates to divinyl ether synthase genes, proteins, and methods of their use. The present invention encompasses both native and recombinant wild-type forms of the synthase, as well as mutants and variant forms, some of which possess altered characteristics relative to the wild-type synthase. The present invention also relates to methods of using divinyl ether synthase genes and proteins, including in their expression in transgenic organisms and in the production of divinyl ether fatty acids, and to methods of suing divinyl ether fatty acids, including in the protection of plants from pathogens.

  12. Evolutinoary Consideration on 5-Aminolevulinate Synthase in Nature

    NASA Astrophysics Data System (ADS)

    Oh-Hama, Tamiko

    1997-08-01

    5-Aminolevulinic acid (ALA), a universal precursor of tetrapyrrole compounds can be synthesized by two pathways: the C5 (glutamate) pathway and ALA synthase. From the phylogenetic distribution it is shown that distribution of ALA synthase is restricted to the α subclass of purple bacteria in prokaryotes, and further distributed to mitochondria of eukaryotes. The monophyletic origin of bacterial and eukaryotic ALA synthase is shown by sequence analysis of the enzyme. Evolution of ALA synthase in the α subclass of purple bacteria is discussed in relation to the energy-generating and biosynthetic devices in subclasses of this bacteria.

  13. Vitis vinifera terpenoid cyclases: functional identification of two sesquiterpene synthase cDNAs encoding (+)-valencene synthase and (-)-germacrene D synthase and expression of mono- and sesquiterpene synthases in grapevine flowers and berries.

    PubMed

    Lücker, Joost; Bowen, Pat; Bohlmann, Jörg

    2004-10-01

    Valencene is a volatile sesquiterpene emitted from flowers of grapevine, Vitis vinifera L. A full-length cDNA from the cultivar Gewürztraminer was functionally expressed in Escherichia coli and found to encode valencene synthase (VvVal). The two major products formed by recombinant VvVal enzyme activity with farnesyl diphosphate (FPP) as substrate are (+)-valencene and (-)-7-epi-alpha-selinene. Grapevine valencene synthase is closely related to a second sesquiterpene synthase from this species, (-)-germacrene D synthase (VvGerD). VvVal and VvGerD cDNA probes revealed strong signals in Northern hybridizations with RNA isolated from grapevine flower buds. Transcript levels were lower in open pre-anthesis flowers, flowers after anthesis, or at early onset of fruit development. Similar results were obtained using a third probe, (-)-alpha-terpineol synthase, a monoterpenol synthase. Sesquiterpene synthase and monoterpene synthase transcripts were not detected in the mesocarp and exocarp during early stages of fruit development, but transcripts hybridizing with VvVal appeared during late ripening of the berries. Sesquiterpene synthase transcripts were also detected in young seeds. PMID:15464152

  14. Functional Prostacyclin Synthase Promoter Polymorphisms. Impact in Pulmonary Arterial Hypertension

    PubMed Central

    Cornelius, Amber R.; Lu, Xiao; Conklin, David S.; Del Rosario, Mark J.; Lowe, Anita M.; Elos, Mihret T.; Fettig, Lynsey M.; Wong, Randall E.; Hara, Naoko; Cogan, Joy D.; Phillips, John A.; Taylor, Matthew R.; Graham, Brian B.; Tuder, Rubin M.; Loyd, James E.; Geraci, Mark W.

    2014-01-01

    Rationale: Pulmonary arterial hypertension (PAH) is a progressive disease characterized by elevated pulmonary artery pressure, vascular remodeling, and ultimately right ventricular heart failure. PAH can have a genetic component (heritable PAH), most often through mutations of bone morphogenetic protein receptor 2, and idiopathic and associated forms. Heritable PAH is not completely penetrant within families, with approximately 20% concurrence of inactivating bone morphogenetic protein receptor 2 mutations and delayed onset of PAH disease. Because one of the treatment options is using prostacyclin analogs, we hypothesized that prostacyclin synthase promoter sequence variants associated with increased mRNA expression may play a protective role in the bone morphogenetic protein receptor 2 unaffected carriers. Objectives: To characterize the range of prostacyclin synthase promoter variants and assess their transcriptional activities in PAH-relevant cell types. To determine the distribution of prostacyclin synthase promoter variants in PAH, unaffected carriers in heritable PAH families, and control populations. Methods: Polymerase chain reaction approaches were used to genotype prostacyclin synthase promoter variants in more than 300 individuals. Prostacyclin synthase promoter haplotypes’ transcriptional activities were determined with luciferase reporter assays. Measurements and Main Results: We identified a comprehensive set of prostacyclin synthase promoter variants and tested their transcriptional activities in PAH-relevant cell types. We demonstrated differences of prostacyclin synthase promoter activities dependent on their haplotype. Conclusions: Prostacyclin synthase promoter sequence variants exhibit a range of transcriptional activities. We discovered a significant bias for more active prostacyclin synthase promoter variants in unaffected carriers as compared with affected patients with PAH. PMID:24605778

  15. Molecular characterization of the homo-phytochelatin synthase of soybean Glycine max: relation to phytochelatin synthase.

    PubMed

    Oven, Matjaz; Page, Jonathan E; Zenk, Meinhart H; Kutchan, Toni M

    2002-02-15

    The phytochelatin homologs homo-phytochelatins are heavy metal-binding peptides present in many legumes. To study the biosynthesis of these compounds, we have isolated and functionally expressed a cDNA GmhPCS1 encoding homo-phytochelatin synthase from Glycine max, a plant known to accumulate homo-phytochelatins rather than phytochelatins upon the exposure to heavy metals. The catalytic properties of GmhPCS1 were compared with the phytochelatin synthase AtPCS1 from Arabidopsis thaliana. When assayed only in the presence of glutathione, both enzymes catalyzed phytochelatin formation. GmhPCS1 accepted homoglutathione as the sole substrate for the synthesis of homo-phytochelatins whereas AtPCS1 did not. Homo-phytochelatin synthesis activity of both recombinant enzymes was significantly higher when glutathione was included in the reaction mixture. The incorporation of both glutathione and homoglutathione into homo-phytochelatin, n = 2, was demonstrated using GmhPCS1 and AtPCS1. In addition to bis(glutathionato)-metal complexes, various other metal-thiolates were shown to contribute to the activation of phytochelatin synthase. These complexes were not accepted as substrates by the enzyme, thereby suggesting that a recently proposed model of activation cannot fully explain the catalytic mechanism of phytochelatin synthase (Vatamaniuk, O. K., Mari, S., Lu, Y. P., and Rea, P. A. (2000) J. Biol. Chem. 275, 31451-31459). PMID:11706029

  16. Thymidylate synthase gene of herpesvirus ateles.

    PubMed Central

    Richter, J; Puchtler, I; Fleckenstein, B

    1988-01-01

    The putative thymidylate synthase (TS) gene of herpesvirus ateles, a T-lymphotropic tumor virus of New World primates, has a single large open reading frame encoding a polypeptide of 32.9 kilodaltons. The gene is transcribed into an unspliced 2.4-kilobase mRNA that is abundantly expressed late in virus replication. The AT-rich 5' untranslated leader sequence of TS mRNA in herpesvirus ateles-infected cells is remarkable in length (1,184 nucleotides), containing 29 minicistrons; this may indicate a role in translation regulation. Images PMID:3404583

  17. Engineering of chimeric class II polyhydroxyalkanoate synthases.

    PubMed

    Niamsiri, Nuttawee; Delamarre, Soazig C; Kim, Young-Rok; Batt, Carl A

    2004-11-01

    PHA synthase is a key enzyme involved in the biosynthesis of polyhydroxyalkanoates (PHAs). Using a combinatorial genetic strategy to create unique chimeric class II PHA synthases, we have obtained a number of novel chimeras which display improved catalytic properties. To engineer the chimeric PHA synthases, we constructed a synthetic phaC gene from Pseudomonas oleovorans (phaC1Po) that was devoid of an internal 540-bp fragment. Randomly amplified PCR products (created with primers based on conserved phaC sequences flanking the deleted internal fragment) were generated using genomic DNA isolated from soil and were substituted for the 540-bp internal region. The chimeric genes were expressed in a PHA-negative strain of Ralstonia eutropha, PHB(-)4 (DSM 541). Out of 1,478 recombinant clones screened for PHA production, we obtained five different chimeric phaC1Po genes that produced more PHA than the native phaC1Po. Chimeras S1-71, S4-8, S5-58, S3-69, and S3-44 exhibited 1.3-, 1.4-, 2.0-, 2.1-, and 3.0-fold-increased levels of in vivo activity, respectively. All of the mutants mediated the synthesis of PHAs with a slightly increased molar fraction of 3-hydroxyoctanoate; however, the weight-average molecular weights (Mw) of the PHAs in all cases remained almost the same. Based upon DNA sequence analyses, the various phaC fragments appear to have originated from Pseudomonas fluorescens and Pseudomonas aureofaciens. The amino acid sequence analyses showed that the chimeric proteins had 17 to 20 amino acid differences from the wild-type phaC1Po, and these differences were clustered in the same positions in the five chimeric clones. A threading model of PhaC1Po, developed based on homology of the enzyme to the Burkholderia glumae lipase, suggested that the amino acid substitutions found in the active chimeras were located mostly on the protein model surface. Thus, our combinatorial genetic engineering strategy proved to be broadly useful for improving the catalytic

  18. Novel family of terpene synthases evolved from trans-isoprenyl diphosphate synthases in a flea beetle

    PubMed Central

    Beran, Franziska; Rahfeld, Peter; Luck, Katrin; Nagel, Raimund; Vogel, Heiko; Wielsch, Natalie; Irmisch, Sandra; Ramasamy, Srinivasan; Gershenzon, Jonathan; Heckel, David G.; Köllner, Tobias G.

    2016-01-01

    Sesquiterpenes play important roles in insect communication, for example as pheromones. However, no sesquiterpene synthases, the enzymes involved in construction of the basic carbon skeleton, have been identified in insects to date. We investigated the biosynthesis of the sesquiterpene (6R,7S)-himachala-9,11-diene in the crucifer flea beetle Phyllotreta striolata, a compound previously identified as a male-produced aggregation pheromone in several Phyllotreta species. A (6R,7S)-himachala-9,11-diene–producing sesquiterpene synthase activity was detected in crude beetle protein extracts, but only when (Z,E)-farnesyl diphosphate [(Z,E)-FPP] was offered as a substrate. No sequences resembling sesquiterpene synthases from plants, fungi, or bacteria were found in the P. striolata transcriptome, but we identified nine divergent putative trans-isoprenyl diphosphate synthase (trans-IDS) transcripts. Four of these putative trans-IDSs exhibited terpene synthase (TPS) activity when heterologously expressed. Recombinant PsTPS1 converted (Z,E)-FPP to (6R,7S)-himachala-9,11-diene and other sesquiterpenes observed in beetle extracts. RNAi-mediated knockdown of PsTPS1 mRNA in P. striolata males led to reduced emission of aggregation pheromone, confirming a significant role of PsTPS1 in pheromone biosynthesis. Two expressed enzymes showed genuine IDS activity, with PsIDS1 synthesizing (E,E)-FPP, whereas PsIDS3 produced neryl diphosphate, (Z,Z)-FPP, and (Z,E)-FPP. In a phylogenetic analysis, the PsTPS enzymes and PsIDS3 were clearly separated from a clade of known coleopteran trans-IDS enzymes including PsIDS1 and PsIDS2. However, the exon–intron structures of IDS and TPS genes in P. striolata are conserved, suggesting that this TPS gene family evolved from trans-IDS ancestors. PMID:26936952

  19. Novel family of terpene synthases evolved from trans-isoprenyl diphosphate synthases in a flea beetle.

    PubMed

    Beran, Franziska; Rahfeld, Peter; Luck, Katrin; Nagel, Raimund; Vogel, Heiko; Wielsch, Natalie; Irmisch, Sandra; Ramasamy, Srinivasan; Gershenzon, Jonathan; Heckel, David G; Köllner, Tobias G

    2016-03-15

    Sesquiterpenes play important roles in insect communication, for example as pheromones. However, no sesquiterpene synthases, the enzymes involved in construction of the basic carbon skeleton, have been identified in insects to date. We investigated the biosynthesis of the sesquiterpene (6R,7S)-himachala-9,11-diene in the crucifer flea beetle Phyllotreta striolata, a compound previously identified as a male-produced aggregation pheromone in several Phyllotreta species. A (6R,7S)-himachala-9,11-diene-producing sesquiterpene synthase activity was detected in crude beetle protein extracts, but only when (Z,E)-farnesyl diphosphate [(Z,E)-FPP] was offered as a substrate. No sequences resembling sesquiterpene synthases from plants, fungi, or bacteria were found in the P. striolata transcriptome, but we identified nine divergent putative trans-isoprenyl diphosphate synthase (trans-IDS) transcripts. Four of these putative trans-IDSs exhibited terpene synthase (TPS) activity when heterologously expressed. Recombinant PsTPS1 converted (Z,E)-FPP to (6R,7S)-himachala-9,11-diene and other sesquiterpenes observed in beetle extracts. RNAi-mediated knockdown of PsTPS1 mRNA in P. striolata males led to reduced emission of aggregation pheromone, confirming a significant role of PsTPS1 in pheromone biosynthesis. Two expressed enzymes showed genuine IDS activity, with PsIDS1 synthesizing (E,E)-FPP, whereas PsIDS3 produced neryl diphosphate, (Z,Z)-FPP, and (Z,E)-FPP. In a phylogenetic analysis, the PsTPS enzymes and PsIDS3 were clearly separated from a clade of known coleopteran trans-IDS enzymes including PsIDS1 and PsIDS2. However, the exon-intron structures of IDS and TPS genes in P. striolata are conserved, suggesting that this TPS gene family evolved from trans-IDS ancestors. PMID:26936952

  20. Torque generation mechanism of ATP synthase

    NASA Astrophysics Data System (ADS)

    Miller, John; Maric, Sladjana; Scoppa, M.; Cheung, M.

    2010-03-01

    ATP synthase is a rotary motor that produces adenosine triphosphate (ATP), the chemical currency of life. Our proposed electric field driven torque (EFT) model of FoF1-ATP synthase describes how torque, which scales with the number of c-ring proton binding sites, is generated by the proton motive force (pmf) across the mitochondrial inner membrane. When Fo is coupled to F1, the model predicts a critical pmf to drive ATP production. In order to fully understand how the electric field resulting from the pmf drives the c-ring to rotate, it is important to examine the charge distributions in the protonated c-ring and a-subunit containing the proton channels. Our calculations use a self-consistent field approach based on a refinement of reported structural data. The results reveal changes in pKa for key residues on the a-subunit and c-ring, as well as titration curves and protonation state energy diagrams. Health implications will be briefly discussed.

  1. ATP synthase: a tentative structural model.

    PubMed

    Engelbrecht, S; Junge, W

    1997-09-15

    Adenosine triphosphate (ATP) synthase produces ATP from ADP and inorganic phosphate at the expense of proton- or sodium-motive force across the respective coupling membrane in Archaea, Bacteria and Eucarya. Cation flow through the intrinsic membrane portion of this enzyme (Fo, subunits ab2c9-12) and substrate turnover in the headpiece (F1, subunits alpha3beta3 gammadeltaepsilon) are mechanically coupled by the rotation of subunit gamma in the center of the catalytic hexagon of subunits (alphabeta)3 in F1. ATP synthase is the smallest rotatory engine in nature. With respect to the headpiece alone, it probably operates with three steps. Partial structures of six out of its at least eight different subunits have been published and a 3-dimensional structure is available for the assembly (alphabeta)3gamma. In this article, we review the available structural data and build a tentative topological model of the holoenzyme. The rotor portion is proposed to consist of a wheel of at least nine copies of subunits c, epsilon and a portion of gamma as a spoke, and another portion of gamma as a crankshaft. The stator is made up from a, the transmembrane portion of b2, delta and the catalytic hexagon of (alphabeta)3. As an educated guess, the model may be of heuristic value for ongoing studies on this fascinating electrochemical-to-mechanical-to-chemical transducer. PMID:9323021

  2. Loss of LRPPRC causes ATP synthase deficiency.

    PubMed

    Mourier, Arnaud; Ruzzenente, Benedetta; Brandt, Tobias; Kühlbrandt, Werner; Larsson, Nils-Göran

    2014-05-15

    Defects of the oxidative phosphorylation system, in particular of cytochrome-c oxidase (COX, respiratory chain complex IV), are common causes of Leigh syndrome (LS), which is a rare neurodegenerative disorder with severe progressive neurological symptoms that usually present during infancy or early childhood. The COX-deficient form of LS is commonly caused by mutations in genes encoding COX assembly factors, e.g. SURF1, SCO1, SCO2 or COX10. However, other mutations affecting genes that encode proteins not directly involved in COX assembly can also cause LS. The leucine-rich pentatricopeptide repeat containing protein (LRPPRC) regulates mRNA stability, polyadenylation and coordinates mitochondrial translation. In humans, mutations in Lrpprc cause the French Canadian type of LS. Despite the finding that LRPPRC deficiency affects the stability of most mitochondrial mRNAs, its pathophysiological effect has mainly been attributed to COX deficiency. Surprisingly, we show here that the impaired mitochondrial respiration and reduced ATP production observed in Lrpprc conditional knockout mouse hearts is caused by an ATP synthase deficiency. Furthermore, the appearance of inactive subassembled ATP synthase complexes causes hyperpolarization and increases mitochondrial reactive oxygen species production. Our findings shed important new light on the bioenergetic consequences of the loss of LRPPRC in cardiac mitochondria. PMID:24399447

  3. Activities and regulation of peptidoglycan synthases

    PubMed Central

    Egan, Alexander J. F.; Biboy, Jacob; van't Veer, Inge; Breukink, Eefjan; Vollmer, Waldemar

    2015-01-01

    Peptidoglycan (PG) is an essential component in the cell wall of nearly all bacteria, forming a continuous, mesh-like structure, called the sacculus, around the cytoplasmic membrane to protect the cell from bursting by its turgor. Although PG synthases, the penicillin-binding proteins (PBPs), have been studied for 70 years, useful in vitro assays for measuring their activities were established only recently, and these provided the first insights into the regulation of these enzymes. Here, we review the current knowledge on the glycosyltransferase and transpeptidase activities of PG synthases. We provide new data showing that the bifunctional PBP1A and PBP1B from Escherichia coli are active upon reconstitution into the membrane environment of proteoliposomes, and that these enzymes also exhibit DD-carboxypeptidase activity in certain conditions. Both novel features are relevant for their functioning within the cell. We also review recent data on the impact of protein–protein interactions and other factors on the activities of PBPs. As an example, we demonstrate a synergistic effect of multiple protein–protein interactions on the glycosyltransferase activity of PBP1B, by its cognate lipoprotein activator LpoB and the essential cell division protein FtsN. PMID:26370943

  4. Discovery of DF-461, a Potent Squalene Synthase Inhibitor

    PubMed Central

    2013-01-01

    We report the development of a new trifluoromethyltriazolobenzoxazepine series of squalene synthase inhibitors. Structure–activity studies and pharmacokinetics optimization on this series led to the identification of compound 23 (DF-461), which exhibited potent squalene synthase inhibitory activity, high hepatic selectivity, excellent rat hepatic cholesterol synthesis inhibitory activity, and plasma lipid lowering efficacy in nonrodent repeated dose studies. PMID:24900587

  5. Probing myo-inositol 1-phosphate synthase with multisubstrate adducts

    PubMed Central

    Deranieh, Rania M.; Greenberg, Miriam L.; Le Calvez, Pierre-B.; Mooney, Maura C.; Migaud, Marie E.

    2015-01-01

    The synthesis of a series of carbohydrate-nucleotide hybrids, designed to be multisubstrate adducts mimicking myo-inositol 1-phosphate synthase first oxidative transition state, is reported. Their ability to inhibit the synthase has been assessed and results have been rationalised computationally to estimate their likely binding mode. PMID:23132282

  6. Ligand-induced formation of a transient tryptophan synthase complex with αββ subunit stoichiometry.

    PubMed

    Ehrmann, Alexander; Richter, Klaus; Busch, Florian; Reimann, Julia; Albers, Sonja-Verena; Sterner, Reinhard

    2010-12-28

    The prototypical tryptophan synthases form a stable heterotetrameric αββα complex in which the constituting TrpA and TrpB1 subunits activate each other in a bidirectional manner. The hyperthermophilic archaeon Sulfolobus solfataricus does not contain a TrpB1 protein but instead two members of the phylogenetically distinct family of TrpB2 proteins, which are encoded within (sTrpB2i) and outside (sTrpB2a) the tryptophan operon. It has previously been shown that sTrpB2a does not functionally or structurally interact with sTrpA, whereas sTrpB2i substantially activates sTrpA in a unidirectional manner. However, in the absence of catalysis, no physical complex between sTrpB2i and sTrpA could be detected. In order to elucidate the structural requirements for complex formation, we have analyzed the interaction between sTrpA (α-monomer) and sTrpB2i (ββ-dimer) by means of spectroscopy, analytical gel filtration, and analytical ultracentrifugation, as well as isothermal titration calorimetry. In the presence of the TrpA ligand glycerol 3-phosphate (GP) and the TrpB substrate l-serine, sTrpA and sTrpB2i formed a physical complex with a thermodynamic dissociation constant of about 1 μM, indicating that the affinity between the α- and ββ-subunits is weaker by at least 1 order of magnitude than the affinity between the corresponding subunits of prototypical tryptophan synthases. The observed stoichiometry of the complex was 1 subunit of sTrpA per 2 subunits of sTrpB2i, which corresponds to a αββ quaternary structure and testifies to a strong negative cooperativity for the binding of the α-monomers to the ββ-dimer. The analysis of the interaction between sTrpB2i and sTrpA in the presence of several substrate, transition state, and product analogues suggests that the αββ complex remains stable during the whole catalytic cycle and disintegrates into α- and ββ-subunits upon the release of the reaction product tryptophan. The formation of a transient tryptophan

  7. Ubiquitination and filamentous structure of cytidine triphosphate synthase.

    PubMed

    Pai, Li-Mei; Wang, Pei-Yu; Lin, Wei-Cheng; Chakraborty, Archan; Yeh, Chau-Ting; Lin, Yu-Hung

    2016-07-01

    Living organisms respond to nutrient availability by regulating the activity of metabolic enzymes. Therefore, the reversible post-translational modification of an enzyme is a common regulatory mechanism for energy conservation. Recently, cytidine-5'-triphosphate (CTP) synthase was discovered to form a filamentous structure that is evolutionarily conserved from flies to humans. Interestingly, induction of the formation of CTP synthase filament is responsive to starvation or glutamine depletion. However, the biological roles of this structure remain elusive. We have recently shown that ubiquitination regulates CTP synthase activity by promoting filament formation in Drosophila ovaries during endocycles. Intriguingly, although the ubiquitination process was required for filament formation induced by glutamine depletion, CTP synthase ubiquitination was found to be inversely correlated with filament formation in Drosophila and human cell lines. In this article, we discuss the putative dual roles of ubiquitination, as well as its physiological implications, in the regulation of CTP synthase structure. PMID:27116391

  8. A Comparison of the Effects of Neuronal Nitric Oxide Synthase and Inducible Nitric Oxide Synthase Inhibition on Cartilage Damage

    PubMed Central

    Gokay, Nevzat Selim; Yilmaz, Ibrahim; Demiroz, Ahu Senem; Gokce, Alper; Dervisoglu, Sergülen; Gokay, Banu Vural

    2016-01-01

    The objective of this study was to investigate the effects of selective inducible nitric oxide synthase and neuronal nitric oxide synthase inhibitors on cartilage regeneration. The study involved 27 Wistar rats that were divided into five groups. On Day 1, both knees of 3 rats were resected and placed in a formalin solution as a control group. The remaining 24 rats were separated into 4 groups, and their right knees were surgically damaged. Depending on the groups, the rats were injected with intra-articular normal saline solution, neuronal nitric oxide synthase inhibitor 7-nitroindazole (50 mg/kg), inducible nitric oxide synthase inhibitor amino-guanidine (30 mg/kg), or nitric oxide precursor L-arginine (200 mg/kg). After 21 days, the right and left knees of the rats were resected and placed in formalin solution. The samples were histopathologically examined by a blinded evaluator and scored on 8 parameters. Although selective neuronal nitric oxide synthase inhibition exhibited significant (P = 0.044) positive effects on cartilage regeneration following cartilage damage, it was determined that inducible nitric oxide synthase inhibition had no statistically significant effect on cartilage regeneration. It was observed that the nitric oxide synthase activation triggered advanced arthrosis symptoms, such as osteophyte formation. The fact that selective neuronal nitric oxide synthase inhibitors were observed to have mitigating effects on the severity of the damage may, in the future, influence the development of new agents to be used in the treatment of cartilage disorders. PMID:27382570

  9. Light/Dark Profiles of Sucrose Phosphate Synthase, Sucrose Synthase, and Acid Invertase in Leaves of Sugar Beets

    PubMed Central

    Vassey, Terry L.

    1989-01-01

    The activity of sucrose phosphate synthase, sucrose synthase, and acid invertase was monitored in 1- to 2-month-old sugar beet (Beta vulgaris L.) leaves. Sugar beet leaves achieve full laminar length in 13 days. Therefore, leaves were harvested at 2-day intervals for 15 days. Sucrose phosphate synthase activity was not detectable for 6 days in the dark-grown leaves. Once activity was measurable, sucrose phosphate synthase activity never exceeded half that observed in the light-grown leaves. After 8 days in the dark, leaves which were illuminated for 30 minutes showed no significant change in sucrose phosphate synthase activity. Leaves illuminated for 24 hours after 8 days in darkness, however, recovered sucrose phosphate synthase activity to 80% of that of normally grown leaves. Sucrose synthase and acid invertase activity in the light-grown leaves both increased for the first 7 days and then decreased as the leaves matured. In contrast, the activity of sucrose synthase oscillated throughout the growth period in the dark-grown leaves. Acid invertase activity in the dark-grown leaves seemed to be the same as the activity found in the light-grown leaves. PMID:16666537

  10. Functional Contribution of Chorismate Synthase, Anthranilate Synthase, and Chorismate Mutase to Penetration Resistance in Barley-Powdery Mildew Interactions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant processes resulting from primary or secondary metabolism have been hypothesized to contribute to defense against microbial attack. Barley chorismate synthase (HvCS), anthranilate synthase alpha subunit 2 (HvASa2) and chorismate mutase 1 (HvCM1) occupy pivotal branch-points downstream of the s...

  11. Hyaluronan synthases and hyaluronidases in nasal polyps.

    PubMed

    Panogeorgou, T; Tserbini, E; Filou, S; Vynios, D H; Naxakis, S S; Papadas, T A; Goumas, P D; Mastronikolis, N S

    2016-07-01

    Nasal polyps (NPs) are benign lesions of nasal and paranasal sinuses mucosa affecting 1-4 % of all adults. Nasal polyposis affects the quality of patient's life as it causes nasal obstruction, postnasal drainage, purulent nasal discharge, hyposmia or anosmia, chronic sinusitis, facial pain and snoring. Without treatment, the disease can alter the craniofacial skeleton in cases of extended growth of polyps. The development of NPs is caused by the hyperplasia of nasal or paranasal sinuses mucosa, and edema of extracellular matrix. This is usually the result of high concentration of high molecular mass hyaluronan (HA) which is either overproduced or accumulated from blood supply. The size of HA presents high diversity and, especially in pathologic conditions, chains of low molecular mass can be observed. In NPs, chains of about 200 kDa have been identified and considered to be responsible for the inflammation. The purpose of the present study was the investigation, in NPs and normal nasal mucosa (NM), of the expression of the wild-type and alternatively spliced forms of hyaluronidases, their immunolocalization, and the expression of HA synthases to examine the isoform(s) responsible for the increased amounts of HA in NPs. Hyaluronidases' presence was examined on mRNA (RT-PCR analysis) and protein (immunohistochemistry) levels. Hyaluronan synthases' presence was examined on mRNA levels. Hyaluronidases were localized in the cytoplasm of epithelial and inflammatory cells, as well as in the matrix. On mRNA level, it was found that hyal-1-wt was decreased in NPs compared to NM and hyal-1-v3, -v4 and -v5 were substantially increased. Moreover, HAS2 and HAS3 were the only hyaluronan synthases detected, the expression of which was almost similar in NPs and NM. Overall, the results of the present study support that hyaluronidases are the main enzymes responsible for the decreased size of hyaluronan observed in NPs; thus they behave as inflammatory agents. Therefore, they

  12. Heterologous expression in Saccharopolyspora erythraea of a pentaketide synthase derived from the spinosyn polyketide synthase.

    PubMed

    Martin, Christine J; Timoney, Máire C; Sheridan, Rose M; Kendrew, Steven G; Wilkinson, Barrie; Staunton, James C; Leadlay, Peter F

    2003-12-01

    A truncated version of the spinosyn polyketide synthase comprising the loading module and the first four extension modules fused to the erythromycin thioesterase domain was expressed in Saccharopolyspora erythraea. A novel pentaketide lactone product was isolated, identifying cryptic steps of spinosyn biosynthesis and indicating the potential of this approach for the biosynthetic engineering of spinosyn analogues. A pathway for the formation of the tetracyclic spinosyn aglycone is proposed. PMID:14685317

  13. Substrate Controlled Divergence in Polyketide Synthase Catalysis

    PubMed Central

    2015-01-01

    Biochemical characterization of polyketide synthases (PKSs) has relied on synthetic substrates functionalized as electrophilic esters to acylate the enzyme and initiate the catalytic cycle. In these efforts, N-acetylcysteamine thioesters have typically been employed for in vitro studies of full PKS modules as well as excised domains. However, substrate engineering approaches to control the catalytic cycle of a full PKS module harboring multiple domains remain underexplored. This study examines a series of alternatively activated native hexaketide substrates on the catalytic outcome of PikAIV, the sixth and final module of the pikromycin (Pik) pathway. We demonstrate the ability to control product formation with greater than 10:1 selectivity for either full module catalysis, leading to a 14-membered macrolactone, or direct cyclization to a 12-membered ring. This outcome was achieved through modifying the type of hexaketide ester employed, demonstrating the utility of substrate engineering in PKS functional studies and biocatalysis. PMID:25730816

  14. Endothelial nitric oxide synthase in the microcirculation.

    PubMed

    Shu, Xiaohong; Keller, T C Stevenson; Begandt, Daniela; Butcher, Joshua T; Biwer, Lauren; Keller, Alexander S; Columbus, Linda; Isakson, Brant E

    2015-12-01

    Endothelial nitric oxide synthase (eNOS, NOS3) is responsible for producing nitric oxide (NO)--a key molecule that can directly (or indirectly) act as a vasodilator and anti-inflammatory mediator. In this review, we examine the structural effects of regulation of the eNOS enzyme, including post-translational modifications and subcellular localization. After production, NO diffuses to surrounding cells with a variety of effects. We focus on the physiological role of NO and NO-derived molecules, including microvascular effects on vessel tone and immune response. Regulation of eNOS and NO action is complicated; we address endogenous and exogenous mechanisms of NO regulation with a discussion of pharmacological agents used in clinical and laboratory settings and a proposed role for eNOS in circulating red blood cells. PMID:26390975

  15. Bacterial phytoene synthase: molecular cloning, expression, and characterization of Erwinia herbicola phytoene synthase.

    PubMed

    Iwata-Reuyl, Dirk; Math, Shivanand K; Desai, Shrivallabh B; Poulter, C Dale

    2003-03-25

    Phytoene synthase (PSase) catalyzes the condensation of two molecules of geranylgeranyl diphosphate (GGPP) to give prephytoene diphosphate (PPPP) and the subsequent rearrangement of the cyclopropylcarbinyl intermediate to phytoene. These reactions constitute the first pathway specific step in carotenoid biosynthesis. The crtB gene encoding phytoene synthase was isolated from a plasmid containing the carotenoid gene cluster in Erwinia herbicola and cloned into an Escherichia coli expression system. Upon induction, recombinant phytoene synthase constituted 5-10% of total soluble protein. To facilitate purification of the recombinant enzyme, the structural gene for PSase was modified by site-directed mutagenesis to incorporate a C-terminal Glu-Glu-Phe (EEF) tripepetide to allow purification by immunoaffinity chromatography on an immobilized monoclonal anti-alpha-tubulin antibody YL1/2 column. Purified recombinant PSase-EEF gave a band at 34.5 kDa upon SDS-PAGE. Recombinant PSase-EEF was then purified to >90% homogeneity in two steps by ion-exchange and immunoaffinity chromatography. The enzyme required Mn(2+) for activity, had a pH optimum of 8.2, and was strongly stimulated by detergent. The concentration of GGPP needed for half-maximal activity was approximately 35 microM, and a significant inhibition of activity was seen at GGPP concentrations above 100 microM. The sole product of the reaction was 15,15'-Z-phytoene. PMID:12641468

  16. CLYBL is a polymorphic human enzyme with malate synthase and β-methylmalate synthase activity

    PubMed Central

    Strittmatter, Laura; Li, Yang; Nakatsuka, Nathan J.; Calvo, Sarah E.; Grabarek, Zenon; Mootha, Vamsi K.

    2014-01-01

    CLYBL is a human mitochondrial enzyme of unknown function that is found in multiple eukaryotic taxa and conserved to bacteria. The protein is expressed in the mitochondria of all mammalian organs, with highest expression in brown fat and kidney. Approximately 5% of all humans harbor a premature stop polymorphism in CLYBL that has been associated with reduced levels of circulating vitamin B12. Using comparative genomics, we now show that CLYBL is strongly co-expressed with and co-evolved specifically with other components of the mitochondrial B12 pathway. We confirm that the premature stop polymorphism in CLYBL leads to a loss of protein expression. To elucidate the molecular function of CLYBL, we used comparative operon analysis, structural modeling and enzyme kinetics. We report that CLYBL encodes a malate/β-methylmalate synthase, converting glyoxylate and acetyl-CoA to malate, or glyoxylate and propionyl-CoA to β-methylmalate. Malate synthases are best known for their established role in the glyoxylate shunt of plants and lower organisms and are traditionally described as not occurring in humans. The broader role of a malate/β-methylmalate synthase in human physiology and its mechanistic link to vitamin B12 metabolism remain unknown. PMID:24334609

  17. Structure and Function of Fusicoccadiene Synthase, a Hexameric Bifunctional Diterpene Synthase.

    PubMed

    Chen, Mengbin; Chou, Wayne K W; Toyomasu, Tomonobu; Cane, David E; Christianson, David W

    2016-04-15

    Fusicoccin A is a diterpene glucoside phytotoxin generated by the fungal pathogen Phomopsis amygdali that causes the plant disease constriction canker, first discovered in New Jersey peach orchards in the 1930s. Fusicoccin A is also an emerging new lead in cancer chemotherapy. The hydrocarbon precursor of fusicoccin A is the tricyclic diterpene fusicoccadiene, which is generated by a bifunctional terpenoid synthase. Here, we report X-ray crystal structures of the individual catalytic domains of fusicoccadiene synthase: the C-terminal domain is a chain elongation enzyme that generates geranylgeranyl diphosphate, and the N-terminal domain catalyzes the cyclization of geranylgeranyl diphosphate to form fusicoccadiene. Crystal structures of each domain complexed with bisphosphonate substrate analogues suggest that three metal ions and three positively charged amino acid side chains trigger substrate ionization in each active site. While in vitro incubations reveal that the cyclase domain can utilize farnesyl diphosphate and geranyl diphosphate as surrogate substrates, these shorter isoprenoid diphosphates are mainly converted into acyclic alcohol or hydrocarbon products. Gel filtration chromatography and analytical ultracentrifugation experiments indicate that full-length fusicoccadiene synthase adopts hexameric quaternary structure, and small-angle X-ray scattering data yield a well-defined molecular envelope illustrating a plausible model for hexamer assembly. PMID:26734760

  18. Biosynthetic potential of sesquiterpene synthases: product profiles of Egyptian Henbane premnaspirodiene synthase and related mutants.

    PubMed

    Koo, Hyun Jo; Vickery, Christopher R; Xu, Yi; Louie, Gordon V; O'Maille, Paul E; Bowman, Marianne; Nartey, Charisse M; Burkart, Michael D; Noel, Joseph P

    2016-07-01

    The plant terpene synthase (TPS) family is responsible for the biosynthesis of a variety of terpenoid natural products possessing diverse biological functions. TPSs catalyze the ionization and, most commonly, rearrangement and cyclization of prenyl diphosphate substrates, forming linear and cyclic hydrocarbons. Moreover, a single TPS often produces several minor products in addition to a dominant product. We characterized the catalytic profiles of Hyoscyamus muticus premnaspirodiene synthase (HPS) and compared it with the profile of a closely related TPS, Nicotiana tabacum 5-epi-aristolochene synthase (TEAS). The profiles of two previously studied HPS and TEAS mutants, each containing nine interconverting mutations, dubbed HPS-M9 and TEAS-M9, were also characterized. All four TPSs were compared under varying temperature and pH conditions. In addition, we solved the X-ray crystal structures of TEAS and a TEAS quadruple mutant complexed with substrate and products to gain insight into the enzymatic features modulating product formation. These informative structures, along with product profiles, provide new insight into plant TPS catalytic promiscuity. PMID:27328867

  19. Cellulose in Cyanobacteria. Origin of Vascular Plant Cellulose Synthase?

    PubMed Central

    Nobles, David R.; Romanovicz, Dwight K.; Brown, R. Malcolm

    2001-01-01

    Although cellulose biosynthesis among the cyanobacteria has been suggested previously, we present the first conclusive evidence, to our knowledge, of the presence of cellulose in these organisms. Based on the results of x-ray diffraction, electron microscopy of microfibrils, and cellobiohydrolase I-gold labeling, we report the occurrence of cellulose biosynthesis in nine species representing three of the five sections of cyanobacteria. Sequence analysis of the genomes of four cyanobacteria revealed the presence of multiple amino acid sequences bearing the DDD35QXXRW motif conserved in all cellulose synthases. Pairwise alignments demonstrated that CesAs from plants were more similar to putative cellulose synthases from Anabaena sp. Pasteur Culture Collection 7120 and Nostoc punctiforme American Type Culture Collection 29133 than any other cellulose synthases in the database. Multiple alignments of putative cellulose synthases from Anabaena sp. Pasteur Culture Collection 7120 and N. punctiforme American Type Culture Collection 29133 with the cellulose synthases of other prokaryotes, Arabidopsis, Gossypium hirsutum, Populus alba × Populus tremula, corn (Zea mays), and Dictyostelium discoideum showed that cyanobacteria share an insertion between conserved regions U1 and U2 found previously only in eukaryotic sequences. Furthermore, phylogenetic analysis indicates that the cyanobacterial cellulose synthases share a common branch with CesAs of vascular plants in a manner similar to the relationship observed with cyanobacterial and chloroplast 16s rRNAs, implying endosymbiotic transfer of CesA from cyanobacteria to plants and an ancient origin for cellulose synthase in eukaryotes. PMID:11598227

  20. Class IV polyhydroxyalkanoate (PHA) synthases and PHA-producing Bacillus.

    PubMed

    Tsuge, Takeharu; Hyakutake, Manami; Mizuno, Kouhei

    2015-08-01

    This review highlights the recent investigations of class IV polyhydroxyalkanoate (PHA) synthases, the newest classification of PHA synthases. Class IV synthases are prevalent in organisms of the Bacillus genus and are composed of a catalytic subunit PhaC (approximately 40 kDa), which has a PhaC box sequence ([GS]-X-C-X-[GA]-G) at the active site, and a second subunit PhaR (approximately 20 kDa). The representative PHA-producing Bacillus strains are Bacillus megaterium and Bacillus cereus; the nucleotide sequence of phaC and the genetic organization of the PHA biosynthesis gene locus are somewhat different between these two strains. It is generally considered that class IV synthases favor short-chain-length monomers such as 3-hydroxybutyrate (C4) and 3-hydroxyvalerate (C5) for polymerization, but can polymerize some unusual monomers as minor components. In Escherichia coli expressing PhaRC from B. cereus YB-4, the biosynthesized PHA undergoes synthase-catalyzed alcoholytic cleavage using endogenous and exogenous alcohols. This alcoholysis is thought to be shared among class IV synthases, and this reaction is useful not only for the regulation of PHA molecular weight but also for the modification of the PHA carboxy terminus. The novel properties of class IV synthases will open up the possibility for the design of new PHA materials. PMID:26135986

  1. Acetolactate Synthase Activity in Developing Maize (Zea mays L.) Kernels

    PubMed Central

    Muhitch, Michael J.

    1988-01-01

    Acetolactate synthase (EC 4.1.3.18) activity was examined in maize (Zea mays L.) endosperm and embryos as a function of kernel development. When assayed using unpurified homogenates, embryo acetolactate synthase activity appeared less sensitive to inhibition by leucine + valine and by the imidazolinone herbicide imazapyr than endosperm acetolactate synthase activity. Evidence is presented to show that pyruvate decarboxylase contributes to apparent acetolactate synthase activity in crude embryo extracts and a modification of the acetolactate synthase assay is proposed to correct for the presence of pyruvate decarboxylase in unpurified plant homogenates. Endosperm acetolactate synthase activity increased rapidly during early kernel development, reaching a maximum of 3 micromoles acetoin per hour per endosperm at 25 days after pollination. In contrast, embryo activity was low in young kernels and steadily increased throughout development to a maximum activity of 0.24 micromole per hour per embryo by 45 days after pollination. The sensitivity of both endosperm and embryo acetolactate synthase activities to feedback inhibition by leucine + valine did not change during kernel development. The results are compared to those found for other enzymes of nitrogen metabolism and discussed with respect to the potential roles of the embryo and endosperm in providing amino acids for storage protein synthesis. PMID:16665871

  2. Generation and Functional Evaluation of Designer Monoterpene Synthases.

    PubMed

    Srividya, N; Lange, I; Lange, B M

    2016-01-01

    Monoterpene synthases are highly versatile enzymes that catalyze the first committed step in the pathways toward terpenoids, the structurally most diverse class of plant natural products. Recent advancements in our understanding of the reaction mechanism have enabled engineering approaches to develop mutant monoterpene synthases that produce specific monoterpenes. In this chapter, we are describing protocols to introduce targeted mutations, express mutant enzyme catalysts in heterologous hosts, and assess their catalytic properties. Mutant monoterpene synthases have the potential to contribute significantly to synthetic biology efforts aimed at producing larger amounts of commercially attractive monoterpenes. PMID:27480686

  3. The Pseudouridine Synthases Proceed through a Glycal Intermediate

    PubMed Central

    2016-01-01

    The pseudouridine synthases isomerize (U) in RNA to pseudouridine (Ψ), and the mechanism that they follow has long been a question of interest. The recent elucidation of a product of the mechanistic probe 5-fluorouridine that had been epimerized to the arabino isomer suggested that the Ψ synthases might operate through a glycal intermediate formed by deprotonation of C2′. When that position in substrate U is deuterated, a primary kinetic isotope effect is observed, which indisputably indicates that the proposed deprotonation occurs during the isomerization of U to Ψ and establishes the mechanism followed by the Ψ synthases. PMID:27292228

  4. Computational design and selections for an engineered, thermostable terpene synthase

    PubMed Central

    Diaz, Juan E; Lin, Chun-Shi; Kunishiro, Kazuyoshi; Feld, Birte K; Avrantinis, Sara K; Bronson, Jonathan; Greaves, John; Saven, Jeffery G; Weiss, Gregory A

    2011-01-01

    Terpenoids include structurally diverse antibiotics, flavorings, and fragrances. Engineering terpene synthases for control over the synthesis of such compounds represents a long sought goal. We report computational design, selections, and assays of a thermostable mutant of tobacco 5-epi-aristolochene synthase (TEAS) for the catalysis of carbocation cyclization reactions at elevated temperatures. Selection for thermostability included proteolytic digestion followed by capture of intact proteins. Unlike the wild-type enzyme, the mutant TEAS retains enzymatic activity at 65°C. The thermostable terpene synthase variant denatures above 80°C, approximately twice the temperature of the wild-type enzyme. PMID:21739507

  5. The Pseudouridine Synthases Proceed through a Glycal Intermediate.

    PubMed

    Veerareddygari, Govardhan Reddy; Singh, Sanjay K; Mueller, Eugene G

    2016-06-29

    The pseudouridine synthases isomerize (U) in RNA to pseudouridine (Ψ), and the mechanism that they follow has long been a question of interest. The recent elucidation of a product of the mechanistic probe 5-fluorouridine that had been epimerized to the arabino isomer suggested that the Ψ synthases might operate through a glycal intermediate formed by deprotonation of C2'. When that position in substrate U is deuterated, a primary kinetic isotope effect is observed, which indisputably indicates that the proposed deprotonation occurs during the isomerization of U to Ψ and establishes the mechanism followed by the Ψ synthases. PMID:27292228

  6. Geranylfarnesyl diphosphate synthase from Methanosarcina mazei: Different role, different evolution

    SciTech Connect

    Ogawa, Takuya; Yoshimura, Tohru; Hemmi, Hisashi

    2010-02-26

    The gene of (all-E) geranylfarnesyl diphosphate synthase that is responsible for the biosynthesis of methanophenazine, an electron carrier utilized for methanogenesis, was cloned from a methanogenic archaeon Methanosarcina mazei Goe1. The properties of the recombinant enzyme and the results of phylogenetic analysis suggest that the enzyme is closely related to (all-E) prenyl diphosphate synthases that are responsible for the biosynthesis of respiratory quinones, rather than to the enzymes involved in the biosynthesis of archaeal membrane lipids, including (all-E) geranylfarnesyl diphosphate synthase from a thermophilic archaeon.

  7. Science to practice: what do molecular biologic studies in rodent models add to our understanding of interventional oncologic procedures including percutaneous ablation by using glyceraldehyde-3-phosphate dehydrogenase antagonists?

    PubMed

    Goldberg, S Nahum

    2012-03-01

    In this basic research study, Ganapathy-Kanniappan et al advance our understanding of how to block the glycolytic pathway to inhibit tumor progression by using image guided procedures (1). This was accomplished by demonstrating their ability to perform molecular targeting of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in human hepatocellular carcinoma (HCC) by using percutaneous injection of either inhibitor--3-bromopyruvate (3-BrPA) or short hairpin RNA (shRNA). They take the critical step of providing further rationale for potentially advancing this therapy into clinical trials by demonstrating that GAPDH expression strongly correlates with c-jun, a proto-oncogene involved in liver tumorigenesis in human HCC (2). PMID:22357877

  8. A Pollen Protein, NaPCCP, That Binds Pistil Arabinogalactan Proteins Also Binds Phosphatidylinositol 3-Phosphate and Associates with the Pollen Tube Endomembrane System1[C][W][OA

    PubMed Central

    Lee, Christopher B.; Kim, Sunran; McClure, Bruce

    2009-01-01

    As pollen tubes grow toward the ovary, they are in constant contact with the pistil extracellular matrix (ECM). ECM components are taken up during growth, and some pistil molecules exert their effect inside the pollen tube. For instance, the Nicotiana alata 120-kD glycoprotein (120K) is an abundant arabinogalactan protein that is taken up from the ECM; it has been detected in association with pollen tube vacuoles, but the transport pathway between these compartments is unknown. We recently identified a pollen C2 domain-containing protein (NaPCCP) that binds to the carboxyl-terminal domain of 120K. As C2 domain proteins mediate protein-lipid interactions, NaPCCP could function in intracellular transport of 120K in pollen tubes. Here, we describe binding studies showing that the NaPCCP C2 domain is functional and that binding is specific for phosphatidylinositol 3-phosphate. Subcellular fractionation, immunolocalization, and live imaging results show that NaPCCP is associated with the plasma membrane and internal pollen tube vesicles. Colocalization between an NaPCCP∷green fluorescent protein fusion and internalized FM4-64 suggest an association with the endosomal system. NaPCCP localization is altered in pollen tubes rejected by the self-incompatibility mechanism, but our hypothesis is that it has a general function in the transport of endocytic cargo rather than a specific function in self-incompatibility. NaPCCP represents a bifunctional protein with both phosphatidylinositol 3-phosphate- and arabinogalactan protein-binding domains. Therefore, it could function in the transport of pistil ECM proteins in the pollen tube endomembrane system. PMID:19098095

  9. The FAD-dependent glycerol-3-phosphate dehydrogenase of Giardia duodenalis: an unconventional enzyme that interacts with the g14-3-3 and it is a target of the antitumoral compound NBDHEX

    PubMed Central

    Lalle, Marco; Camerini, Serena; Cecchetti, Serena; Finelli, Renata; Sferra, Gabriella; Müller, Joachim; Ricci, Giorgio; Pozio, Edoardo

    2015-01-01

    The flagellated protozoan Giardia duodenalis is a worldwide parasite causing giardiasis, an acute and chronic diarrheal disease. Metabolism in G. duodenalis has a limited complexity thus making metabolic enzymes ideal targets for drug development. However, only few metabolic pathways (i.e., carbohydrates) have been described so far. Recently, the parasite homolog of the mitochondrial-like glycerol-3-phosphate dehydrogenase (gG3PD) has been identified among the interactors of the g14-3-3 protein. G3PD is involved in glycolysis, electron transport, glycerophospholipids metabolism, and hyperosmotic stress response, and is emerging as promising target in tumor treatment. In this work, we demonstrate that gG3PD is a functional flavoenzyme able to convert glycerol-3-phosphate into dihydroxyacetone phosphate and that its activity and the intracellular glycerol level increase during encystation. Taking advantage of co-immunoprecipitation assays and deletion mutants, we provide evidence that gG3PD and g14-3-3 interact at the trophozoite stage, the intracellular localization of gG3PD is stage dependent and it partially co-localizes with mitosomes during cyst development. Finally, we demonstrate that the gG3PD activity is affected by the antitumoral compound 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol, that results more effective in vitro at killing G. duodenalis trophozoites than the reference drug metronidazole. Overall, our results highlight the involvement of gG3PD in processes crucial for the parasite survival thus proposing this enzyme as target for novel antigiardial interventions. PMID:26082764

  10. Dimer ribbons of ATP synthase shape the inner mitochondrial membrane

    PubMed Central

    Strauss, Mike; Hofhaus, Götz; Schröder, Rasmus R; Kühlbrandt, Werner

    2008-01-01

    ATP synthase converts the electrochemical potential at the inner mitochondrial membrane into chemical energy, producing the ATP that powers the cell. Using electron cryo-tomography we show that the ATP synthase of mammalian mitochondria is arranged in long ∼1-μm rows of dimeric supercomplexes, located at the apex of cristae membranes. The dimer ribbons enforce a strong local curvature on the membrane with a 17-nm outer radius. Calculations of the electrostatic field strength indicate a significant increase in charge density, and thus in the local pH gradient of ∼0.5 units in regions of high membrane curvature. We conclude that the mitochondrial cristae act as proton traps, and that the proton sink of the ATP synthase at the apex of the compartment favours effective ATP synthesis under proton-limited conditions. We propose that the mitochondrial ATP synthase organises itself into dimer ribbons to optimise its own performance. PMID:18323778

  11. Thymoquinone Inhibits Escherichia coli ATP Synthase and Cell Growth

    PubMed Central

    Ahmad, Zulfiqar; Laughlin, Thomas F.; Kady, Ismail O.

    2015-01-01

    We examined the thymoquinone induced inhibition of purified F1 or membrane bound F1FO E. coli ATP synthase. Both purified F1 and membrane bound F1FO were completely inhibited by thymoquinone with no residual ATPase activity. The process of inhibition was fully reversible and identical in both membrane bound F1Fo and purified F1 preparations. Moreover, thymoquinone induced inhibition of ATP synthase expressing wild-type E. coli cell growth and non-inhibition of ATPase gene deleted null control cells demonstrates that ATP synthase is a molecular target for thymoquinone. This also links the beneficial dietary based antimicrobial and anticancer effects of thymoquinone to its inhibitory action on ATP synthase. PMID:25996607

  12. A new family of zinc finger proteins in petunia: structure, DNA sequence recognition, and floral organ-specific expression.

    PubMed

    Takatsuji, H; Nakamura, N; Katsumoto, Y

    1994-07-01

    We have previously cloned a gene for a zinc finger protein (EPF1) that is expressed specifically in petals and interacts with the promoter region of the 5-enolpyruvylshikimate-3-phosphate synthase gene in petunia. In an attempt to isolate genes encoding additional factors that interact with this promoter, we cloned four novel genes encoding zinc finger proteins (EPF2-5a, EPF2-5b, EPF2-4, and EPF2-7). Sequence analyses revealed that overall similarity between the EPF1 and the EPF2 protein family, except in the zinc finger motifs and the basic amino acid cluster, was very low, suggesting that the two groups belong to different subfamilies. DNA binding specificities of EPF1, EPF2-5, and EPF2-4 were very similar, as expected from the conserved zinc finger motifs. However, EPF2-7 showed no binding to the probes tested in spite of having the conserved motifs. DNA binding studies using a series of spacing mutant probes suggested a binding mechanism in which the EPF proteins recognize spacings in target DNA. RNA gel blot analyses and histochemical analyses with a promoter and beta-glucuronidase fusion revealed that expression of the EPF2-5 gene (EPF2-5) was petal and stamen specific. Expression of the EPF2-7 gene (EPF2-7) was sepal and petal specific and localized in vascular tissues. The preferential expression in two adjacent floral organs raises the possibility that these genes are downstream transcription factors of floral homeotic genes. PMID:8069106

  13. Non-target Site Tolerance Mechanisms Describe Tolerance to Glyphosate in Avena sterilis

    PubMed Central

    Fernández-Moreno, Pablo T.; Alcantara-de la Cruz, Ricardo; Cruz-Hipólito, Hugo E.; Rojano-Delgado, Antonia M.; Travlos, Ilias; De Prado, Rafael

    2016-01-01

    Sterile wild oat (Avena sterilis L.) is an autogamous grass established in warm climate regions. This species has been used as a cover crop in Mediterranean perennial crops during the spring period prior to initiating competition with the main crop for water and nutrients. However, such cover crops need to be controlled (by glyphosate or tillage) before the beginning of summer period (due to the possibility of intense drought stress). In 2011, the olive grove farmers of southern Spain expressed dissatisfaction because of the ineffective control with glyphosate on A. sterilis. Experiments were conducted to determine whether the continued use of glyphosate over a 5 year period had selected a new resistant or tolerant species. The GR50 values obtained for A. sterilis were 297.12 and 245.23 g ae ha−1 for exposed (E) and un-exposed (UE) glyphosate accessions, respectively. The spray retention and shikimic acid accumulation exhibited a non-significant difference between the two accessions. The results of 14C- glyphosate absorption was the same in the two accessions (E and UE), while the translocation from the treated leaf to the rest of the shoots and roots was similar in A. sterilis accessions. Glyphosate metabolism to aminomethylphosphonic acid (AMPA) and glyoxylate was similar in both accessions, but increased after treatment with glyphosate, indicating that metabolism plays an important role in tolerance. Both A. sterilis accessions, present similarity in the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) activity enzyme with different glyphosate concentrations and without glyphosate, confirming that both accessions present the same genomic characteristics. The above-mentioned results indicate that innate tolerance to glyphosate in A. sterilis is probably and partly due to reduced herbicide absorption and translocation and metabolism compared to the susceptibility of other grasses weeds like Chloris inflata, Eleusine indica, and Lolium rigidum. PMID:27570531

  14. Improving Glyphosate Oxidation Activity of Glycine Oxidase from Bacillus cereus by Directed Evolution

    PubMed Central

    Zhan, Tao; Zhang, Kai; Chen, Yangyan; Lin, Yongjun; Wu, Gaobing; Zhang, Lili; Yao, Pei; Shao, Zongze; Liu, Ziduo

    2013-01-01

    Glyphosate, a broad spectrum herbicide widely used in agriculture all over the world, inhibits 5-enolpyruvylshikimate-3-phosphate synthase in the shikimate pathway, and glycine oxidase (GO) has been reported to be able to catalyze the oxidative deamination of various amines and cleave the C-N bond in glyphosate. Here, in an effort to improve the catalytic activity of the glycine oxidase that was cloned from a glyphosate-degrading marine strain of Bacillus cereus (BceGO), we used a bacteriophage T7 lysis-based method for high-throughput screening of oxidase activity and engineered the gene encoding BceGO by directed evolution. Six mutants exhibiting enhanced activity toward glyphosate were screened from two rounds of error-prone PCR combined with site directed mutagenesis, and the beneficial mutations of the six evolved variants were recombined by DNA shuffling. Four recombinants were generated and, when compared with the wild-type BceGO, the most active mutant B3S1 showed the highest activity, exhibiting a 160-fold increase in substrate affinity, a 326-fold enhancement in catalytic efficiency against glyphosate, with little difference between their pH and temperature stabilities. The role of these mutations was explored through structure modeling and molecular docking, revealing that the Arg51 mutation is near the active site and could be an important residue contributing to the stabilization of glyphosate binding, while the role of the remaining mutations is unclear. These results provide insight into the application of directed evolution in optimizing glycine oxidase function and have laid a foundation for the development of glyphosate-tolerant crops. PMID:24223901

  15. Expression of CP4 EPSPS in microspores and tapetum cells of cotton (Gossypium hirsutum) is critical for male reproductive development in response to late-stage glyphosate applications.

    PubMed

    Chen, Yun-Chia Sophia; Hubmeier, Christopher; Tran, Minhtien; Martens, Amy; Cerny, R Eric; Sammons, R Doug; CaJacob, Claire

    2006-09-01

    Plants expressing Agrobacterium sp. strain CP4 5-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) are known to be resistant to glyphosate, a potent herbicide that inhibits the activity of the endogenous plant EPSPS. The RR1445 transgenic cotton line (current commercial line for Roundup Ready Cotton) was generated using the figwort mosaic virus (FMV) 35S promoter to drive the expression of the CP4 EPSPS gene, and has excellent vegetative tolerance to glyphosate. However, with high glyphosate application rates at developmental stages later than the four-leaf stage (late-stage applications: applications that are inconsistent with the Roundup labels), RR1445 shows male sterility. Another transgenic cotton line, RR60, was generated using the FMV 35S promoter and the Arabidopsis elongation factor-1alpha promoter (AtEF1alpha) for the expression of CP4 EPSPS. RR60 has excellent vegetative and reproductive tolerance to applications of glyphosate at all developmental stages. Histochemical analyses were conducted to examine the male reproductive development at the cellular level of these cotton lines in response to glyphosate applications, and to investigate the correlation between glyphosate injury and the expression of CP4 EPSPS in male reproductive tissues. The expression of CP4 EPSPS in RR60 was found to be strong in all male reproductive cell types. Conversely, CP4 EPSPS expression in RR1445 was low in pollen mother cells, male gametophytes and tapetum, three crucial male reproductive cell types. Our results indicate that the FMV 35S promoter, although expressing strongly in most vegetative tissues in plants, has extremely low activity in these cell types. PMID:17309724

  16. Glyphosate-Resistant and Conventional Canola (Brassica napus L.) Responses to Glyphosate and Aminomethylphosphonic Acid (AMPA) Treatment.

    PubMed

    Corrêa, Elza Alves; Dayan, Franck E; Owens, Daniel K; Rimando, Agnes M; Duke, Stephen O

    2016-05-11

    Glyphosate-resistant (GR) canola contains two transgenes that impart resistance to the herbicide glyphosate: (1) the microbial glyphosate oxidase gene (gox) encoding the glyphosate oxidase enzyme (GOX) that metabolizes glyphosate to aminomethylphosphonic acid (AMPA) and (2) cp4 that encodes a GR form of the glyphosate target enzyme 5-enolpyruvylshikimic acid-3-phosphate synthase. The objectives of this research were to determine the phytotoxicity of AMPA to canola, the relative metabolism of glyphosate to AMPA in GR and conventional non-GR (NGR) canola, and AMPA pool sizes in glyphosate-treated GR canola. AMPA applied at 1.0 kg ha(-1) was not phytotoxic to GR or NGR. At this AMPA application rate, NGR canola accumulated a higher concentration of AMPA in its tissues than GR canola. At rates of 1 and 3.33 kg ae ha(-1) of glyphosate, GR canola growth was stimulated. This stimulatory effect is similar to that of much lower doses of glyphosate on NGR canola. Both shikimate and AMPA accumulated in tissues of these glyphosate-treated plants. In a separate experiment in which young GR and NGR canola plants were treated with non-phytotoxic levels of [(14)C]-glyphosate, very little glyphosate was metabolized in NGR plants, whereas most of the glyphosate was metabolized to AMPA in GR plants at 7 days after application. Untreated leaves of GR plants accumulated only metabolites (mostly AMPA) of glyphosate, indicating that GOX activity is very high in the youngest leaves. These data indicate that more glyphosate is transformed to AMPA rapidly in GR canola and that the accumulated AMPA is not toxic to the canola plant. PMID:27092715

  17. A Built-In Mechanism to Mitigate the Spread of Insect-Resistance and Herbicide-Tolerance Transgenes into Weedy Rice Populations

    PubMed Central

    Liu, Chengyi; Li, Jingjing; Gao, Jianhua; Shen, Zhicheng; Lu, Bao-Rong; Lin, Chaoyang

    2012-01-01

    Background The major challenge of cultivating genetically modified (GM) rice (Oryza sativa) at the commercial scale is to prevent the spread of transgenes from GM cultivated rice to its coexisting weedy rice (O. sativa f. spontanea). The strategic development of GM rice with a built-in control mechanism can mitigate transgene spread in weedy rice populations. Methodology/Principal Findings An RNAi cassette suppressing the expression of the bentazon detoxifying enzyme CYP81A6 was constructed into the T-DNA which contained two tightly linked transgenes expressing the Bt insecticidal protein Cry1Ab and the glyphosate tolerant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), respectively. GM rice plants developed from this T-DNA were resistant to lepidopteran pests and tolerant to glyphosate, but sensitive to bentazon. The application of bentazon of 2000 mg/L at the rate of 40 mL/m2, which is approximately the recommended dose for the field application to control common rice weeds, killed all F2 plants containing the transgenes generated from the Crop-weed hybrids between a GM rice line (CGH-13) and two weedy rice strains (PI-63 and PI-1401). Conclusions/Significance Weedy rice plants containing transgenes from GM rice through gene flow can be selectively killed by the spray of bentazon when a non-GM rice variety is cultivated alternately in a few-year interval. The built-in control mechanism in combination of cropping management is likely to mitigate the spread of transgenes into weedy rice populations. PMID:22359609

  18. Length of time in tissue culture can affect the selected glyphosate resistance mechanism.

    PubMed

    Papanikou, Efstratia; Brotherton, Jeffrey E; Widholm, Jack M

    2004-02-01

    Usually, stepwise selection of plant suspension cultures with gradually increasing concentrations of the herbicide glyphosate results in the amplification of the target enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19) gene that leads to resistance by increasing EPSPS mRNA and enzyme activity. We show that glyphosate selection with newly initiated suspension cultures can produce resistant lines with resistance mechanisms other than gene amplification and that usually as the cultures age gene amplification becomes the predominant mechanism. Gene amplification did not occur in 3 lines selected from 5-month-old Datura innoxia Mill. cultures but did occur in all 10 lines selected after 52 months. Selection with Nicotiana tabacum L. (tobacco) less than 5 months old produced 2 lines out of 24 with no EPSPS amplification while all 17 lines selected from older cultures contained amplified genes. Lines selected from the oldest culture (35 years) also exhibited amplification of several different genes, indicating the expression of different EPSPS genes or an enhanced gene amplification incidence. None of the 15 lines selected from 2 different 5-month-old Daucus carota L. (carrot) lines exhibited amplification while amplification led to the resistance of all 7 lines selected from one of the original carrot lines (DHL) after 3 years. However, the other line (Car4) was exceptional and produced only non-amplified lines (9 of 9) after 8 years in culture. These results show that plant tissue cultures change with time in culture and that several different new mechanisms can result in glyphosate resistance. PMID:14566562

  19. Non-target Site Tolerance Mechanisms Describe Tolerance to Glyphosate in Avena sterilis.

    PubMed

    Fernández-Moreno, Pablo T; Alcantara-de la Cruz, Ricardo; Cruz-Hipólito, Hugo E; Rojano-Delgado, Antonia M; Travlos, Ilias; De Prado, Rafael

    2016-01-01

    Sterile wild oat (Avena sterilis L.) is an autogamous grass established in warm climate regions. This species has been used as a cover crop in Mediterranean perennial crops during the spring period prior to initiating competition with the main crop for water and nutrients. However, such cover crops need to be controlled (by glyphosate or tillage) before the beginning of summer period (due to the possibility of intense drought stress). In 2011, the olive grove farmers of southern Spain expressed dissatisfaction because of the ineffective control with glyphosate on A. sterilis. Experiments were conducted to determine whether the continued use of glyphosate over a 5 year period had selected a new resistant or tolerant species. The GR50 values obtained for A. sterilis were 297.12 and 245.23 g ae ha(-1) for exposed (E) and un-exposed (UE) glyphosate accessions, respectively. The spray retention and shikimic acid accumulation exhibited a non-significant difference between the two accessions. The results of (14)C- glyphosate absorption was the same in the two accessions (E and UE), while the translocation from the treated leaf to the rest of the shoots and roots was similar in A. sterilis accessions. Glyphosate metabolism to aminomethylphosphonic acid (AMPA) and glyoxylate was similar in both accessions, but increased after treatment with glyphosate, indicating that metabolism plays an important role in tolerance. Both A. sterilis accessions, present similarity in the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) activity enzyme with different glyphosate concentrations and without glyphosate, confirming that both accessions present the same genomic characteristics. The above-mentioned results indicate that innate tolerance to glyphosate in A. sterilis is probably and partly due to reduced herbicide absorption and translocation and metabolism compared to the susceptibility of other grasses weeds like Chloris inflata, Eleusine indica, and Lolium rigidum. PMID:27570531

  20. Selection and characterization of glyphosate tolerance in birdsfoot trefoil (Lotus corniculatus)

    SciTech Connect

    Boerboom, C.M.

    1989-01-01

    If birdsfoot trefoil (Lotus corniculatus L.) was tolerant to glyphosate (N-(phosphonomethyl)glycine), Canada thistle (Cirsium arvense (L.) Scop.) and other dicot weeds could be selectively controlled in certified seed production fields. Glyphosate tolerance in birdsfoot trefoil was identified in plants from the cultivar Leo, plants regenerated from tolerant callus, and selfed progeny of plants regenerated from callus. Plants from the three sources were evaluated in field studies for tolerance to glyphosate at rates up to 1.6 kg ae/ha. Plants of Leo selected for tolerance exhibited a twofold range in the rate required to reduce shoot weight 50% (I{sub 50}s from 0.6 to 1.2 kg/ha glyphosate). Plants regenerated from tolerant callus had tolerance up to 66% greater than plants regenerated from unselected callus. Transgressive segregation for glyphosate tolerance was observed in the selfed progeny of two regenerated plants that both had I{sub 50}s of 0.7 kg/ha glyphosate. The selfed progeny ranged from highly tolerant (I{sub 50} of 1.5 kg/ha) to susceptible (I{sub 50} of 0.5 kg/ha). Spray retention, {sup 14}C-glyphosate absorption and translocation did not account for the differential tolerance of nine plants that were evaluated from the three sources. The specific activity of 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase ranged from 1.3 to 3.5 nmol/min{sm bullet}mg among the nine plants and was positively correlated with glyphosate tolerance. Leo birdsfoot trefoil was found to have significant variation in glyphosate tolerance which made it possible to initiate a recurrent selection program to select for glyphosate tolerance in birdsfoot trefoil. Two cycles of selection for glyphosate tolerance were practiced in three birdsfoot trefoil populations, Leo, Norcen, and MU-81.

  1. Biosynthesis of riboflavin: an unusual riboflavin synthase of Methanobacterium thermoautotrophicum.

    PubMed Central

    Eberhardt, S; Korn, S; Lottspeich, F; Bacher, A

    1997-01-01

    Riboflavin synthase was purified by a factor of about 1,500 from cell extract of Methanobacterium thermoautotrophicum. The enzyme had a specific activity of about 2,700 nmol mg(-1) h(-1) at 65 degrees C, which is relatively low compared to those of riboflavin synthases of eubacteria and yeast. Amino acid sequences obtained after proteolytic cleavage had no similarity with known riboflavin synthases. The gene coding for riboflavin synthase (designated ribC) was subsequently cloned by marker rescue with a ribC mutant of Escherichia coli. The ribC gene of M. thermoautotrophicum specifies a protein of 153 amino acid residues. The predicted amino acid sequence agrees with the information gleaned from Edman degradation of the isolated protein and shows 67% identity with the sequence predicted for the unannotated reading frame MJ1184 of Methanococcus jannaschii. The ribC gene is adjacent to a cluster of four genes with similarity to the genes cbiMNQO of Salmonella typhimurium, which form part of the cob operon (this operon contains most of the genes involved in the biosynthesis of vitamin B12). The amino acid sequence predicted by the ribC gene of M. thermoautotrophicum shows no similarity whatsoever to the sequences of riboflavin synthases of eubacteria and yeast. Most notably, the M. thermoautotrophicum protein does not show the internal sequence homology characteristic of eubacterial and yeast riboflavin synthases. The protein of M. thermoautotrophicum can be expressed efficiently in a recombinant E. coli strain. The specific activity of the purified, recombinant protein is 1,900 nmol mg(-1) h(-1) at 65 degrees C. In contrast to riboflavin synthases from eubacteria and fungi, the methanobacterial enzyme has an absolute requirement for magnesium ions. The 5' phosphate of 6,7-dimethyl-8-ribityllumazine does not act as a substrate. The findings suggest that riboflavin synthase has evolved independently in eubacteria and methanobacteria. PMID:9139911

  2. Nitric oxide synthase in ferret brain: localization and characterization.

    PubMed Central

    Matsumoto, T.; Mitchell, J. A.; Schmidt, H. H.; Kohlhaas, K. L.; Warner, T. D.; Förstermann, U.; Murad, F.

    1992-01-01

    1. In the present study, we have investigated the distribution of nitric oxide synthase in the ferret brain. Nitric oxide synthase was determined biochemically and immunochemically. 2. In the rat brain, the highest nitric oxide synthase activity has been detected in the cerebellum. However, in the ferret brain, the highest activity was found in the striatum and the lowest in the cerebellum and cerebral cortex. The enzymatic activity was localized predominantly in the cytosolic fractions, it was dependent on NADPH and Ca2+, and inhibited by NG-nitro-L-arginine or NG-methyl-L-arginine. 3. Western blot analysis revealed that all regions of the ferret brain contained a 160 kD protein crossreacting with an antibody to nitric oxide synthase purified from the rat cerebellum, and the levels of relative intensity of staining by the antibody correlated with the distribution of nitric oxide synthase activity. 4. These results indicate that the ferret brain contains a nitric oxide synthase similar to the rat brain, but the distribution of enzymatic activity in the ferret brain differs markedly from the rat brain. Images Figure 1 PMID:1282076

  3. Understanding structure, function, and mutations in the mitochondrial ATP synthase

    PubMed Central

    Xu, Ting; Pagadala, Vijayakanth; Mueller, David M.

    2015-01-01

    The mitochondrial ATP synthase is a multimeric enzyme complex with an overall molecular weight of about 600,000 Da. The ATP synthase is a molecular motor composed of two separable parts: F1 and Fo. The F1 portion contains the catalytic sites for ATP synthesis and protrudes into the mitochondrial matrix. Fo forms a proton turbine that is embedded in the inner membrane and connected to the rotor of F1. The flux of protons flowing down a potential gradient powers the rotation of the rotor driving the synthesis of ATP. Thus, the flow of protons though Fo is coupled to the synthesis of ATP. This review will discuss the structure/function relationship in the ATP synthase as determined by biochemical, crystallographic, and genetic studies. An emphasis will be placed on linking the structure/function relationship with understanding how disease causing mutations or putative single nucleotide polymorphisms (SNPs) in genes encoding the subunits of the ATP synthase, will affect the function of the enzyme and the health of the individual. The review will start by summarizing the current understanding of the subunit composition of the enzyme and the role of the subunits followed by a discussion on known mutations and their effect on the activity of the ATP synthase. The review will conclude with a summary of mutations in genes encoding subunits of the ATP synthase that are known to be responsible for human disease, and a brief discussion on SNPs. PMID:25938092

  4. Protein preparation, crystallization and preliminary X-ray analysis of Polygonum cuspidatum bifunctional chalcone synthase/benzalacetone synthase.

    PubMed

    Lu, Heshu; Yang, Mingfeng; Liu, Chunmei; Lu, Ping; Cang, Huaixing; Ma, Lanqing

    2013-08-01

    The chalcone synthase (CHS) superfamily of type III polyketide synthases (PKSs) generate the backbones of a variety of plant secondary metabolites. An active bifunctional chalcone synthase/benzalacetone synthase (CHS/BAS) from Polygonum cuspidatum was overexpressed in Escherichia coli as a C-terminally polyhistidine-tagged fusion protein, purified to homogeneity and crystallized using polyethylene glycol 4000 as a precipitant. The production of well shaped crystals of the complex between PcPKS1 and benzalacetone was dependent on the presence of sorbitol and barium chloride as additives. The crystals belonged to the orthorhombic space group P2₁2₁2₁, with unit-cell parameters a = 80.23, b = 81.01, c = 122.89 Å, and diffracted X-rays to at least 2.0 Å resolution. PMID:23908031

  5. Nitric Oxide Synthases in Heart Failure

    PubMed Central

    Carnicer, Ricardo; Crabtree, Mark J.; Sivakumaran, Vidhya

    2013-01-01

    Abstract Significance: The regulation of myocardial function by constitutive nitric oxide synthases (NOS) is important for the maintenance of myocardial Ca2+ homeostasis, relaxation and distensibility, and protection from arrhythmia and abnormal stress stimuli. However, sustained insults such as diabetes, hypertension, hemodynamic overload, and atrial fibrillation lead to dysfunctional NOS activity with superoxide produced instead of NO and worse pathophysiology. Recent Advances: Major strides in understanding the role of normal and abnormal constitutive NOS in the heart have revealed molecular targets by which NO modulates myocyte function and morphology, the role and nature of post-translational modifications of NOS, and factors controlling nitroso-redox balance. Localized and differential signaling from NOS1 (neuronal) versus NOS3 (endothelial) isoforms are being identified, as are methods to restore NOS function in heart disease. Critical Issues: Abnormal NOS signaling plays a key role in many cardiac disorders, while targeted modulation may potentially reverse this pathogenic source of oxidative stress. Future Directions: Improvements in the clinical translation of potent modulators of NOS function/dysfunction may ultimately provide a powerful new treatment for many hearts diseases that are fueled by nitroso-redox imbalance. Antioxid. Redox Signal. 18, 1078–1099. PMID:22871241

  6. Human Isoprenoid Synthase Enzymes as Therapeutic Targets

    NASA Astrophysics Data System (ADS)

    Park, Jaeok; Matralis, Alexios; Berghuis, Albert; Tsantrizos, Youla

    2014-07-01

    The complex biochemical network known as the mevalonate pathway is responsible for the biosynthesis of all isoprenoids in the human body, which consists of a vast array of metabolites that are vital for proper cellular functions. Two key isoprenoids, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) are responsible for the post-translational prenylation of small GTP-binding proteins, and serve as the biosynthetic precursors to numerous other biomolecules. The down-stream metabolite of FPP and GGPP is squalene, the precursor to steroids, bile acids, lipoproteins and vitamin D. In the past, interest in prenyl synthase inhibitors focused mainly on the role of the FPP in lytic bone diseases. More recently, pre-clinical and clinical studies have strongly implicated high levels of protein prenylation in a plethora of human diseases, including non-skeletal cancers, the progression of neurodegenerative diseases and cardiovascular diseases. In this review, we focus mainly on the potential therapeutic value of down-regulating the biosynthesis of FPP, GGPP and squalene. We summarize the most recent drug discovery efforts and the structural data available that support the current on-going studies.

  7. Human isoprenoid synthase enzymes as therapeutic targets

    PubMed Central

    Park, Jaeok; Matralis, Alexios N.; Berghuis, Albert M.; Tsantrizos, Youla S.

    2014-01-01

    In the human body, the complex biochemical network known as the mevalonate pathway is responsible for the biosynthesis of all isoprenoids, which consists of a vast array of metabolites that are vital for proper cellular functions. Two key isoprenoids, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) are responsible for the post-translational prenylation of small GTP-binding proteins, and serve as the biosynthetic precursors to numerous other biomolecules. The down-stream metabolite of FPP and GGPP is squalene, the precursor to steroids, bile acids, lipoproteins, and vitamin D. In the past, interest in prenyl synthase inhibitors focused mainly on the role of the FPP in lytic bone diseases. More recently pre-clinical and clinical studies have strongly implicated high levels of protein prenylation in a plethora of human diseases, including non-skeletal cancers, the progression of neurodegenerative diseases and cardiovascular diseases. In this review, we focus mainly on the potential therapeutic value of down-regulating the biosynthesis of FPP, GGPP, and squalene. We summarize the most recent drug discovery efforts and the structural data available that support the current on-going studies. PMID:25101260

  8. Tapentadol and nitric oxide synthase systems.

    PubMed

    Bujalska-Zadrożny, Magdalena; Wolińska, Renata; Gąsińska, Emilia; Nagraba, Łukasz

    2015-04-01

    Tapentadol, a new analgesic drug with a dual mechanism of action (μ-opioid receptor agonism and norepinephrine reuptake inhibition), is indicated for the treatment of moderate to severe acute and chronic pain. In this paper, the possible additional involvement of the nitric oxide synthase (NOS) system in the antinociceptive activity of tapentadol was investigated using an unspecific inhibitor of NOS, L-NOArg, a relatively specific inhibitor of neuronal NOS, 7-NI, a relatively selective inhibitor of inducible NOS, L-NIL, and a potent inhibitor of endothelial NOS, L-NIO. Tapentadol (1-10 mg/kg, intraperitoneal) increased the threshold for mechanical (Randall-Selitto test) and thermal (tail-flick test) nociceptive stimuli in a dose-dependent manner. All four NOS inhibitors, administered intraperitoneally in the dose range 0.1-10 mg/kg, potentiated the analgesic action of tapentadol at a low dose of 2 mg/kg in both models of pain. We conclude that NOS systems participate in tapentadol analgesia. PMID:25485639

  9. Undecaprenyl diphosphate synthase inhibitors: antibacterial drug leads.

    PubMed

    Sinko, William; Wang, Yang; Zhu, Wei; Zhang, Yonghui; Feixas, Ferran; Cox, Courtney L; Mitchell, Douglas A; Oldfield, Eric; McCammon, J Andrew

    2014-07-10

    There is a significant need for new antibiotics due to the rise in drug resistance. Drugs such as methicillin and vancomycin target bacterial cell wall biosynthesis, but methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE) have now arisen and are of major concern. Inhibitors acting on new targets in cell wall biosynthesis are thus of particular interest since they might also restore sensitivity to existing drugs, and the cis-prenyl transferase undecaprenyl diphosphate synthase (UPPS), essential for lipid I, lipid II, and thus, peptidoglycan biosynthesis, is one such target. We used 12 UPPS crystal structures to validate virtual screening models and then assayed 100 virtual hits (from 450,000 compounds) against UPPS from S. aureus and Escherichia coli. The most promising inhibitors (IC50 ∼2 μM, Ki ∼300 nM) had activity against MRSA, Listeria monocytogenes, Bacillus anthracis, and a vancomycin-resistant Enterococcus sp. with MIC or IC50 values in the 0.25-4 μg/mL range. Moreover, one compound (1), a rhodanine with close structural similarity to the commercial diabetes drug epalrestat, exhibited good activity as well as a fractional inhibitory concentration index (FICI) of 0.1 with methicillin against the community-acquired MRSA USA300 strain, indicating strong synergism. PMID:24827744

  10. Electric Field Driven Torque in ATP Synthase

    PubMed Central

    Miller, John H.; Rajapakshe, Kimal I.; Infante, Hans L.; Claycomb, James R.

    2013-01-01

    FO-ATP synthase (FO) is a rotary motor that converts potential energy from ions, usually protons, moving from high- to low-potential sides of a membrane into torque and rotary motion. Here we propose a mechanism whereby electric fields emanating from the proton entry and exit channels act on asymmetric charge distributions in the c-ring, due to protonated and deprotonated sites, and drive it to rotate. The model predicts a scaling between time-averaged torque and proton motive force, which can be hindered by mutations that adversely affect the channels. The torque created by the c-ring of FO drives the γ-subunit to rotate within the ATP-producing complex (F1) overcoming, with the aid of thermal fluctuations, an opposing torque that rises and falls with angular position. Using the analogy with thermal Brownian motion of a particle in a tilted washboard potential, we compute ATP production rates vs. proton motive force. The latter shows a minimum, needed to drive ATP production, which scales inversely with the number of proton binding sites on the c-ring. PMID:24040370

  11. Catalytic site interactions in yeast OMP synthase.

    PubMed

    Hansen, Michael Riis; Barr, Eric W; Jensen, Kaj Frank; Willemoës, Martin; Grubmeyer, Charles; Winther, Jakob R

    2014-01-15

    The enigmatic kinetics, half-of-the-sites binding, and structural asymmetry of the homodimeric microbial OMP synthases (orotate phosphoribosyltransferase, EC 2.4.2.10) have been proposed to result from an alternating site mechanism in these domain-swapped enzymes [R.W. McClard et al., Biochemistry 45 (2006) 5330-5342]. This behavior was investigated in the yeast enzyme by mutations in the conserved catalytic loop and 5-phosphoribosyl-1-diphosphate (PRPP) binding motif. Although the reaction is mechanistically sequential, the wild-type (WT) enzyme shows parallel lines in double reciprocal initial velocity plots. Replacement of Lys106, the postulated intersubunit communication device, produced intersecting lines in kinetic plots with a 2-fold reduction of kcat. Loop (R105G K109S H111G) and PRPP-binding motif (D131N D132N) mutant proteins, each without detectable enzymatic activity and ablated ability to bind PRPP, complemented to produce a heterodimer with a single fully functional active site showing intersecting initial velocity plots. Equilibrium binding of PRPP and orotidine 5'-monophosphate showed a single class of two binding sites per dimer in WT and K106S enzymes. Evidence here shows that the enzyme does not follow half-of-the-sites cooperativity; that interplay between catalytic sites is not an essential feature of the catalytic mechanism; and that parallel lines in steady-state kinetics probably arise from tight substrate binding. PMID:24262852

  12. Binding Modes of Zaragozic Acid A to Human Squalene Synthase and Staphylococcal Dehydrosqualene Synthase*

    PubMed Central

    Liu, Chia-I; Jeng, Wen-Yih; Chang, Wei-Jung; Ko, Tzu-Ping; Wang, Andrew H.-J.

    2012-01-01

    Zaragozic acids (ZAs) belong to a family of fungal metabolites with nanomolar inhibitory activity toward squalene synthase (SQS). The enzyme catalyzes the committed step of sterol synthesis and has attracted attention as a potential target for antilipogenic and antiinfective therapies. Here, we have determined the structure of ZA-A complexed with human SQS. ZA-A binding induces a local conformational change in the substrate binding site, and its C-6 acyl group also extends over to the cofactor binding cavity. In addition, ZA-A effectively inhibits a homologous bacterial enzyme, dehydrosqualene synthase (CrtM), which synthesizes the precursor of staphyloxanthin in Staphylococcus aureus to cope with oxidative stress. Size reduction at Tyr248 in CrtM further increases the ZA-A binding affinity, and it reveals a similar overall inhibitor binding mode to that of human SQS/ZA-A except for the C-6 acyl group. These structures pave the way for further improving selectivity and development of a new generation of anticholesterolemic and antimicrobial inhibitors. PMID:22474324

  13. Binding modes of zaragozic acid A to human squalene synthase and staphylococcal dehydrosqualene synthase.

    PubMed

    Liu, Chia-I; Jeng, Wen-Yih; Chang, Wei-Jung; Ko, Tzu-Ping; Wang, Andrew H-J

    2012-05-25

    Zaragozic acids (ZAs) belong to a family of fungal metabolites with nanomolar inhibitory activity toward squalene synthase (SQS). The enzyme catalyzes the committed step of sterol synthesis and has attracted attention as a potential target for antilipogenic and antiinfective therapies. Here, we have determined the structure of ZA-A complexed with human SQS. ZA-A binding induces a local conformational change in the substrate binding site, and its C-6 acyl group also extends over to the cofactor binding cavity. In addition, ZA-A effectively inhibits a homologous bacterial enzyme, dehydrosqualene synthase (CrtM), which synthesizes the precursor of staphyloxanthin in Staphylococcus aureus to cope with oxidative stress. Size reduction at Tyr(248) in CrtM further increases the ZA-A binding affinity, and it reveals a similar overall inhibitor binding mode to that of human SQS/ZA-A except for the C-6 acyl group. These structures pave the way for further improving selectivity and development of a new generation of anticholesterolemic and antimicrobial inhibitors. PMID:22474324

  14. Characterisation of the tryptophan synthase alpha subunit in maize

    PubMed Central

    Kriechbaumer, Verena; Weigang, Linda; Fießelmann, Andreas; Letzel, Thomas; Frey, Monika; Gierl, Alfons; Glawischnig, Erich

    2008-01-01

    Background In bacteria, such as Salmonella typhimurium, tryptophan is synthesized from indole-3-glycerole phosphate (IGP) by a tryptophan synthase αββα heterotetramer. Plants have evolved multiple α (TSA) and β (TSB) homologs, which have probably diverged in biological function and their ability of subunit interaction. There is some evidence for a tryptophan synthase (TS) complex in Arabidopsis. On the other hand maize (Zea mays) expresses the TSA-homologs BX1 and IGL that efficiently cleave IGP, independent of interaction with TSB. Results In order to clarify, how tryptophan is synthesized in maize, two TSA homologs, hitherto uncharacterized ZmTSA and ZmTSAlike, were functionally analyzed. ZmTSA is localized in plastids, the major site of tryptophan biosynthesis in plants. It catalyzes the tryptophan synthase α-reaction (cleavage of IGP), and forms a tryptophan synthase complex with ZmTSB1 in vitro. The catalytic efficiency of the α-reaction is strongly enhanced upon complex formation. A 160 kD tryptophan synthase complex was partially purified from maize leaves and ZmTSA was identified as native α-subunit of this complex by mass spectrometry. ZmTSAlike, for which no in vitro activity was detected, is localized in the cytosol. ZmTSAlike, BX1, and IGL were not detectable in the native tryptophan synthase complex in leaves. Conclusion It was demonstrated in vivo and in vitro that maize forms a tryptophan synthase complex and ZmTSA functions as α-subunit in this complex. PMID:18430213

  15. The Tomato Terpene Synthase Gene Family1[W][OA

    PubMed Central

    Falara, Vasiliki; Akhtar, Tariq A.; Nguyen, Thuong T.H.; Spyropoulou, Eleni A.; Bleeker, Petra M.; Schauvinhold, Ines; Matsuba, Yuki; Bonini, Megan E.; Schilmiller, Anthony L.; Last, Robert L.; Schuurink, Robert C.; Pichersky, Eran

    2011-01-01

    Compounds of the terpenoid class play numerous roles in the interactions of plants with their environment, such as attracting pollinators and defending the plant against pests. We show here that the genome of cultivated tomato (Solanum lycopersicum) contains 44 terpene synthase (TPS) genes, including 29 that are functional or potentially functional. Of these 29 TPS genes, 26 were expressed in at least some organs or tissues of the plant. The enzymatic functions of eight of the TPS proteins were previously reported, and here we report the specific in vitro catalytic activity of 10 additional tomato terpene synthases. Many of the tomato TPS genes are found in clusters, notably on chromosomes 1, 2, 6, 8, and 10. All TPS family clades previously identified in angiosperms are also present in tomato. The largest clade of functional TPS genes found in tomato, with 12 members, is the TPS-a clade, and it appears to encode only sesquiterpene synthases, one of which is localized to the mitochondria, while the rest are likely cytosolic. A few additional sesquiterpene synthases are encoded by TPS-b clade genes. Some of the tomato sesquiterpene synthases use z,z-farnesyl diphosphate in vitro as well, or more efficiently than, the e,e-farnesyl diphosphate substrate. Genes encoding monoterpene synthases are also prevalent, and they fall into three clades: TPS-b, TPS-g, and TPS-e/f. With the exception of two enzymes involved in the synthesis of ent-kaurene, the precursor of gibberellins, no other tomato TPS genes could be demonstrated to encode diterpene synthases so far. PMID:21813655

  16. The distribution of acetohydroxyacid synthase in soil bacteria.

    PubMed

    Nelson, Darryl R; Duxbury, Trevor

    2008-01-01

    Most bacteria possess the enzyme acetohydroxyacid synthase, which is used to produce branched-chain amino acids. Enteric bacteria contain several isozymes suited to different conditions, but the distribution of acetohydroxyacid synthase in soil bacteria is largely unknown. Growth experiments confirmed that Escherichia coli, Salmonella enterica serotype Typhimurium, and Enterobacter aerogenes contain isozymes of acetohydroxyacid synthase, allowing the bacteria to grow in the presence of valine (which causes feedback inhibition of AHAS I) or the sulfonylurea herbicide triasulfuron (which inhibits AHAS II) although a slight lag phase was observed in growth in the latter case. Several common soil isolates were inhibited by triasulfuron, but Pseudomonas fluorescens and Rhodococcus erythropolis were not inhibited by any combination of triasulfuron and valine. The extent of sulfonylurea-sensitive acetohydroxyacid synthase in soil was revealed when 21 out of 27 isolated bacteria in pure culture were inhibited by triasulfuron, the addition of isoleucine and/or valine reversing the effect in 19 cases. Primers were designed to target the genes encoding the large subunits (ilvB, ilvG and ilvI) of acetohydroxyacid synthase from available sequence data and a approximately 355 bp fragment in Bacillus subtilis, Arthrobacter globiformis, E. coli and S. enterica was subsequently amplified. The primers were used to create a small clone library of sequences from an agricultural soil. Phylogenetic analysis revealed significant sequence variation, but all 19 amino acid sequences were most closely related to published large subunit acetohydroxyacid synthase amino acid sequences within several phyla including the Proteobacteria and Actinobacteria. The results suggested the majority of soil microorganisms contain only one functional acetohydroxyacid synthase enzyme sensitive to sulfonylurea herbicides. PMID:17624809

  17. Characterization of Lipoyl Synthase from Mycobacterium tuberculosis.

    PubMed

    Lanz, Nicholas D; Lee, Kyung-Hoon; Horstmann, Abigail K; Pandelia, Maria-Eirini; Cicchillo, Robert M; Krebs, Carsten; Booker, Squire J

    2016-03-01

    The prevalence of multiple and extensively drug-resistant strains of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is on the rise, necessitating the identification of new targets to combat an organism that has infected one-third of the world's population, according to the World Health Organization. The biosynthesis of the lipoyl cofactor is one possible target, given its critical importance in cellular metabolism and the apparent lack of functional salvage pathways in Mtb that are found in humans and many other organisms. The lipoyl cofactor is synthesized de novo in two committed steps, involving the LipB-catalyzed transfer of an octanoyl chain derived from fatty acid biosynthesis to a lipoyl carrier protein and the LipA-catalyzed insertion of sulfur atoms at C6 and C8 of the octanoyl chain. A number of in vitro studies of lipoyl synthases from Escherichia coli, Sulfolobus solfataricus, and Thermosynechococcus elongatus have been conducted, but the enzyme from Mtb has not been characterized. Herein, we show that LipA from Mtb contains two [4Fe-4S] clusters and converts an octanoyl peptide substrate to the corresponding lipoyl peptide product via the same C6-monothiolated intermediate as that observed in the E. coli LipA reaction. In addition, we show that LipA from Mtb forms a complex with the H protein of the glycine cleavage system and that the strength of association is dependent on the presence of S-adenosyl-l-methionine. We also show that LipA from Mtb can complement a lipA mutant of E. coli, demonstrating the commonalities of the two enzymes. Lastly, we show that the substrate for LipA, which normally acts on a post-translationally modified protein, can be reduced to carboxybenzyl-octanoyllysine. PMID:26841001

  18. Nitric oxide synthases: structure, function and inhibition.

    PubMed Central

    Alderton, W K; Cooper, C E; Knowles, R G

    2001-01-01

    This review concentrates on advances in nitric oxide synthase (NOS) structure, function and inhibition made in the last seven years, during which time substantial advances have been made in our understanding of this enzyme family. There is now information on the enzyme structure at all levels from primary (amino acid sequence) to quaternary (dimerization, association with other proteins) structure. The crystal structures of the oxygenase domains of inducible NOS (iNOS) and vascular endothelial NOS (eNOS) allow us to interpret other information in the context of this important part of the enzyme, with its binding sites for iron protoporphyrin IX (haem), biopterin, L-arginine, and the many inhibitors which interact with them. The exact nature of the NOS reaction, its mechanism and its products continue to be sources of controversy. The role of the biopterin cofactor is now becoming clearer, with emerging data implicating one-electron redox cycling as well as the multiple allosteric effects on enzyme activity. Regulation of the NOSs has been described at all levels from gene transcription to covalent modification and allosteric regulation of the enzyme itself. A wide range of NOS inhibitors have been discussed, interacting with the enzyme in diverse ways in terms of site and mechanism of inhibition, time-dependence and selectivity for individual isoforms, although there are many pitfalls and misunderstandings of these aspects. Highly selective inhibitors of iNOS versus eNOS and neuronal NOS have been identified and some of these have potential in the treatment of a range of inflammatory and other conditions in which iNOS has been implicated. PMID:11463332

  19. Tertiary model of a plant cellulose synthase

    PubMed Central

    Sethaphong, Latsavongsakda; Haigler, Candace H.; Kubicki, James D.; Zimmer, Jochen; Bonetta, Dario; DeBolt, Seth; Yingling, Yaroslava G.

    2013-01-01

    A 3D atomistic model of a plant cellulose synthase (CESA) has remained elusive despite over forty years of experimental effort. Here, we report a computationally predicted 3D structure of 506 amino acids of cotton CESA within the cytosolic region. Comparison of the predicted plant CESA structure with the solved structure of a bacterial cellulose-synthesizing protein validates the overall fold of the modeled glycosyltransferase (GT) domain. The coaligned plant and bacterial GT domains share a six-stranded β-sheet, five α-helices, and conserved motifs similar to those required for catalysis in other GT-2 glycosyltransferases. Extending beyond the cross-kingdom similarities related to cellulose polymerization, the predicted structure of cotton CESA reveals that plant-specific modules (plant-conserved region and class-specific region) fold into distinct subdomains on the periphery of the catalytic region. Computational results support the importance of the plant-conserved region and/or class-specific region in CESA oligomerization to form the multimeric cellulose–synthesis complexes that are characteristic of plants. Relatively high sequence conservation between plant CESAs allowed mapping of known mutations and two previously undescribed mutations that perturb cellulose synthesis in Arabidopsis thaliana to their analogous positions in the modeled structure. Most of these mutation sites are near the predicted catalytic region, and the confluence of other mutation sites supports the existence of previously undefined functional nodes within the catalytic core of CESA. Overall, the predicted tertiary structure provides a platform for the biochemical engineering of plant CESAs. PMID:23592721

  20. Highly Divergent Mitochondrial ATP Synthase Complexes in Tetrahymena thermophila

    PubMed Central

    Balabaskaran Nina, Praveen; Dudkina, Natalya V.; Kane, Lesley A.; van Eyk, Jennifer E.; Boekema, Egbert J.; Mather, Michael W.; Vaidya, Akhil B.

    2010-01-01

    The F-type ATP synthase complex is a rotary nano-motor driven by proton motive force to synthesize ATP. Its F1 sector catalyzes ATP synthesis, whereas the Fo sector conducts the protons and provides a stator for the rotary action of the complex. Components of both F1 and Fo sectors are highly conserved across prokaryotes and eukaryotes. Therefore, it was a surprise that genes encoding the a and b subunits as well as other components of the Fo sector were undetectable in the sequenced genomes of a variety of apicomplexan parasites. While the parasitic existence of these organisms could explain the apparent incomplete nature of ATP synthase in Apicomplexa, genes for these essential components were absent even in Tetrahymena thermophila, a free-living ciliate belonging to a sister clade of Apicomplexa, which demonstrates robust oxidative phosphorylation. This observation raises the possibility that the entire clade of Alveolata may have invented novel means to operate ATP synthase complexes. To assess this remarkable possibility, we have carried out an investigation of the ATP synthase from T. thermophila. Blue native polyacrylamide gel electrophoresis (BN-PAGE) revealed the ATP synthase to be present as a large complex. Structural study based on single particle electron microscopy analysis suggested the complex to be a dimer with several unique structures including an unusually large domain on the intermembrane side of the ATP synthase and novel domains flanking the c subunit rings. The two monomers were in a parallel configuration rather than the angled configuration previously observed in other organisms. Proteomic analyses of well-resolved ATP synthase complexes from 2-D BN/BN-PAGE identified orthologs of seven canonical ATP synthase subunits, and at least 13 novel proteins that constitute subunits apparently limited to the ciliate lineage. A mitochondrially encoded protein, Ymf66, with predicted eight transmembrane domains could be a substitute for the subunit a

  1. Argininosuccinate synthase: at the center of arginine metabolism

    PubMed Central

    Haines, Ricci J.; Pendleton, Laura C.; Eichler, Duane C.

    2011-01-01

    The levels of L-arginine, a cationic, semi-essential amino acid, are often controlled within a cell at the level of local availability through biosynthesis. The importance of this temporal and spatial control of cellular L-arginine is highlighted by the tissue specific roles of argininosuccinate synthase (argininosuccinate synthetase) (EC 6.3.4.5), as the rate-limiting step in the conversion of L-citrulline to L-arginine. Since its discovery, the function of argininosuccinate synthase has been linked almost exclusively to hepatic urea production despite the fact that alternative pathways involving argininosuccinate synthase were defined, such as its role in providing arginine for creatine and for polyamine biosynthesis. However, it was the discovery of nitric oxide that meaningfully extended our understanding of the metabolic importance of non-hepatic argininosuccinate synthase. Indeed, our knowledge of the number of tissues that manage distinct pools of arginine under the control of argininosuccinate synthase has expanded significantly. PMID:21494411

  2. Sucrose Phosphate Synthase and Sucrose Accumulation at Low Temperature 1

    PubMed Central

    Guy, Charles L.; Huber, Joan L. A.; Huber, Steven C.

    1992-01-01

    The influence of growth temperature on the free sugar and sucrose phosphate synthase content and activity of spinach (Spinacia oleracea) leaf tissue was studied. When plants were grown at 25°C for 3 weeks and then transferred to a constant 5°C, sucrose, glucose, and fructose accumulated to high levels during a 14-d period. Predawn sugar levels increased from 14- to 20-fold over the levels present at the outset of the low-temperature treatment. Sucrose was the most abundant free sugar before, during, and after exposure to 5°C. Leaf sucrose phosphate synthase activity was significantly increased by the low-temperature treatment, whereas sucrose synthase and invertases were not. Synthesis of the sucrose phosphate synthase subunit was increased during and after low-temperature exposure and paralleled an increase in the steady-state level of the subunit. The increases in sucrose and its primary biosynthetic enzyme, sucrose phosphate synthase, are discussed in relation to adjustment of metabolism to low nonfreezing temperature and freezing stress tolerance. Images Figure 1 Figure 2 Figure 3 PMID:16652990

  3. Plasticity and Evolution of (+)-3-Carene Synthase and (−)-Sabinene Synthase Functions of a Sitka Spruce Monoterpene Synthase Gene Family Associated with Weevil Resistance*

    PubMed Central

    Roach, Christopher R.; Hall, Dawn E.; Zerbe, Philipp; Bohlmann, Jörg

    2014-01-01

    The monoterpene (+)-3-carene is associated with resistance of Sitka spruce against white pine weevil, a major North American forest insect pest of pine and spruce. High and low levels of (+)-3-carene in, respectively, resistant and susceptible Sitka spruce genotypes are due to variation of (+)-3-carene synthase gene copy number, transcript and protein expression levels, enzyme product profiles, and enzyme catalytic efficiency. A family of multiproduct (+)-3-carene synthase-like genes of Sitka spruce include the three (+)-3-carene synthases, PsTPS-3car1, PsTPS-3car2, PsTPS-3car3, and the (−)-sabinene synthase PsTPS-sab. Of these, PsTPS-3car2 is responsible for the relatively higher levels of (+)-3-carene in weevil-resistant trees. Here, we identified features of the PsTPS-3car1, PsTPS-3car2, PsTPS-3car3, and PsTPS-sab proteins that determine different product profiles. A series of domain swap and site-directed mutations, supported by structural comparisons, identified the amino acid in position 596 as critical for product profiles dominated by (+)-3-carene in PsTPS-3car1, PsTPS-3car2, and PsTPS-3car3, or (−)-sabinene in PsTPS-sab. A leucine in this position promotes formation of (+)-3-carene, whereas phenylalanine promotes (−)-sabinene. Homology modeling predicts that position 596 directs product profiles through differential stabilization of the reaction intermediate. Kinetic analysis revealed position 596 also plays a role in catalytic efficiency. Mutations of position 596 with different side chain properties resulted in a series of enzymes with different product profiles, further highlighting the inherent plasticity and potential for evolution of alternative product profiles of these monoterpene synthases of conifer defense against insects. PMID:25016016

  4. Subcellular localization of dinoflagellate polyketide synthases and fatty acid synthase activity.

    PubMed

    Van Dolah, Frances M; Zippay, Mackenzie L; Pezzolesi, Laura; Rein, Kathleen S; Johnson, Jillian G; Morey, Jeanine S; Wang, Zhihong; Pistocchi, Rossella

    2013-12-01

    Dinoflagellates are prolific producers of polyketide secondary metabolites. Dinoflagellate polyketide synthases (PKSs) have sequence similarity to Type I PKSs, megasynthases that encode all catalytic domains on a single polypeptide. However, in dinoflagellate PKSs identified to date, each catalytic domain resides on a separate transcript, suggesting multiprotein complexes similar to Type II PKSs. Here, we provide evidence through coimmunoprecipitation that single-domain ketosynthase and ketoreductase proteins interact, suggesting a predicted multiprotein complex. In Karenia brevis (C.C. Davis) Gert Hansen & Ø. Moestrup, previously observed chloroplast localization of PKSs suggested that brevetoxin biosynthesis may take place in the chloroplast. Here, we report that PKSs are present in both cytosol and chloroplast. Furthermore, brevetoxin is not present in isolated chloroplasts, raising the question of what chloroplast-localized PKS enzymes might be doing. Antibodies to K. brevis PKSs recognize cytosolic and chloroplast proteins in Ostreopsis cf. ovata Fukuyo, and Coolia monotis Meunier, which produce different suites of polyketide toxins, suggesting that these PKSs may share common pathways. Since PKSs are closely related to fatty acid synthases (FAS), we sought to determine if fatty acid biosynthesis colocalizes with either chloroplast or cytosolic PKSs. [(3) H]acetate labeling showed fatty acids are synthesized in the cytosol, with little incorporation in chloroplasts, consistent with a Type I FAS system. However, although 29 sequences in a K. brevis expressed sequence tag database have similarity (BLASTx e-value <10(-10) ) to PKSs, no transcripts for either Type I (cytosolic) or Type II (chloroplast) FAS are present. Further characterization of the FAS complexes may help to elucidate the functions of the PKS enzymes identified in dinoflagellates. PMID:27007632

  5. Understanding Plant Cellulose Synthases through a Comprehensive Investigation of the Cellulose Synthase Family Sequences

    PubMed Central

    Carroll, Andrew; Specht, Chelsea D.

    2011-01-01

    The development of cellulose as an organizing structure in the plant cell wall was a key event in both the initial colonization and the subsequent domination of the terrestrial ecosystem by vascular plants. A wealth of experimental data has demonstrated the complicated genetic interactions required to form the large synthetic complex that synthesizes cellulose. However, these results are lacking an extensive analysis of the evolution, specialization, and regulation of the proteins that compose this complex. Here we perform an in-depth analysis of the sequences in the cellulose synthase (CesA) family. We investigate the phylogeny of the CesA family, with emphasis on evolutionary specialization. We define specialized clades and identify the class-specific regions within the CesA sequence that may explain this specialization. We investigate changes in regulation of CesAs by looking at the conservation of proposed phosphorylation sites. We investigate the conservation of sites where mutations have been documented that impair CesA function, and compare these sites to those observed in the closest cellulose synthase-like (Csl) families to better understand what regions may separate the CesAs from other Csls. Finally we identify two positions with strong conservation of the aromatic trait, but lacking conservation of amino acid identity, which may represent residues important for positioning the sugar substrate for catalysis. These analyses provide useful tools for understanding characterized mutations and post-translational modifications, and for informing further experiments to probe CesA assembly, regulation, and function through site-directed mutagenesis or domain swapping experiments. PMID:22629257

  6. Deprotonations in the Reaction of Flavin-Dependent Thymidylate Synthase.

    PubMed

    Stull, Frederick W; Bernard, Steffen M; Sapra, Aparna; Smith, Janet L; Zuiderweg, Erik R P; Palfey, Bruce A

    2016-06-14

    Many microorganisms use flavin-dependent thymidylate synthase (FDTS) to synthesize the essential nucleotide 2'-deoxythymidine 5'-monophosphate (dTMP) from 2'-deoxyuridine 5'-monophosphate (dUMP), 5,10-methylenetetrahydrofolate (CH2THF), and NADPH. FDTSs have a structure that is unrelated to the thymidylate synthase used by humans and a very different mechanism. Here we report nuclear magnetic resonance evidence that FDTS ionizes N3 of dUMP using an active-site arginine. The ionized form of dUMP is largely responsible for the changes in the flavin absorbance spectrum of FDTS upon dUMP binding. dUMP analogues also suggest that the phosphate of dUMP acts as the base that removes the proton from C5 of the dUMP-methylene intermediate in the FDTS-catalyzed reaction. These findings establish additional differences between the mechanisms of FDTS and human thymidylate synthase. PMID:27214228

  7. Evolutionary history of the chitin synthases of eukaryotes.

    PubMed

    Morozov, Alexey A; Likhoshway, Yelena V

    2016-06-01

    Chitin synthases are widespread among eukaryotes and known to have a complex evolutionary history in some of the groups. We have reconstructed the chitin synthase phylogeny using the most taxonomically comprehensive dataset currently available and have shown the presence of independently formed paralogous groups in oomycetes, ciliates, fungi, and all diatoms except raphid pennates. There were also two cases of horizontal gene transfer (HGT): transfer from fungus to early diatoms gave rise to diatom paralogous group, while transfer from raphid pennate diatom to Acantamoeba ancestor is, to our knowledge, restricted to a single gene in amoeba. Early evolution of chitin synthases is heavily obscured by paralogy, and further sequencing effort is necessary. PMID:26887391

  8. Divergence of multimodular polyketide synthases revealed by a didomain structure.

    PubMed

    Zheng, Jianting; Gay, Darren C; Demeler, Borries; White, Mark A; Keatinge-Clay, Adrian T

    2012-07-01

    The enoylreductase (ER) is the final common enzyme from modular polyketide synthases (PKSs) to be structurally characterized. The 3.0 Å-resolution structure of the didomain comprising the ketoreductase (KR) and ER from the second module of the spinosyn PKS reveals that ER shares an ∼600-Å(2) interface with KR distinct from that of the related mammalian fatty acid synthase (FAS). In contrast to the ER domains of the mammalian FAS, the ER domains of the second module of the spinosyn PKS do not make contact across the two-fold axis of the synthase. This monomeric organization may have been necessary in the evolution of multimodular PKSs to enable acyl carrier proteins to access each of their cognate enzymes. The isolated ER domain showed activity toward a substrate analog, enabling us to determine the contributions of its active site residues. PMID:22634636

  9. Divergence of multimodular polyketide synthases revealed by a didomain structure

    PubMed Central

    Zheng, Jianting; Gay, Darren C.; Demeler, Borries; White, Mark A.; Keatinge-Clay, Adrian T.

    2012-01-01

    The enoylreductase (ER) is the final common enzyme from modular polyketide synthases (PKSs) to be structurally characterized. The 3.0 Å resolution structure of the didomain comprised of the ketoreductase (KR) and ER from the second module of the spinosyn PKS reveals that ER shares an ~600 Å2 interface with KR distinct from that of the related mammalian fatty acid synthase (FAS). In contrast to the ER domains of the mammalian FAS, the ER domains of the second module of the spinosyn PKS do not make contact across the twofold axis of the synthase. This monomeric organization may have been necessary in the evolution of multimodular PKSs to enable acyl carrier proteins (ACPs) to access each of their cognate enzymes. The isolated ER domain showed activity towards a substrate analog, enabling the contributions of its active site residues to be determined. PMID:22634636

  10. Plant terpenoid synthases: Molecular biology and phylogenetic analysis

    PubMed Central

    Bohlmann, Jörg; Meyer-Gauen, Gilbert; Croteau, Rodney

    1998-01-01

    This review focuses on the monoterpene, sesquiterpene, and diterpene synthases of plant origin that use the corresponding C10, C15, and C20 prenyl diphosphates as substrates to generate the enormous diversity of carbon skeletons characteristic of the terpenoid family of natural products. A description of the enzymology and mechanism of terpenoid cyclization is followed by a discussion of molecular cloning and heterologous expression of terpenoid synthases. Sequence relatedness and phylogenetic reconstruction, based on 33 members of the Tps gene family, are delineated, and comparison of important structural features of these enzymes is provided. The review concludes with an overview of the organization and regulation of terpenoid metabolism, and of the biotechnological applications of terpenoid synthase genes. PMID:9539701

  11. Synthase-dependent exopolysaccharide secretion in Gram-negative bacteria

    PubMed Central

    Whitney, J.C.; Howell, P.L.

    2014-01-01

    The biosynthesis and export of bacterial cell-surface polysaccharides is known to occur through several distinct mechanisms. Recent advances in the biochemistry and structural biology of several proteins in synthase-dependent polysaccharide secretion systems have identified key conserved components of this pathway in Gram-negative bacteria. These components include an inner-membrane-embedded polysaccharide synthase, a periplasmic tetratricopeptide repeat (TPR)-containing scaffold protein, and an outer-membrane β-barrel porin. There is also increasing evidence that many synthase-dependent systems are post-translationally regulated by the bacterial second messenger bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP). Here, we compare these core proteins in the context of the alginate, cellulose, and poly-β-D-N-acetylglucosamine (PNAG) secretion systems. PMID:23117123

  12. The beta subunit of glyceraldehyde 3-phosphate dehydrogenase is an important factor for maintaining photosynthesis and plant development under salt stress-Based on an integrative analysis of the structural, physiological and proteomic changes in chloroplasts in Thellungiella halophila.

    PubMed

    Chang, Lili; Guo, Anping; Jin, Xiang; Yang, Qian; Wang, Dan; Sun, Yong; Huang, Qixing; Wang, Limin; Peng, Cunzhi; Wang, Xuchu

    2015-07-01

    Thellungiella halophila, a new model halophyte, can survive under highly saline conditions. We performed comparative proteomics of chloroplasts from plants grown under different saline conditions. Seventy-five salt-responsive proteins were positively identified by mass spectrometry, which represented 43 unique ones. These proteins were categorized into 7 main pathways: light reaction, carbon fixation, energy metabolism, antenna proteins, cell structure, and protein degradation and folding. Saline conditions increased the abundance of proteins involved in photosynthesis, energy metabolism and cell structure. The results indicated that Thellungiella could withstand high salinity by maintaining normal or high photosynthetic capacity, reducing ROS production, as well as enhancing energy usage. Meanwhile, the ultrastructural and physiological data also agree with chloroplast proteomics results. Subsequently, the glyceraldehydes 3-phosphate dehydrogenase beta subunit (GAPB) involved in carbon fixation was selected and its role in salt tolerance was clarified by over-expressing it in Arabidopsis. ThGAPB-overexpressing plants had higher total chlorophyll contents, dry weights, water contents and survival rates than that of wild type plants. These results indicated that ThGAPB might improve plant salt tolerance by maintaining higher recycling rates of ADP and NADP(+) to decrease ROS production, helping to maintain photosynthetic efficiency and plant development under saline conditions. PMID:26025536

  13. Insulin-induced activation of glycerol-3-phosphate acyltransferase by a chiro-inositol-containing insulin mediator is defective in adipocytes of insulin-resistant, type II diabetic, Goto-Kakizaki rats.

    PubMed Central

    Farese, R V; Standaert, M L; Yamada, K; Huang, L C; Zhang, C; Cooper, D R; Wang, Z; Yang, Y; Suzuki, S; Toyota, T

    1994-01-01

    Type II diabetic Goto-Kakizaki (GK) rats were insulin-resistant in euglycemic-hyperinsulinemic clamp studies. We therefore examined insulin signaling systems in control Wistar and diabetic GK rats. Glycerol-3-phosphate acyltransferase (G3PAT), which is activated by headgroup mediators released from glycosyl-phosphatidylinositol (GPI), was activated by insulin in intact and cell-free adipocyte preparations of control, but not diabetic, rats. A specific chiro-inositol-containing inositol phosphoglycan (IPG) mediator, prepared from beef liver, bypassed this defect and comparably activated G3PAT in cell-free adipocyte preparations of both diabetic GK and control rats. A myo-inositol-containing IPG mediator did not activate G3PAT. Relative to control adipocytes, labeling of GPI by [3H]glucosamine was diminished by 50% and insulin failed to stimulate GPI hydrolysis in GK adipocytes. In contrast to GPI-dependent G3PAT activation, insulin-stimulated hexose transport was intact in adipocytes and soleus and gastrocnemius muscles of the GK rat, as was insulin-induced activation of mitogen-activated protein kinase and protein kinase C. We conclude that (i) chiro-inositol-containing IPG mediator activates G3PAT during insulin action, (ii) diabetic GK rats have a defect in synthesizing or releasing functional chiro-inositol-containing IPG, and (iii) defective IPG-regulated intracellular glucose metabolism contributes importantly to insulin resistance in diabetic GK rats. PMID:7972005

  14. Specificities of the Acyl-Acyl Carrier Protein (ACP) Thioesterase and Glycerol-3-Phosphate Acyltransferase for Octadecenoyl-ACP Isomers (Identification of a Petroselinoyl-ACP Thioesterase in Umbelliferae).

    PubMed Central

    Dormann, P.; Frentzen, M.; Ohlrogge, J. B.

    1994-01-01

    This study was designed to address the question: How specific for double bond position and conformation are plant enzymes that act on oleoyl-acyl carrier protein (ACP)? Octadecenoyl-ACPs with cis double bonds at positions [delta]6, [delta]7, [delta]8, [delta]9, [delta]10, [delta]11, or [delta]12 and elaidyl (18:1[delta]9trans)-ACP were synthesized and used to characterize the substrate specificity of the acyl-ACP thioesterase and acyl-ACP:sn-glycerol-3-phosphate acyltransferase. The two enzymes were found to be specific for the [delta]9 position of the double bond. The thioesterase was highly specific for the [delta]9 cis conformation, but the transferase was almost equally active with the cis and the trans isomer of 18:1[delta]9-ACP. In plants such as the Umbelliferae species coriander (Coriandrum sativum L.) that accumulate petroselinic acid (18:1[delta]6cis) in their seed triacylglycerols, a high petroselinoyl-ACP thioesterase activity was found in addition to the oleoyl-ACP thioesterase. The two activities could be separated by anion-exchange chromatography, indicating that the petroselinoyl-ACP thioesterase is represented by a distinct polypeptide. PMID:12232130

  15. Mapping a kingdom-specific functional domain of squalene synthase.

    PubMed

    Linscott, Kristin B; Niehaus, Thomas D; Zhuang, Xun; Bell, Stephen A; Chappell, Joe

    2016-09-01

    Squalene synthase catalyzes the first committed step in sterol biosynthesis and consists of both an amino-terminal catalytic domain and a carboxy-terminal domain tethering the enzyme to the ER membrane. While the overall architecture of this enzyme is identical in eukaryotes, it was previously shown that plant and animal genes cannot complement a squalene synthase knockout mutation in yeast unless the carboxy-terminal domain is swapped for one of fungal origin. This implied a unique component of the fungal carboxy-terminal domain was responsible for the complementation phenotype. To identify this motif, we used Saccharomyces cerevisiae with a squalene synthase knockout mutation, and expressed intact and chimeric squalene synthases originating from fungi, plants, and animals. In contrast to previous observations, all enzymes tested could partially complement the knockout mutation when the genes were weakly expressed. However, when highly expressed, non-fungal squalene synthases could not complement the yeast mutation and instead led to the accumulation of a toxic intermediate(s) as defined by mutations of genes downstream in the ergosterol pathway. Restoration of the complete complementation phenotype was mapped to a 26-amino acid hinge region linking the catalytic and membrane-spanning domains specific to fungal squalene synthases. Over-expression of the C-terminal domain containing a hinge domain from fungi, not from animals or plants, led to growth inhibition of wild-type yeast. Because this hinge region is unique to and highly conserved within each kingdom of life, the data suggests that the hinge domain plays an essential functional role, such as assembly of ergosterol multi-enzyme complexes in fungi. PMID:27320012

  16. Twisting and subunit rotation in single FOF1-ATP synthase

    PubMed Central

    Sielaff, Hendrik; Börsch, Michael

    2013-01-01

    FOF1-ATP synthases are ubiquitous proton- or ion-powered membrane enzymes providing ATP for all kinds of cellular processes. The mechanochemistry of catalysis is driven by two rotary nanomotors coupled within the enzyme. Their different step sizes have been observed by single-molecule microscopy including videomicroscopy of fluctuating nanobeads attached to single enzymes and single-molecule Förster resonance energy transfer. Here we review recent developments of approaches to monitor the step size of subunit rotation and the transient elastic energy storage mechanism in single FOF1-ATP synthases. PMID:23267178

  17. Exploiting the Biosynthetic Potential of Type III Polyketide Synthases.

    PubMed

    Lim, Yan Ping; Go, Maybelle K; Yew, Wen Shan

    2016-01-01

    Polyketides are structurally and functionally diverse secondary metabolites that are biosynthesized by polyketide synthases (PKSs) using acyl-CoA precursors. Recent studies in the engineering and structural characterization of PKSs have facilitated the use of target enzymes as biocatalysts to produce novel functionally optimized polyketides. These compounds may serve as potential drug leads. This review summarizes the insights gained from research on type III PKSs, from the discovery of chalcone synthase in plants to novel PKSs in bacteria and fungi. To date, at least 15 families of type III PKSs have been characterized, highlighting the utility of PKSs in the development of natural product libraries for therapeutic development. PMID:27338328

  18. Analysis of the cercosporin polyketide synthase CTB1 reveals a new fungal thioesterase function

    PubMed Central

    Newman, Adam G.; Vagstad, Anna L.; Belecki, Katherine; Scheerer, Jonathan R.

    2012-01-01

    The polyketide synthase CTB1 is demonstrated to catalyze pyrone formation thereby expanding the known biosynthetic repertoire of thioesterase domains in iterative, non-reducing polyketide synthases. PMID:23108075

  19. Identification of a cryptic type III polyketide synthase (1,3,6,8-tetrahydroxynaphthalene synthase) from Streptomyces peucetius ATCC 27952.

    PubMed

    Ghimire, Gopal Prasad; Oh, Tae-Jin; Liou, Kwangkyoung; Sohng, Jae Kyung

    2008-10-31

    We identified a 1,134-bp putative type III polyketide synthase from the sequence analysis of Streptomyces peucetius ATCC 27952, named Sp-RppA, which is characterized as 1,3,6,8-tetrahydroxynaphthalene synthase and shares 33% identity with SCO1206 from S. coelicolor A3(2) and 32% identity with RppA from S. griseus. The 1,3,6,8-tetrahydroxynaphthalene synthase is known to catalyze the sequential decarboxylative condensation, intramolecular cyclization, and aromatization of an oligoketide derived from five units of malonyl-CoA to give 1,3,6,8-tetrahydroxynaphthalene, which spontaneously oxidizes to form 2,5,7-trihydroxy-1,4-naphthoquinone (flaviolin). In this study, we report the in vivo expression and in vitro synthesis of flaviolin from purified gene product (Sp-RppA). PMID:18612244

  20. Benzophenone Synthase and Chalcone Synthase Accumulate in the Mesophyll of Hypericum perforatum Leaves at Different Developmental Stages.

    PubMed

    Belkheir, Asma K; Gaid, Mariam; Liu, Benye; Hänsch, Robert; Beerhues, Ludger

    2016-01-01

    The active medicinal constituents in Hypericum perforatum, used to treat depression and skin irritation, include flavonoids and xanthones. The carbon skeletons of these compounds are formed by chalcone synthase (CHS) and benzophenone synthase (BPS), respectively. Polyclonal antisera were raised against the polyketide synthases from Hypericum androsaemum and their IgG fractions were isolated. Immunoblotting and immunotitration were used to test the IgGs for crossreactivity and monospecificity in H. perforatum leaf protein extract. Immunofluorescence localization revealed that both CHS and BPS are located in the mesophyll. The maximum fluorescence levels were observed in approx. 0.5 and 1 cm long leaves, respectively. The fluorescence intensity observed for CHS significantly exceeded that for BPS. Using histochemical staining, flavonoids were detected in the mesophyll, indicating that the sites of biosynthesis and accumulation coincide. Our results help understand the biosynthesis and underlying regulation of active H. perforatum constituents. PMID:27446151

  1. Benzophenone Synthase and Chalcone Synthase Accumulate in the Mesophyll of Hypericum perforatum Leaves at Different Developmental Stages

    PubMed Central

    Belkheir, Asma K.; Gaid, Mariam; Liu, Benye; Hänsch, Robert; Beerhues, Ludger

    2016-01-01

    The active medicinal constituents in Hypericum perforatum, used to treat depression and skin irritation, include flavonoids and xanthones. The carbon skeletons of these compounds are formed by chalcone synthase (CHS) and benzophenone synthase (BPS), respectively. Polyclonal antisera were raised against the polyketide synthases from Hypericum androsaemum and their IgG fractions were isolated. Immunoblotting and immunotitration were used to test the IgGs for crossreactivity and monospecificity in H. perforatum leaf protein extract. Immunofluorescence localization revealed that both CHS and BPS are located in the mesophyll. The maximum fluorescence levels were observed in approx. 0.5 and 1 cm long leaves, respectively. The fluorescence intensity observed for CHS significantly exceeded that for BPS. Using histochemical staining, flavonoids were detected in the mesophyll, indicating that the sites of biosynthesis and accumulation coincide. Our results help understand the biosynthesis and underlying regulation of active H. perforatum constituents. PMID:27446151

  2. Structure and mechanism of the diterpene cyclase ent-copalyl diphosphate synthase

    SciTech Connect

    Köksal, Mustafa; Hu, Huayou; Coates, Robert M.; Peters, Reuben J.; Christianson, David W.

    2011-09-20

    The structure of ent-copalyl diphosphate synthase reveals three {alpha}-helical domains ({alpha}, {beta} and {gamma}), as also observed in the related diterpene cyclase taxadiene synthase. However, active sites are located at the interface of the {beta}{gamma} domains in ent-copalyl diphosphate synthase but exclusively in the {alpha} domain of taxadiene synthase. Modular domain architecture in plant diterpene cyclases enables the evolution of alternative active sites and chemical strategies for catalyzing isoprenoid cyclization reactions.

  3. Loop residues and catalysis in OMP synthase.

    PubMed

    Wang, Gary P; Hansen, Michael Riis; Grubmeyer, Charles

    2012-06-01

    Residue-to-alanine mutations and a two-amino acid deletion have been made in the highly conserved catalytic loop (residues 100-109) of Salmonella typhimurium OMP synthase (orotate phosphoribosyltransferase, EC 2.4.2.10). As described previously, the K103A mutant enzyme exhibited a 10(4)-fold decrease in k(cat)/K(M) for PRPP; the K100A enzyme suffered a 50-fold decrease. Alanine mutations at His105 and Glu107 produced 40- and 7-fold decreases in k(cat)/K(M), respectively, and E101A, D104A, and G106A were slightly faster than the wild-type (WT) in terms of k(cat), with minor effects on k(cat)/K(M). Equilibrium binding of OMP or PRPP in binary complexes was affected little by loop mutation, suggesting that the energetics of ground-state binding have little contribution from the catalytic loop, or that a favorable binding energy is offset by costs of loop reorganization. Pre-steady-state kinetics for mutants showed that K103A and E107A had lost the burst of product formation in each direction that indicated rapid on-enzyme chemistry for WT, but that the burst was retained by H105A. Δ102Δ106, a loop-shortened enzyme with Ala102 and Gly106 deleted, showed a 10(4)-fold reduction of k(cat) but almost unaltered K(D) values for all four substrate molecules. The 20% (i.e., 1.20) intrinsic [1'-(3)H]OMP kinetic isotope effect (KIE) for WT is masked because of high forward and reverse commitment factors. K103A failed to express intrinsic KIEs fully (1.095 ± 0.013). In contrast, H105A, which has a smaller catalytic lesion, gave a [1'-(3)H]OMP KIE of 1.21 ± 0.0005, and E107A (1.179 ± 0.0049) also gave high values. These results are interpreted in the context of the X-ray structure of the complete substrate complex for the enzyme [Grubmeyer, C., Hansen, M. R., Fedorov, A. A., and Almo, S. C. (2012) Biochemistry 51 (preceding paper in this issue, DOI 10.1021/bi300083p )]. The full expression of KIEs by H105A and E107A may result from a less secure closure of the catalytic loop

  4. SUGARBEET ROOT SUCROSE SYNTHASE ISOFORMS DIFFER IN DEVELOPMENTAL EXPRESSION, SUBUNIT COMPOSITION AND RESPONSE TO PH.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two sucrose synthase isoforms have been identified by activity stained isoelectric focused polyacrylamide electrophoresis in developing sugarbeet (Beta vulgaris L.) root. Sucrose synthase isoform I (SuSyI) was present from the early stages of development to maturity. Sucrose synthase isoform II (S...

  5. Malate Synthase Activity in Cotton and Other Ungerminated Oilseeds

    PubMed Central

    Miernyk, Jan A.; Trelease, Richard N.; Choinski, John S.

    1979-01-01

    Extracts from several species and varieties of ungerminated cotton seeds plus homogenates from 18 other oilseeds (representing 11 different families) were examined for malate synthase and isocitrate lyase activity. Malate synthase activities in the various cotton seeds ranged from 35 to 129% of the units per dry seed weight found in Deltapine 16 cotton. For other oilseeds, the range was from 0.3 to 58% of Deltapine 16 cotton. Castor bean (Ricinus communis L.) had the least activity per mg dry weight (12-fold lower than the next lowest species), while Pima cotton (Gossypium barbadense L.) had the highest level (8.53 units). On a per seed basis, these values were 15 and 747 nanomoles per minute. Malate synthase activity was measurable in all seed types examined, whereas isocitrate lyase could not be detected in any of the seeds. We suggest that synthesis of malate synthase during seed development is universal among oilseeds in the absence of glyoxylate-cycle-associated isocitrate lyase activity. PMID:16660858

  6. Subcellular targeting and trafficking of nitric oxide synthases

    PubMed Central

    Oess, Stefanie; Icking, Ann; Fulton, David; Govers, Roland; Müller-Esterl, Werner

    2006-01-01

    Unlike most other endogenous messengers that are deposited in vesicles, processed on demand and/or secreted in a regulated fashion, NO (nitric oxide) is a highly active molecule that readily diffuses through cell membranes and thus cannot be stored inside the producing cell. Rather, its signalling capacity must be controlled at the levels of biosynthesis and local availability. The importance of temporal and spatial control of NO production is highlighted by the finding that differential localization of NO synthases in cardiomyocytes translates into distinct effects of NO in the heart. Thus NO synthases belong to the most tightly controlled enzymes, being regulated at transcriptional and translational levels, through co- and post-translational modifications, by substrate availability and not least via specific sorting to subcellular compartments, where they are in close proximity to their target proteins. Considerable efforts have been made to elucidate the molecular mechanisms that underlie the intracellular targeting and trafficking of NO synthases, to ultimately understand the cellular pathways controlling the formation and function of this powerful signalling molecule. In the present review, we discuss the mechanisms and triggers for subcellular routing and dynamic redistribution of NO synthases and the ensuing consequences for NO production and action. PMID:16722822

  7. Genetics Home Reference: N-acetylglutamate synthase deficiency

    MedlinePlus

    ... of reactions that occurs in liver cells. This cycle processes excess nitrogen, generated when protein is used by the body, to make a compound called urea that is excreted by the kidneys. The ... cycle. In people with N-acetylglutamate synthase deficiency , N- ...

  8. Incremental truncation of PHA synthases results in altered product specificity.

    PubMed

    Wang, Qian; Xia, Yongzhen; Chen, Quan; Qi, Qingsheng

    2012-05-10

    PHA synthase is the key enzyme involved in the biosynthesis of microbial polymers, polyhydroxyalkanoates (PHA). In this study, we created a hybrid library of PHA synthase gene with different crossover points by an incremental truncation method between the C-terminal fragments of the phaC(Cn) (phaC from Cupriavidus necator) and the N-terminal fragments of the phaC1(Pa) (phaC from Pseudomonas aeruginosa). As the truncation of the hybrid enzyme increased, the in vivo PHB synthesis ability of the hybrids declined gradually. PHA synthase PhaC(Cn) with a deletion on N-terminal up to 83 amino acid residues showed no synthase activity. While with the removal of up to 270 amino acids from the N-terminus, the activity of the truncated PhaC(Cn) could be complemented by the N-terminus of PhaC1(Pa). Three of the hybrid enzymes W188, W235 and W272 (named by the deleted nucleic acid number) were found to have altered product specificities. PMID:22500895

  9. Polyhydroyxalkanoate synthase fusions as a strategy for oriented enzyme immobilisation.

    PubMed

    Hooks, David O; Venning-Slater, Mark; Du, Jinping; Rehm, Bernd H A

    2014-01-01

    Polyhydroxyalkanoate (PHA) is a carbon storage polymer produced by certain bacteria in unbalanced nutrient conditions. The PHA forms spherical inclusions surrounded by granule associate proteins including the PHA synthase (PhaC). Recently, the intracellular formation of PHA granules with covalently attached synthase from Ralstonia eutropha has been exploited as a novel strategy for oriented enzyme immobilisation. Fusing the enzyme of interest to PHA synthase results in a bifunctional protein able to produce PHA granules and immobilise the active enzyme of choice to the granule surface. Functionalised PHA granules can be isolated from the bacterial hosts, such as Escherichia coli, and maintain enzymatic activity in a wide variety of assay conditions. This approach to oriented enzyme immobilisation has produced higher enzyme activities and product levels than non-oriented immobilisation techniques such as protein inclusion based particles. Here, enzyme immobilisation via PHA synthase fusion is reviewed in terms of the genetic designs, the choices of enzymes, the control of enzyme orientations, as well as their current and potential applications. PMID:24962396

  10. A geraniol-synthase gene from Cinnamomum tenuipilum.

    PubMed

    Yang, Tao; Li, Jing; Wang, Hao-Xin; Zeng, Ying

    2005-02-01

    Geraniol may accumulate up to 86-98% of the leaf essential oils in geraniol chemotypes of the evergreen camphor tree Cinnamomum tenuipilum. A similarity-based cloning strategy yielded a cDNA clone that appeared to encode a terpene synthase and which could be phylogenetically grouped within the angiosperm monoterpene synthase/subfamily. After its expression in Escherichia coli and enzyme assay with prenyl diphosphates as substrates, the enzyme encoded by the putative C. tenuipilum monoterpene synthase gene was shown to specifically convert geranyl diphosphate to geraniol as a single product by GC-MS analysis. Biochemical characterization of the partially purified recombinant protein revealed a strong dependency for Mg2+ and Mn2+, and an apparent Michaelis constant of 55.8 microM for geranyl diphosphate. Thus, a new member of the monoterpene synthase family was identified and designated as CtGES. The genome contains a single copy of CtGES gene. Expression of CtGES was exclusively observed in the geraniol chemotype of C. tenuipilum. Furthermore, in situ hybridization analysis demonstrated that CtGES mRNA was localized in the oil cells of the leaves. PMID:15680985

  11. A particular phenotype in a girl with aldosterone synthase deficiency.

    PubMed

    Williams, Tracy A; Mulatero, Paolo; Bosio, Maurizio; Lewicka, Sabina; Palermo, Mario; Veglio, Franco; Armanini, Decio

    2004-07-01

    Aldosterone synthase deficiency (ASD) usually presents in infancy as a life-threatening electrolyte imbalance. A 4-wk-old child of unrelated parents was examined for failure to thrive and salt-wasting. Notable laboratory findings were hyperkalemia, high plasma renin, and low-normal aldosterone levels. Urinary metabolite ratios of corticosterone/18-hydroxycorticosterone and 18-hydroxycorticosterone/aldosterone were intermediate between ASD type I and type II. Sequence analysis of CYP11B2, the gene encoding aldosterone synthase (P450c11AS), revealed that the patient was a compound heterozygote carrying a previously described mutation located in exon 4 causing a premature stop codon (E255X) and a further, novel mutation in exon 5 that also causes a premature stop codon (Q272X). The patient's unaffected father was a heterozygous carrier of the E255X mutation, whereas the unaffected mother was a heterozygous carrier of the Q272X mutation. Therefore, the patient's CYP11B2 encodes two truncated forms of aldosterone synthase predicted to be inactive because they lack critical active site residues as well as the heme-binding site. This case of ASD is of particular interest because despite the apparent lack of aldosterone synthase activity, the patient displays low-normal aldosterone levels, thus raising the question of its source. PMID:15240589

  12. Inhibition of ATP Synthase by Chlorinated Adenosine Analogue

    PubMed Central

    Chen, Lisa S.; Nowak, Billie J.; Ayres, Mary L.; Krett, Nancy L.; Rosen, Steven T.; Zhang, Shuxing; Gandhi, Varsha

    2009-01-01

    8-Chloroadenosine (8-Cl-Ado) is a ribonucleoside analogue that is currently in clinical trial for chronic lymphocytic leukemia. Based on the decline in cellular ATP pool following 8-Cl-Ado treatment, we hypothesized that 8-Cl-ADP and 8-Cl-ATP may interfere with ATP synthase, a key enzyme in ATP production. Mitochondrial ATP synthase is composed of two major parts; FO intermembrane base and F1 domain, containing α and β subunits. Crystal structures of both α and β subunits that bind to the substrate, ADP, are known in tight binding (αdpβdp) and loose binding (αtpβtp) states. Molecular docking demonstrated that 8-Cl-ADP/8-Cl-ATP occupied similar binding modes as ADP/ATP in the tight and loose binding sites of ATP synthase, respectively, suggesting that the chlorinated nucleotide metabolites may be functional substrates and inhibitors of the enzyme. The computational predictions were consistent with our whole cell biochemical results. Oligomycin, an established pharmacological inhibitor of ATP synthase, decreased both ATP and 8-Cl-ATP formation from exogenous substrates, however, did not affect pyrimidine nucleoside analogue triphosphate accumulation. Synthesis of ATP from ADP was inhibited in cells loaded with 8-Cl-ATP. These biochemical studies are in consent with the computational modeling; in the αtpβtp state 8-Cl-ATP occupies similar binding as ANP, a non-hydrolyzable ATP mimic that is a known inhibitor. Similarly, in the substrate binding site (αdpβdp) 8-Cl-ATP occupies a similar position as ATP mimic ADP-BeF3 −. Collectively, our current work suggests that 8-Cl-ADP may serve as a substrate and the 8-Cl-ATP may be an inhibitor of ATP synthase. PMID:19477165

  13. Purification of a dichlorophenol-indophenol oxidoreductase from rat and bovine synaptic membranes: tight complex association of a glyceraldehyde-3-phosphate dehydrogenase isoform, TOAD64, enolase-gamma and aldolase C.

    PubMed Central

    Bulliard, C; Zurbriggen, R; Tornare, J; Faty, M; Dastoor, Z; Dreyer, J L

    1997-01-01

    NADH-dichlorophenol-indophenol oxidoreductases (PMOs) were purified from synaptic plasma membranes or synaptic vesicles (small recycling vesicles) from both bovine and rat brains and from a neuroblastoma cell line, NB41A3. Several isoforms could be identified in purified plasma membranes and vesicles. Purification of the enzyme activity involved protein extraction with detergents, (NH4)2SO4 precipitation, chromatography under stringent conditions and native PAGE. PMO activity could be attributed to a very tight complex of several proteins that could not be separated except by SDS/PAGE. SDS/PAGE resolved the purified complex into at least five proteins, which could be micro-sequenced and identified unambiguously as hsc70, TOAD64 and glyceraldehyde-3-phosphate dehydrogenase tightly associated with the brain-specific proteins aldolase C and enolase-gamma. Enzyme activity could be purified from both synaptic plasma membranes and recycling vesicles, yields being much greater from the latter source. Highly purified plasma membranes (prepared from a neuroblastoma cell line NB41A3 by iminobiotinylation of intact cells and affinity purification with avidin and anti-avidin antibodies under very stringent conditions) also displayed PMO activity tightly associated with TOAD64. The association of PMO in a tight complex was confirmed by its immunoprecipitation from cellular and membrane extracts of NB41A3 using antibodies directed against any component protein of the complex followed by immunodetection with antibodies directed against the other members. Antibodies also inhibited the enzyme activity synergistically. In addition, induction of the different components of the complex during dichlorophenol-indophenol stress was demonstrated by the S1 RNase-protection assay in synchronized NB41A3 cells. The role of the complex in membrane fusion and cellular response to extracellular oxidative stress during growth and development is discussed. PMID:9182718

  14. Molecular and immunological characterisation of recombinant Brucella abortus glyceraldehyde-3-phosphate-dehydrogenase, a T- and B-cell reactive protein that induces partial protection when co-administered with an interleukin-12-expressing plasmid in a DNA vaccine formulation.

    PubMed

    Rosinha, Gracia M S; Myioshi, Anderson; Azevedo, Vasco; Splitter, Gary A; Oliveira, Sergio C

    2002-08-01

    To identify antigens of Brucella spp. that are potentially involved in stimulating a protective T-cell-mediated immune response, previous studies identified 10 clones from a Brucella abortus 2308 genomic library with primed lymphocytes as probes. One selected positive clone (182) contained an insert of 1.2 kb which was identified, sequenced and characterised. The deduced amino acid sequence of the open reading frame (ORF) revealed 82% and 81% identity to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) enzymes from Agrobacterium tumefaciens and Xanthobacter flavus, respectively. Southern blot analysis demonstrated that the gap gene is present in only one copy in the Brucella genome. B. abortus GAPDH was then expressed in Escherichia coli as a fusion protein with the maltose-binding protein (MBP). To demonstrate the functional activity of Brucella GAPDH, E. coli gap mutants were transformed with a Brucella pMAL-gap construct. Genetic complementation was achieved and as a result E. coli mutants were able to grow on glucose or other carbon source medium. The humoral and cellular immune responses to the recombinant (r) GAPDH were characterised. In Western blots, sera from naturally infected cattle and sheep showed antibody reactivity against rGAPDH. In response to in-vitro stimulation by rGAPDH, splenocytes from mice vaccinated with rGAPDH or B. abortus S19 were able to produce gamma-interferon and tumour necrosis factor-a but not interleukin (IL)-4. Furthermore, gap associated with murine IL-12 gene in a DNA vaccine formulation partially protected mice against experimental infection. PMID:12171297

  15. The Central Polybasic Region of the Soluble SNARE (Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor) Vam7 Affects Binding to Phosphatidylinositol 3-Phosphate by the PX (Phox Homology) Domain.

    PubMed

    Miner, Gregory E; Starr, Matthew L; Hurst, Logan R; Sparks, Robert P; Padolina, Mark; Fratti, Rutilio A

    2016-08-19

    The yeast vacuole requires four SNAREs to trigger membrane fusion including the soluble Qc-SNARE Vam7. The N-terminal PX domain of Vam7 binds to the lipid phosphatidylinositol 3-phosphate (PI3P) and the tethering complex HOPS (homotypic fusion and vacuole protein sorting complex), whereas the C-terminal SNARE motif forms SNARE complexes. Vam7 also contains an uncharacterized middle domain that is predicted to be a coiled-coil domain with multiple helices. One helix contains a polybasic region (PBR) composed of Arg-164, Arg-168, Lys-172, Lys-175, Arg-179, and Lys-186. Polybasic regions are often associated with nonspecific binding to acidic phospholipids including phosphoinositides. Although the PX (phox homology) domain alone binds PI3P, we theorized that the Vam7 PBR could bind to additional acidic phospholipids enriched at fusion sites. Mutating each of the basic residues in the PBR to an alanine (Vam7-6A) led to attenuated vacuole fusion. The defective fusion of Vam7-6A was due in part to inefficient association with its cognate SNAREs and HOPS, yet the overall vacuole association of Vam7-6A was similar to wild type. Experiments testing the binding of Vam7 to specific signaling lipids showed that mutating the PBR to alanines augmented binding to PI3P. The increased binding to PI3P by Vam7-6A likely contributed to the observed wild type levels of vacuole association, whereas protein-protein interactions were diminished. PI3P binding was inhibited when the PX domain mutant Y42A was introduced into Vam7-6A to make Vam7-7A. Thus the Vam7 PBR affects PI3P binding by the PX domain and in turn affects binding to SNAREs and HOPS to support efficient fusion. PMID:27365394

  16. Cannabidiolic-acid synthase, the chemotype-determining enzyme in the fiber-type Cannabis sativa.

    PubMed

    Taura, Futoshi; Sirikantaramas, Supaart; Shoyama, Yoshinari; Yoshikai, Kazuyoshi; Shoyama, Yukihiro; Morimoto, Satoshi

    2007-06-26

    Cannabidiolic-acid (CBDA) synthase is the enzyme that catalyzes oxidative cyclization of cannabigerolic-acid into CBDA, the dominant cannabinoid constituent of the fiber-type Cannabis sativa. We cloned a novel cDNA encoding CBDA synthase by reverse transcription and polymerase chain reactions with degenerate and gene-specific primers. Biochemical characterization of the recombinant enzyme demonstrated that CBDA synthase is a covalently flavinylated oxidase. The structural and functional properties of CBDA synthase are quite similar to those of tetrahydrocannabinolic-acid (THCA) synthase, which is responsible for the biosynthesis of THCA, the major cannabinoid in drug-type Cannabis plants. PMID:17544411

  17. Transgene silencing of sucrose synthase in alfalfa stem vascular tissue by a truncated phosphoenolpyruvate carboxylase: sucrose synthase construct

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An important role of sucrose synthase (SUS, EC 2.4.1.13) in plants is to provide UDP-glucose needed for cellulose synthesis in cell walls. We examined if over-expressing SUS in alfalfa (Medicago sativa L.) would increase cellulose content of stem cell walls. Alfalfa plants were transformed with two ...

  18. Identifying the catalytic components of cellulose synthase and the maize mixed-linkage beta-glucan synthase

    SciTech Connect

    Nicholas C Carpita

    2009-04-20

    Five specific objectives of this project are to develop strategies to identify the genes that encode the catalytic components of "mixed-linkage" (1→3),(1→4)-beta-D-glucans in grasses, to determine the protein components of the synthase complex, and determine the biochemical mechanism of synthesis. We have used proteomic approaches to define intrinsic and extrinsic polypeptides of Golgi membranes that are associated with polysaccharide synthesis and trafficking. We were successful in producing recombinant catalytic domains of cellulose synthase genes and discovered that they dimerize upon concentration, indicating that two CesA proteins form the catalytic unit. We characterized a brittle stalk2 mutant as a defect in a COBRA-like protein that results in compromised lignin-cellulose interactions that decrease tissue flexibility. We used virus-induced gene silencing of barley cell wall polysaccharide synthesis by BSMV in an attempt to silence specific members of the cellulose synthase-like gene family. However, we unexpectedly found that regardless of the specificity of the target gene, whole gene interaction networks were silenced. We discovered the cause to be an antisense transcript of the cellulose synthase gene initiated small interfering RNAs that spread silencing to related genes.

  19. The three tricarboxylate synthase activities of Corynebacterium glutamicum and increase of L-lysine synthesis.

    PubMed

    Radmacher, Eva; Eggeling, Lothar

    2007-09-01

    Corynebacterium glutamicum owns a citrate synthase and two methylcitrate synthases. Characterization of the isolated enzymes showed that the two methylcitrate synthases have comparable catalytic efficiency, k (cat)/K (m), as the citrate synthase with acetyl-CoA as substrate, although these enzymes are only synthesized during growth on propionate-containing media. Thus, the methylcitrate synthases have a relaxed substrate specifity, as also demonstrated by their activity with butyryl-CoA, whereas the citrate synthase does not accept acyl donors other than acetyl-CoA. A double mutant deleted of the citrate synthase gene gltA and one of the methylcitrate synthase genes, prpC1, was made unable to grow on glucose. From this mutant, a collection of suppressor mutants could be isolated which were demonstrated to have regained citrate synthase activity due to the relaxed specificity of the methylcitrate synthase PrpC2. Molecular characterization of these mutants showed that the regulator PrpR (Cg0800) located downstream of prpC1 is mutated with mutations likely to effect the secondary structure of the regulator, thus, resulting in expression of prpC2. This expression results in a citrate synthase activity, which is lower than that due to gltA in the original strain and results in increased L-lysine accumulation. PMID:17653710

  20. Synthesis of delta-aminolaevulinate synthase by isolated liver polyribosomes.

    PubMed Central

    Whiting, M J

    1976-01-01

    1. Postmitochondrial supernatants were prepared from the livers of chick embryos and were incubated under conditions that supported protein synthesis. delta-Aminolaevulinate synthase (EC 2.3.1.37) was synthesized by supernatants from livers treated with the porphyrinogenic drugs 2-allyl-2-isopropylacetamide and/or 3,5-diethoxycarbonyl-1,4-dihydrocollidine, but synthesis by supernatants from normal livers could not be detected. Synthesis of enzyme released from polyribosomes was measured by immunoprecipitation with specific antibody to the mitochondrial enzyme, and the specificity of the reaction was established by electrophoresis of dissociated immunoprecipitates on sodium dodecyl sulphate/polyacrylamide gels. 2. The relative synthesis of delta-aminolaevulinate synthase in vitro was comparable with that previously measured in vivo, and was correlated with the enzyme activity of the liver. 3. Enzyme synthesis in vitro occurred predominantly on free rather than membrane-bound polyribosomes. 4. The mol.wt. of the product synthesized in vitro was 7000 +/- 7000 by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. However, pulse-labelling of the enzyme in vivo confirmed its mol.wt. to be 49000 +/- 5000 when isolated from the mitochondrion. A small amount of immunoprecipitable enzyme of mol.wt. 70000 was detected in the cytosol in vivo. In chick embryo liver, delta-aminolaevulinate synthase therefore appears to be synthesized on cytoplasmic polyribosomes as a polypeptide of mol.wt. 70000, which in vivo is rapidly incorporated into the mitochondrion, and is then extracted as a lower-molecular-weight form. 5. Haemin added to the postmitochondrial supernatant-containing incubation mixture at concentrations up to 10 muM had no effect on general protein synthesis or the synthesis of delta-aminolaevulinate synthase. On the other hand, haemin treatment of induced chick embryo livers in vivo for 3h markedly decreased the relative synthesis of delta

  1. Mechanism of Germacradien-4-ol Synthase-Controlled Water Capture.

    PubMed

    Grundy, Daniel J; Chen, Mengbin; González, Verónica; Leoni, Stefano; Miller, David J; Christianson, David W; Allemann, Rudolf K

    2016-04-12

    The sesquiterpene synthase germacradiene-4-ol synthase (GdolS) from Streptomyces citricolor is one of only a few known high-fidelity terpene synthases that convert farnesyl diphosphate (FDP) into a single hydroxylated product. Crystals of unliganded GdolS-E248A diffracted to 1.50 Å and revealed a typical class 1 sesquiterpene synthase fold with the active site in an open conformation. The metal binding motifs were identified as D(80)DQFD and N(218)DVRSFAQE. Some bound water molecules were evident in the X-ray crystal structure, but none were obviously positioned to quench a putative final carbocation intermediate. Incubations in H2(18)O generated labeled product, confirming that the alcohol functionality arises from nucleophilic capture of the final carbocation by water originating from solution. Site-directed mutagenesis of amino acid residues from both within the metal binding motifs and without identified by sequence alignment with aristolochene synthase from Aspergillus terreus generated mostly functional germacradien-4-ol synthases. Only GdolS-N218Q generated radically different products (∼50% germacrene A), but no direct evidence of the mechanism of incorporation of water into the active site was obtained. Fluorinated FDP analogues 2F-FDP and 15,15,15-F3-FDP were potent noncompetitive inhibitors of GdolS. 12,13-DiF-FDP generated 12,13-(E)-β-farnesene upon being incubated with GdolS, suggesting stepwise formation of the germacryl cation during the catalytic cycle. Incubation of GdolS with [1-(2)H2]FDP and (R)-[1-(2)H]FDP demonstrated that following germacryl cation formation a [1,3]-hydride shift generates the final carbocation prior to nucleophilic capture. The stereochemistry of this shift is not defined, and the deuteron in the final product was scrambled. Because no clear candidate residue for binding of a nucleophilic water molecule in the active site and no significant perturbation of product distribution from the replacement of active site residues

  2. Immunoaffinity purification and characterization of thromboxane synthase from porcine lung.

    PubMed

    Shen, R F; Tai, H H

    1986-09-01

    Thromboxane synthase has been purified 620-fold from porcine lung microsomes by a three-step purification procedure including Lubrol-PX solubilization, reactive blue-agarose chromatography, and immunoaffinity chromatography. The purified enzyme exhibited a single protein band (53,000 daltons) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Rabbit antiserum raised against the purified enzyme immunoprecipitated thromboxane synthase activity from crude enzyme preparations of porcine lung, cow lung, and human platelets, indicating the existence of structural homology of the enzyme in these species. Immunoblotting experiment identified the same polypeptide (53,000 daltons) in porcine lung and a polypeptide of 50,000 daltons in human platelets, confirming the identity of the enzyme and the specificity of the antiserum. Purified thromboxane synthase is a hemoprotein with a Soret-like absorption peak at 418 nm. The enzyme reaction has a Km for 15-hydroxy-9 alpha, 11 alpha-peroxidoprosta-5, 13-dienoic acid of 12 microM, an optimal pH of 7.5, and an optimal temperature of reaction at 30 degrees C. Purified thromboxane synthase catalyzed the formation of both thromboxane B2 and 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT). The ratios of HHT to thromboxane B2 varied from 1.6 to 2.1 dependent on the reaction conditions. Except that HHT was formed at a greater rate, the formation of HHT and that of thromboxane responded identically to pH, temperature, substrate concentration, kinetics of formation, metal ions, and inhibitors suggesting that the two products are probably formed at the same active site via a common intermediate. Thromboxane synthase was irreversibly inactivated by 15-hydroxy-9 alpha, 11 alpha-peroxidoprosta-5,13-dienoic acid during catalysis and by treatment of 15-hydroperoxyeicosatetraenoic acid. The irreversible inactivation, however, could be protected by reversible inhibitors such as sodium (E)-3-[4-(1-imidazolylmethyl)phenyl]-2-propenoate and

  3. Mechanism of Germacradien-4-ol Synthase-Controlled Water Capture

    PubMed Central

    2016-01-01

    The sesquiterpene synthase germacradiene-4-ol synthase (GdolS) from Streptomyces citricolor is one of only a few known high-fidelity terpene synthases that convert farnesyl diphosphate (FDP) into a single hydroxylated product. Crystals of unliganded GdolS-E248A diffracted to 1.50 Å and revealed a typical class 1 sesquiterpene synthase fold with the active site in an open conformation. The metal binding motifs were identified as D80DQFD and N218DVRSFAQE. Some bound water molecules were evident in the X-ray crystal structure, but none were obviously positioned to quench a putative final carbocation intermediate. Incubations in H218O generated labeled product, confirming that the alcohol functionality arises from nucleophilic capture of the final carbocation by water originating from solution. Site-directed mutagenesis of amino acid residues from both within the metal binding motifs and without identified by sequence alignment with aristolochene synthase from Aspergillus terreus generated mostly functional germacradien-4-ol synthases. Only GdolS-N218Q generated radically different products (∼50% germacrene A), but no direct evidence of the mechanism of incorporation of water into the active site was obtained. Fluorinated FDP analogues 2F-FDP and 15,15,15-F3-FDP were potent noncompetitive inhibitors of GdolS. 12,13-DiF-FDP generated 12,13-(E)-β-farnesene upon being incubated with GdolS, suggesting stepwise formation of the germacryl cation during the catalytic cycle. Incubation of GdolS with [1-2H2]FDP and (R)-[1-2H]FDP demonstrated that following germacryl cation formation a [1,3]-hydride shift generates the final carbocation prior to nucleophilic capture. The stereochemistry of this shift is not defined, and the deuteron in the final product was scrambled. Because no clear candidate residue for binding of a nucleophilic water molecule in the active site and no significant perturbation of product distribution from the replacement of active site residues were

  4. Deficiency of sphingomyelin synthase-1 but not sphingomyelin synthase-2 causes hearing impairments in mice.

    PubMed

    Lu, Mei-Hong; Takemoto, Makoto; Watanabe, Ken; Luo, Huan; Nishimura, Masataka; Yano, Masato; Tomimoto, Hidekazu; Okazaki, Toshiro; Oike, Yuichi; Song, Wen-Jie

    2012-08-15

    Sphingomyelin (SM) is a sphingolipid reported to function as a structural component of plasma membranes and to participate in signal transduction. The role of SM metabolism in the process of hearing remains controversial. Here, we examined the role of SM synthase (SMS), which is subcategorized into the family members SMS1 and SMS2, in auditory function. Measurements of auditory brainstem response (ABR) revealed hearing impairment in SMS1−/− mice in a low frequency range (4–16 kHz). As a possible mechanism of this impairment, we found that the stria vascularis (SV) in these mice exhibited atrophy and disorganized marginal cells. Consequently, SMS1−/− mice exhibited significantly smaller endocochlear potentials (EPs). As a possible mechanism for EP reduction, we found altered expression patterns and a reduced level of KCNQ1 channel protein in the SV of SMS1−/− mice. These mice also exhibited reduced levels of distortion product otoacoustic emissions. Quantitative comparison of the SV atrophy, KCNQ1 expression, and outer hair cell density at the cochlear apical and basal turns revealed no location dependence, but more macrophage invasion into the SV was observed in the apical region than the basal region, suggesting a role of cochlear location-dependent oxidative stress in producing the frequency dependence of hearing loss in SMS1−/− mice. Elevated ABR thresholds, decreased EPs, and abnormal KCNQ1 expression patterns in SMS1−/− mice were all found to be progressive with age. Mice lacking SMS2, however, exhibited neither detectable hearing loss nor changes in their EPs. Taken together, our results suggest that hearing impairments occur in SMS1−/− but not SMS2−/− mice. Defects in the SV with subsequent reductions in EPs together with hair cell dysfunction may account, at least partially, for hearing impairments in SMS1−/− mice. PMID:22641779

  5. Hematopoetic prostaglandin D synthase: an ESR1-dependent oviductal epithelial cell synthase.

    PubMed

    Bridges, Phillip J; Jeoung, Myoungkun; Shim, Sarah; Park, Ji Yeon; Lee, Jae Eun; Sapsford, Lindsay A; Trudgen, Kourtney; Ko, Chemyong; Gye, Myung Chan; Jo, Misung

    2012-04-01

    Oviductal disease is a primary cause of infertility, a problem that largely stems from excessive inflammation of this key reproductive organ. Our poor understanding of the mechanisms regulating oviductal inflammation restricts our ability to diagnose, treat, and/or prevent oviductal disease. Using mice, our objective was to determine the spatial localization, regulatory mechanism, and functional attributes of a hypothesized regulator of oviductal inflammation, the hematopoietic form of prostaglandin D synthase (HPGDS). Immunohistochemistry revealed specific localization of HPGDS to the oviduct's epithelium. In the isthmus, expression of HPGDS was consistent. In the ampulla, expression of HPGDS appeared dependent upon stage of the estrous cycle. HPGDS was expressed in the epithelium of immature and cycling mice but not in the oviducts of estrogen receptor α knockouts. Two receptor subtypes bind PGD₂: PGD₂ receptor and G protein-coupled receptor 44. Expression of mRNA for Ptgdr was higher in the epithelial cells (EPI) than in the stroma (P < 0.05), whereas mRNA for Gpr44 was higher in the stroma than epithelium (P < 0.05). Treatment of human oviductal EPI with HQL-79, an inhibitor of HPGDS, decreased cell viability (P < 0.05). Treatment of mice with HQL-79 increased mRNA for chemokine (C-C motif) ligands 3, 4, and 19; chemokine (C-X-C motif) ligands 11 and 12; IL-13 and IL-17B; and TNF receptor superfamily, member 1b (P < 0.02 for each mRNA). Overall, these results suggest that HPGDS may play a role in the regulation of inflammation and EPI health within the oviduct. PMID:22374975

  6. Phasin proteins activate Aeromonas caviae polyhydroxyalkanoate (PHA) synthase but not Ralstonia eutropha PHA synthase.

    PubMed

    Ushimaru, Kazunori; Motoda, Yoko; Numata, Keiji; Tsuge, Takeharu

    2014-05-01

    In this study, we performed in vitro and in vivo activity assays of polyhydroxyalkanoate (PHA) synthases (PhaCs) in the presence of phasin proteins (PhaPs), which revealed that PhaPs are activators of PhaC derived from Aeromonas caviae (PhaCAc). In in vitro assays, among the three PhaCs tested, PhaCAc was significantly activated when PhaPs were added at the beginning of polymerization (prepolymerization PhaCAc), whereas the prepolymerization PhaCRe (derived from Ralstonia eutropha) and PhaCDa (Delftia acidovorans) showed reduced activity with PhaPs. The PhaP-activated PhaCAc showed a slight shift of substrate preference toward 3-hydroxyhexanoyl-CoA (C6). PhaPAc also activated PhaCAc when it was added during polymerization (polymer-elongating PhaCAc), while this effect was not observed for PhaCRe. In an in vivo assay using Escherichia coli TOP10 as the host strain, the effect of PhaPAc expression on PHA synthesis by PhaCAc or PhaCRe was examined. As PhaPAc expression increased, PHA production was increased by up to 2.3-fold in the PhaCAc-expressing strain, whereas it was slightly increased in the PhaCRe-expressing strain. Taken together, this study provides evidence that PhaPs function as activators for PhaCAc both in vitro and in vivo but do not activate PhaCRe. This activating effect may be attributed to the new role of PhaPs in the polymerization reaction by PhaCAc. PMID:24584238

  7. Defining the Product Chemical Space of Monoterpenoid Synthases

    PubMed Central

    Tian, Boxue; Poulter, C. Dale; Jacobson, Matthew P.

    2016-01-01

    Terpenoid synthases create diverse carbon skeletons by catalyzing complex carbocation rearrangements, making them particularly challenging for enzyme function prediction. To begin to address this challenge, we have developed a computational approach for the systematic enumeration of terpenoid carbocations. Application of this approach allows us to systematically define a nearly complete chemical space for the potential carbon skeletons of products from monoterpenoid synthases. Specifically, 18758 carbocations were generated, which we cluster into 74 cyclic skeletons. Five of the 74 skeletons are found in known natural products; some of the others are plausible for new functions, either in nature or engineered. This work systematizes the description of function for this class of enzymes, and provides a basis for predicting functions of uncharacterized enzymes. To our knowledge, this is the first computational study to explore the complete product chemical space of this important class of enzymes. PMID:27517297

  8. Defining the Product Chemical Space of Monoterpenoid Synthases.

    PubMed

    Tian, Boxue; Poulter, C Dale; Jacobson, Matthew P

    2016-08-01

    Terpenoid synthases create diverse carbon skeletons by catalyzing complex carbocation rearrangements, making them particularly challenging for enzyme function prediction. To begin to address this challenge, we have developed a computational approach for the systematic enumeration of terpenoid carbocations. Application of this approach allows us to systematically define a nearly complete chemical space for the potential carbon skeletons of products from monoterpenoid synthases. Specifically, 18758 carbocations were generated, which we cluster into 74 cyclic skeletons. Five of the 74 skeletons are found in known natural products; some of the others are plausible for new functions, either in nature or engineered. This work systematizes the description of function for this class of enzymes, and provides a basis for predicting functions of uncharacterized enzymes. To our knowledge, this is the first computational study to explore the complete product chemical space of this important class of enzymes. PMID:27517297

  9. S-sulfocysteine synthase function in sensing chloroplast redox status

    PubMed Central

    Gotor, Cecilia; Romero, Luis C.

    2013-01-01

    The minor chloroplastic O-acetylserine(thiol)lyase isoform encoded by the CS26 gene in Arabidopsis thaliana has been described as an S-sulfocysteine synthase enzyme that plays an important role in chloroplast function. This enzyme is located in the thylakoid lumen, and its S-sulfocysteine activity is essential for the proper photosynthetic performance of the chloroplast under long-day growth conditions. Based on the present knowledge of this enzyme, we suggest that S-sulfocysteine synthase functions as a protein sensor to detect the accumulation of thiosulfate as a result of the inadequate detoxification of reactive oxygen species generated under conditions of excess light to produce the S-sulfocysteine molecule that triggers protection mechanisms of the photosynthetic apparatus. PMID:23333972

  10. Visualization of cellulose synthases in Arabidopsis secondary cell walls.

    PubMed

    Watanabe, Y; Meents, M J; McDonnell, L M; Barkwill, S; Sampathkumar, A; Cartwright, H N; Demura, T; Ehrhardt, D W; Samuels, A L; Mansfield, S D

    2015-10-01

    Cellulose biosynthesis in plant secondary cell walls forms the basis of vascular development in land plants, with xylem tissues constituting the vast majority of terrestrial biomass. We used plant lines that contained an inducible master transcription factor controlling xylem cell fate to quantitatively image fluorescently tagged cellulose synthase enzymes during cellulose deposition in living protoxylem cells. The formation of secondary cell wall thickenings was associated with a redistribution and enrichment of CESA7-containing cellulose synthase complexes (CSCs) into narrow membrane domains. The velocities of secondary cell wall-specific CSCs were faster than those of primary cell wall CSCs during abundant cellulose production. Dynamic intracellular of endomembranes, in combination with increased velocity and high density of CSCs, enables cellulose to be synthesized rapidly in secondary cell walls. PMID:26450210

  11. Glycerol-3-phosphate acyltransferase-1 upregulation by O-GlcNAcylation of Sp1 protects against hypoxia-induced mouse embryonic stem cell apoptosis via mTOR activation.

    PubMed

    Lee, H J; Ryu, J M; Jung, Y H; Lee, K H; Kim, D I; Han, H J

    2016-01-01

    Oxygen signaling is critical for stem cell regulation, and oxidative stress-induced stem cell apoptosis decreases the efficiency of stem cell therapy. Hypoxia activates O-linked β-N-acetyl glucosaminylation (O-GlcNAcylation) of stem cells, which contributes to regulation of cellular metabolism, as well as cell fate. Our study investigated the role of O-GlcNAcylation via glucosamine in the protection of hypoxia-induced apoptosis of mouse embryonic stem cells (mESCs). Hypoxia increased mESCs apoptosis in a time-dependent manner. Moreover, hypoxia also slightly increased the O-GlcNAc level. Glucosamine treatment further enhanced the O-GlcNAc level and prevented hypoxia-induced mESC apoptosis, which was suppressed by O-GlcNAc transferase inhibitors. In addition, hypoxia regulated several lipid metabolic enzymes, whereas glucosamine increased expression of glycerol-3-phosphate acyltransferase-1 (GPAT1), a lipid metabolic enzyme producing lysophosphatidic acid (LPA). In addition, glucosamine-increased O-GlcNAcylation of Sp1, which subsequently leads to Sp1 nuclear translocation and GPAT1 expression. Silencing of GPAT1 by gpat1 siRNA transfection reduced glucosamine-mediated anti-apoptosis in mESCs and reduced mammalian target of rapamycin (mTOR) phosphorylation. Indeed, LPA prevented mESCs from undergoing hypoxia-induced apoptosis and increased phosphorylation of mTOR and its substrates (S6K1 and 4EBP1). Moreover, mTOR inactivation by rapamycin (mTOR inhibitor) increased pro-apoptotic proteins expressions and mESC apoptosis. Furthermore, transplantation of non-targeting siRNA and glucosamine-treated mESCs increased cell survival and inhibited flap necrosis in mouse skin flap model. Conversely, silencing of GPAT1 expression reversed those glucosamine effects. In conclusion, enhancing O-GlcNAcylation of Sp1 by glucosamine stimulates GPAT1 expression, which leads to inhibition of hypoxia-induced mESC apoptosis via mTOR activation. PMID:27010859

  12. Participation of analogues of lysophosphatidic acid (LPA): oleoyl-sn-glycero-3-phosphate (L-alpha-LPA) and 1-oleoyl-2-O-methyl-rac-glycerophosphothionate (OMPT) in uterine smooth muscle contractility of the pregnant pigs.

    PubMed

    Markiewicz, W; Kamińska, K; Bogacki, M; Maślanka, T; Jaroszewski, J

    2012-01-01

    Recent studies show that a representative of phospholipids, namely lysophosphatidic acid (LPA) and its receptors (LPA1.3) play a significant role in the reproductive processes, i. a, in the modulation of the uterine contractility. The participation of LPA3 in the reproductive processes has been revealed in mice and has not been studied in gilts. Therefore, in the present study we investigated the role/action of LPA and its receptors LPA1, LPA2 and LPA3 on the contraction activity in the porcine uterus. The study was conducted on an experimental model in which the pig uterus consisted of the one whole uterine horn and a part of the second horn, both connected with the uterine corpus. Uterine strips consisting of the endometrium with the myometrium (ENDO/MYO) and myometrium (MYO) alone were collected on days 12-14 of the estrous cycle (control group; n = 5) or pregnancy (experimental group; n = 5). Two analogues of LPA at increasing doses were used: oleoyl-sn-glycero-3-phosphate (L-alpha-LPA, a selective agonist of LPA1 and LPA2 receptors; 10(-7) M; 10(-6) M and 10(-5) M) and 1-oleoyl-2-O-methyl-rac-glycerophosphothionate (OMPT, a selective agonist of LPA3 receptor; 68 nM; 136 nM and 680 nM). L-alpha-LPA caused an increase in the contraction tension, amplitude and frequency of ENDO/MYO from the uterine horn with the developing embryos. This effect was not observed in MYO in both groups examined. In the ENDO/MYO strips of the uterine horn with developing embryos, OMPT significantly increased the contraction tension at the highest dose (680 nM) and amplitude at all doses examined, while frequency of contractions was decreased at doses of 136 nM and 680 nM. In the MYO strips of the uterine horn with embryos a significant increase in the contraction tension and amplitude after the highest dose of OMPT was observed. The results obtained imply the important role of receptors LPA1, LPA2 and LPA3 in the contraction activity of the porcine uterus during early pregnancy. PMID

  13. Glycerol-3-phosphate acyltransferase-1 upregulation by O-GlcNAcylation of Sp1 protects against hypoxia-induced mouse embryonic stem cell apoptosis via mTOR activation

    PubMed Central

    Lee, H J; Ryu, J M; Jung, Y H; Lee, K H; Kim, D I; Han, H J

    2016-01-01

    Oxygen signaling is critical for stem cell regulation, and oxidative stress-induced stem cell apoptosis decreases the efficiency of stem cell therapy. Hypoxia activates O-linked β-N-acetyl glucosaminylation (O-GlcNAcylation) of stem cells, which contributes to regulation of cellular metabolism, as well as cell fate. Our study investigated the role of O-GlcNAcylation via glucosamine in the protection of hypoxia-induced apoptosis of mouse embryonic stem cells (mESCs). Hypoxia increased mESCs apoptosis in a time-dependent manner. Moreover, hypoxia also slightly increased the O-GlcNAc level. Glucosamine treatment further enhanced the O-GlcNAc level and prevented hypoxia-induced mESC apoptosis, which was suppressed by O-GlcNAc transferase inhibitors. In addition, hypoxia regulated several lipid metabolic enzymes, whereas glucosamine increased expression of glycerol-3-phosphate acyltransferase-1 (GPAT1), a lipid metabolic enzyme producing lysophosphatidic acid (LPA). In addition, glucosamine-increased O-GlcNAcylation of Sp1, which subsequently leads to Sp1 nuclear translocation and GPAT1 expression. Silencing of GPAT1 by gpat1 siRNA transfection reduced glucosamine-mediated anti-apoptosis in mESCs and reduced mammalian target of rapamycin (mTOR) phosphorylation. Indeed, LPA prevented mESCs from undergoing hypoxia-induced apoptosis and increased phosphorylation of mTOR and its substrates (S6K1 and 4EBP1). Moreover, mTOR inactivation by rapamycin (mTOR inhibitor) increased pro-apoptotic proteins expressions and mESC apoptosis. Furthermore, transplantation of non-targeting siRNA and glucosamine-treated mESCs increased cell survival and inhibited flap necrosis in mouse skin flap model. Conversely, silencing of GPAT1 expression reversed those glucosamine effects. In conclusion, enhancing O-GlcNAcylation of Sp1 by glucosamine stimulates GPAT1 expression, which leads to inhibition of hypoxia-induced mESC apoptosis via mTOR activation. PMID:27010859

  14. Use of linalool synthase in genetic engineering of scent production

    DOEpatents

    Pichersky, Eran

    1998-01-01

    A purified S-linalool synthase polypeptide from Clarkia breweri is disclosed as is the recombinant polypeptide and nucleic acid sequences encoding the polypeptide. Also disclosed are antibodies immunoreactive with the purified peptide and with recombinant versions of the polypeptide. Methods of using the nucleic acid sequences, as well as methods of enhancing the smell and the flavor of plants expressing the nucleic acid sequences are also disclosed.

  15. Inhibition of Escherichia coli ATP synthase by amphibian antimicrobial peptides.

    PubMed

    Laughlin, Thomas F; Ahmad, Zulfiqar

    2010-04-01

    Previously melittin, the alpha-helical basic honey bee venom peptide, was shown to inhibit F(1)-ATPase by binding at the beta-subunit DELSEED motif of F(1)F(o)-ATP synthase. Herein, we present the inhibitory effects of the basic alpha-helical amphibian antimicrobial peptides, ascaphin-8, aurein 2.2, aurein 2.3, carein 1.8, carein 1.9, citropin 1.1, dermaseptin, maculatin 1.1, maganin II, MRP, or XT-7, on purified F(1) and membrane bound F(1)F(0)Escherichia coli ATP synthase. We found that the extent of inhibition by amphibian peptides is variable. Whereas MRP-amide inhibited ATPase essentially completely (approximately 96% inhibition), carein 1.8 did not inhibit at all (0% inhibition). Inhibition by other peptides was partial with a range of approximately 13-70%. MRP-amide was also the most potent inhibitor on molar scale (IC(50) approximately 3.25 microM). Presence of an amide group at the c-terminal of peptides was found to be critical in exerting potent inhibition of ATP synthase ( approximately 20-40% additional inhibition). Inhibition was fully reversible and found to be identical in both F(1)F(0) membrane preparations as well as in isolated purified F(1). Interestingly, growth of E. coli was abrogated in the presence of ascaphin-8, aurein 2.2, aurein 2.3, citropin 1.1, dermaseptin, magainin II-amide, MRP, MRP-amide, melittin, or melittin-amide but was unaffected in the presence of carein 1.8, carein 1.9, maculatin 1.1, magainin II, or XT-7. Hence inhibition of F(1)-ATPase and E. coli cell growth by amphibian antimicrobial peptides suggests that their antimicrobial/anticancer properties are in part linked to their actions on ATP synthase. PMID:20100509

  16. Structure and Mechanistic Implications of a Tryptophan Synthase Quinonoid Intermediate

    SciTech Connect

    Barends,T.; Domratcheva, T.; Kulik, V.; Blumenstein, L.; Niks, D.; Dunn, M.; Schlichting, I.

    2008-01-01

    Quinonoid intermediates play a key role in the catalytic mechanism of pyridoxal 5'-phosphate (PLP)-dependent enzymes. Whereas structures of other PLP-bound reaction intermediates have been determined, a high-quality structure of a quinonoid species has not been reported. We present the crystal structure of the indoline quinonoid intermediate of tryptophan synthase (see figure) and discuss its implications for the enzymatic mechanism and allosteric regulation.

  17. Purification of geranylgeranyl diphosphate synthase from bovine brain.

    PubMed

    Sagami, H; Morita, Y; Korenaga, T; Ogura, K

    1994-01-01

    Geranylgeranyl diphosphate (GGPP) synthase was purified to homogeneity from bovine brain in a one-step affinity column procedure. For the construction of the affinity column, a farnesyl diphosphate (FPP) analog, O-(6-amino-1-hexyl)-P-farnesylmethyl phosphonophosphate, was synthesized and linked to the spacer of the matrix of Affigel 10 via the amino group. The native enzyme appeared to be homooligomer (150-195 kDa) with a molecular mass of the monomer of 37.5 kDa. The pI for the enzyme was 6.2. The Km values for dimethylallyl diphosphate (DMAPP), geranyl diphosphate (GPP) and FPP were estimated to be 33 microM, 0.80 microM and 0.74 microM, respectively. The Km value for isopentenyl diphosphate (IPP) in the presence of both IPP and FPP mixture was 2 microM. The ratio of the reaction velocity for formation of GGPP from DMAPP, GPP or FPP was 0.004:0.145:1. The intermediate FPP was formed in the reaction with GPP as an allylic primer. FPP synthase catalyzing the formation of FPP from DMAPP and IPP was also purified to homogeneity from the same organ by a similar affinity chromatography procedure using a GPP analog, O-(6-amino-1-hexyl)-P-geranylmethyl phosphonophosphate as a ligand. The enzyme was a homodimer with a monomeric molecular mass of 40.0 kDa. These results indicate that GGPP, a lipid precursor for the biosynthesis of a majority of prenylated proteins, is synthesized from DMAPP and IPP by the action of FPP synthase catalyzing the reactions C5-->C15 followed by the action of GGPP synthase catalyzing the reaction C15-->C20. PMID:7856400

  18. Use of linalool synthase in genetic engineering of scent production

    DOEpatents

    Pichersky, E.

    1998-12-15

    A purified S-linalool synthase polypeptide from Clarkia breweri is disclosed as is the recombinant polypeptide and nucleic acid sequences encoding the polypeptide. Also disclosed are antibodies immunoreactive with the purified peptide and with recombinant versions of the polypeptide. Methods of using the nucleic acid sequences, as well as methods of enhancing the smell and the flavor of plants expressing the nucleic acid sequences are also disclosed. 5 figs.

  19. Expression of prostaglandin E synthases in the bovine oviduct.

    PubMed

    Gauvreau, D; Moisan, V; Roy, M; Fortier, M A; Bilodeau, J-F

    2010-01-01

    The oviduct is a specialized organ responsible for the storage and the transport of male and female gametes. It also provides an optimal environment for final gamete maturation, fertilization, and early embryo development. Prostaglandin (PG) E(2) is involved in many female reproductive functions, including ovulation, fertilization, implantation, and parturition. However, the control of its synthesis in the oviduct is not fully understood. Cyclooxygenases (COXs) are involved in the first step of the transformation of arachidonic acid to PGH(2.) The prostaglandin E synthases (PGESs) constitute a family of enzymes that catalyze the conversion of PGH(2) to PGE(2), the terminal step in the formation of this bioactive prostaglandin. Quantitative real-time PCR was used to determine the expression of COX-1, COX-2, microsomal prostaglandin E synthase-1 (mPGES-1), microsomal prostaglandin E synthase-2 (mPGES-2), and cytosolic prostaglandin E synthase (cPGES) mRNA in various sections of the oviduct, both ipsilateral and contralateral (to the ovary on which ovulation occurred) at various stages of the estrous cycle. Furthermore, protein expression and localization of cPGES, mPGES-1, and mPGES-2 were determined by Western blot and immunohistochemistry. All three PGESs were detected at both mRNA and protein levels in the oviduct. These PGESs were mostly concentrated in the oviductal epithelial layer and primarily expressed in the ampulla section of the oviduct and to a lesser extent in the isthmus and the isthmic-ampullary junction. The mPGES-1 protein was highly expressed in the contralateral oviduct, which contrasted with mPGES-2 mostly expressed in the ipsilateral oviduct. This is apparently the first report documenting that the three PGESs involved in PGE(2) production were present in the Bos taurus oviduct. PMID:19875162

  20. Piriformospora indica requires kaurene synthase activity for successful plant colonization.

    PubMed

    Li, Liang; Chen, Xi; Ma, Chaoyang; Wu, Hongqing; Qi, Shuting

    2016-05-01

    Ent-kaurene (KS) synthases and ent-kaurene-like (KSL) synthases are involved in the biosynthesis of phytoalexins and/or gibberellins which play a role in plant immunity and development. The relationship between expression of five synthase genes (HvKSL1, HvKS2, HvKS4, HvKS5, HvKSL4) and plant colonization by the endophytic fungus Piriformospora indica was assessed in barley (Hordeum vulgare). The KS gene family is differently up-regulated at 1, 3 and 7 day after P. indica inoculation. By comparison, the HvKSL4 gene expression pattern is more significantly affected by UV irradiation and P. indica colonization. The characterizations of two silencing lines (HvKSL1-RNAi, HvKSL4-RNAi) also were analyzed. HvKSL1-RNAi and HvKSL4-RNAi lines in the first generation lead to less dark green leaves and slower plant development. Further, reduced spikelet fertility in progenies of RNAi plants heterozygous for HvKSL1 were observed, but not for HvKSL4. T2 generation of HvKSL1-RNAi line showed semi-dwarf phenotype while the wild type phenotype could be restored by applying GA3. Silencing of HvKSL4 and HvKSL1 resulted in reduced colonization by P. indica especially in the HvKSL1-RNAi line. These results probably suggest the presence of two ent-KS synthase in barley, one (HvKSL1) that participates in the biosynthesis of GAs and another (HvKSL4) that is involved in the biosynthesis of phytoalexins. PMID:26943021

  1. Inhibition of Escherichia coli ATP synthase by amphibian antimicrobial peptides

    PubMed Central

    Laughlin, Thomas F.; Ahmad, Zulfiqar

    2010-01-01

    Previously melittin, the α-helical basic honey bee venom peptide, was shown to inhibit F1-ATPase by binding at the β-subunit DELSEED motif of F1Fo ATP synthase. Herein, we present the inhibitory effects of the basic α-helical amphibian antimicrobial peptides, ascaphin-8, aurein 2.2, aurein 2.3, carein 1.8, carein 1.9, citropin 1.1, dermaseptin, maculatin 1.1, maganin II, MRP, or XT-7, on purified F1 and membrane bound F1Fo E. coli ATP synthase. We found that the extent of inhibition by amphibian peptides is variable. Whereas MRP-amide inhibited ATPase essentially completely (~96% inhibition), carein 1.8 did not inhibit at all (0% inhibition). Inhibition by other peptides was partial with a range of ~13% to 70%. MRP-amide was also the most potent inhibitor on molar scale (IC50 ~3.25 µM). Presence of an amide group at the c-terminal of peptides was found to be critical in exerting potent inhibition of ATP synthase (~20–40% additional inhibition). Inhibition was fully reversible and found to be identical in both F1Fo membrane preparations as well as in isolated purified F1. Interestingly, growth of Escherichia coli was abrogated in the presence of ascaphin-8, aurein 2.2, aurein 2.3, citropin 1.1, dermaseptin, magainin II-amide, MRP, MRP-amide, melittin, or melittin-amide but was unaffected in the presence of carein 1.8, carein 1.9, maculatin 1.1, magainin II, or XT-7. Hence inhibition of F1-ATPase and E. coli cell growth by amphibian antimicrobial peptides suggests that their antimicrobial/anticancer properties are in part linked to their actions on ATP synthase. PMID:20100509

  2. Suites of Terpene Synthases Explain Differential Terpenoid Production in Ginger and Turmeric Tissues

    PubMed Central

    Koo, Hyun Jo; Gang, David R.

    2012-01-01

    The essential oils of ginger (Zingiber officinale) and turmeric (Curcuma longa) contain a large variety of terpenoids, some of which possess anticancer, antiulcer, and antioxidant properties. Despite their importance, only four terpene synthases have been identified from the Zingiberaceae family: (+)-germacrene D synthase and (S)-β-bisabolene synthase from ginger rhizome, and α-humulene synthase and β-eudesmol synthase from shampoo ginger (Zingiber zerumbet) rhizome. We report the identification of 25 mono- and 18 sesquiterpene synthases from ginger and turmeric, with 13 and 11, respectively, being functionally characterized. Novel terpene synthases, (−)-caryolan-1-ol synthase and α-zingiberene/β-sesquiphellandrene synthase, which is responsible for formation of the major sesquiterpenoids in ginger and turmeric rhizomes, were also discovered. These suites of enzymes are responsible for formation of the majority of the terpenoids present in these two plants. Structures of several were modeled, and a comparison of sets of paralogs suggests how the terpene synthases in ginger and turmeric evolved. The most abundant and most important sesquiterpenoids in turmeric rhizomes, (+)-α-turmerone and (+)-β-turmerone, are produced from (−)-α-zingiberene and (−)-β-sesquiphellandrene, respectively, via α-zingiberene/β-sesquiphellandrene oxidase and a still unidentified dehydrogenase. PMID:23272109

  3. [BIOINFORMATIC SEARCH AND PHYLOGENETIC ANALYSIS OF THE CELLULOSE SYNTHASE GENES OF FLAX (LINUM USITATISSIMUM)].

    PubMed

    Pydiura, N A; Bayer, G Ya; Galinousky, D V; Yemets, A I; Pirko, Ya V; Podvitski, T A; Anisimova, N V; Khotyleva, L V; Kilchevsky, A V; Blume, Ya B

    2015-01-01

    A bioinformatic search of sequences encoding cellulose synthase genes in the flax genome, and their comparison to dicots orthologs was carried out. The analysis revealed 32 cellulose synthase gene candidates, 16 of which are highly likely to encode cellulose synthases, and the remaining 16--cellulose synthase-like proteins (Csl). Phylogenetic analysis of gene products of cellulose synthase genes allowed distinguishing 6 groups of cellulose synthase genes of different classes: CesA1/10, CesA3, CesA4, CesA5/6/2/9, CesA7 and CesA8. Paralogous sequences within classes CesA1/10 and CesA5/6/2/9 which are associated with the primary cell wall formation are characterized by a greater similarity within these classes than orthologous sequences. Whereas the genes controlling the biosynthesis of secondary cell wall cellulose form distinct clades: CesA4, CesA7, and CesA8. The analysis of 16 identified flax cellulose synthase gene candidates shows the presence of at least 12 different cellulose synthase gene variants in flax genome which are represented in all six clades of cellulose synthase genes. Thus, at this point genes of all ten known cellulose synthase classes are identify in flax genome, but their correct classification requires additional research. PMID:26638491

  4. Multi-Substrate Terpene Synthases: Their Occurrence and Physiological Significance.

    PubMed

    Pazouki, Leila; Niinemets, Ülo

    2016-01-01

    Terpene synthases are responsible for synthesis of a large number of terpenes in plants using substrates provided by two distinct metabolic pathways, the mevalonate-dependent pathway that is located in cytosol and has been suggested to be responsible for synthesis of sesquiterpenes (C15), and 2-C-methyl-D-erythritol-4-phosphate pathway located in plastids and suggested to be responsible for the synthesis of hemi- (C5), mono- (C10), and diterpenes (C20). Recent advances in characterization of genes and enzymes responsible for substrate and end product biosynthesis as well as efforts in metabolic engineering have demonstrated existence of a number of multi-substrate terpene synthases. This review summarizes the progress in the characterization of such multi-substrate terpene synthases and suggests that the presence of multi-substrate use might have been significantly underestimated. Multi-substrate use could lead to important changes in terpene product profiles upon substrate profile changes under perturbation of metabolism in stressed plants as well as under certain developmental stages. We therefore argue that multi-substrate use can be significant under physiological conditions and can result in complicate modifications in terpene profiles. PMID:27462341

  5. Kinetic Characterization of Spinach Leaf Sucrose-Phosphate Synthase 1

    PubMed Central

    Amir, Jacob; Preiss, Jack

    1982-01-01

    The spinach (Spinacia oleracea) leaf sucrose-phosphate synthase was partially purified via DEAE-cellulose chromatography, and its kinetic properties were studied. Fructose-6-phosphate saturation curves were sigmoidal, while UDPglucose saturation curves were hyperbolic. At subsaturating concentrations of fructose-6-phosphate, 1,5 anhydroglucitol-6-phosphate had a stimulatory effect on enzyme activity, suggesting multiple and interacting fructose-6-phosphate sites on sucrose-phosphate synthase. The concentrations required for 50% of maximal activity were 3.0 millimolar and 1.3 millimolar, respectively, for fructose-6-phosphate and UDPglucose. The enzyme was not stimulated by divalent cations. Inorganic phosphate proved to be a potent inhibitor, particularly at low concentrations of substrate. Phosphate inhibition was competitive with UDPglucose, and its Ki was determined to be 1.75 millimolar. Sucrose phosphate, the product of the reaction, was also shown to be a competitive inhibitor towards UDPglucose concentration and had Ki of 0.4 millimolar. The kinetic results suggest that spinach leaf sucrose-phospahte synthase is a regulatory enzyme and that its activity is modulated by the concentrations of phosphate, fructose-6-phosphate, and UDPglucose occurring in the cytoplasm of the leaf cell. PMID:16662338

  6. Dexmedetomidine inhibits vasoconstriction via activation of endothelial nitric oxide synthase.

    PubMed

    Nong, Lidan; Ma, Jue; Zhang, Guangyan; Deng, Chunyu; Mao, Songsong; Li, Haifeng; Cui, Jianxiu

    2016-09-01

    Despite the complex vascular effects of dexmedetomidine (DEX), its actions on human pulmonary resistance arteries remain unknown. The present study tested the hypothesis that DEX inhibits vascular tension in human pulmonary arteries through the endothelial nitric oxide synthase (eNOS) mediated production of nitric oxide (NO). Pulmonary artery segments were obtained from 62 patients who underwent lung resection. The direct effects of DEX on human pulmonary artery tension and changes in vascular tension were determined by isometric force measurements recorded on a myograph. Arterial contractions caused by increasing concentrations of serotonin with DEX in the presence or absence of L-NAME (endothelial nitric oxide synthase inhibitor), yohimbine (α2-adrenoceptor antagonist) and indomethacin (cyclooxygenase inhibitor) as antagonists were also measured. DEX had no effect on endothelium-intact pulmonary arteries, whereas at concentrations of 10(-8)~10(-6) mol/L, it elicited contractions in endothelium-denuded pulmonary arteries. DEX (0.3, 1, or 3×10(-9) mmol/L) inhibited serotonin-induced contraction in arteries with intact endothelium in a dose-dependent manner. L-NAME and yohimbine abolished DEX-induced inhibition, whereas indomethacin had no effect. No inhibitory effect was observed in endothelium-denuded pulmonary arteries. DEX-induced inhibition of vasoconstriction in human pulmonary arteries is mediated by NO production induced by the activation of endothelial α2-adrenoceptor and nitric oxide synthase. PMID:27610030

  7. [Preparation and crystallization of Polygonum cuspidatum benzalacetone synthase].

    PubMed

    Ma, Wenrui; Liu, Chunmei; Yang, Mingfeng; Xue, Feiyan; Chen, Qing; Ma, Lanqing; Lü, Heshu

    2016-02-01

    The chalcone synthase (CHS) superfamily of the type III polyketide synthases (PKSs) generates backbones of a variety of plant secondary metabolites. Benzalacetone synthase (BAS) catalyzes a condensation reaction of decarboxylation between the substrates of 4-coumaric coenzyme A and malonyl coenzyme A to generate benzylidene acetone, whose derivatives are series of compounds with various biological activities. A BAS gene Pcpks2 and a bifunctional CHS/BAS PcPKSI were isolated from medicinal plant P. cuspidatum. Crystallographic and structure-based mutagenesis studies indicate that the functional diversity of the CHS-superfamily enzymes is principally derived from small modifications of the active site architecture. In order to obtain an understanding of the biosynthesis of polyketides in P. cuspidatum, which has been poorly described, as well as of its activation mechanism, PcPKS2 was overexpressed in Escherichia coli as a C-terminally poly-His-tagged fusion protein, purified to homogeneity and crystallized, which is helpful for the clarification of the catalytic mechanism of the enzyme and lays the foundation for its genetic engineering manipulation. PMID:27382775

  8. Dexmedetomidine inhibits vasoconstriction via activation of endothelial nitric oxide synthase

    PubMed Central

    Nong, Lidan; Ma, Jue; Zhang, Guangyan; Deng, Chunyu; Mao, Songsong; Li, Haifeng

    2016-01-01

    Despite the complex vascular effects of dexmedetomidine (DEX), its actions on human pulmonary resistance arteries remain unknown. The present study tested the hypothesis that DEX inhibits vascular tension in human pulmonary arteries through the endothelial nitric oxide synthase (eNOS) mediated production of nitric oxide (NO). Pulmonary artery segments were obtained from 62 patients who underwent lung resection. The direct effects of DEX on human pulmonary artery tension and changes in vascular tension were determined by isometric force measurements recorded on a myograph. Arterial contractions caused by increasing concentrations of serotonin with DEX in the presence or absence of L-NAME (endothelial nitric oxide synthase inhibitor), yohimbine (α2-adrenoceptor antagonist) and indomethacin (cyclooxygenase inhibitor) as antagonists were also measured. DEX had no effect on endothelium-intact pulmonary arteries, whereas at concentrations of 10–8~10–6 mol/L, it elicited contractions in endothelium-denuded pulmonary arteries. DEX (0.3, 1, or 3×10–9 mmol/L) inhibited serotonin-induced contraction in arteries with intact endothelium in a dose-dependent manner. L-NAME and yohimbine abolished DEX-induced inhibition, whereas indomethacin had no effect. No inhibitory effect was observed in endothelium-denuded pulmonary arteries. DEX-induced inhibition of vasoconstriction in human pulmonary arteries is mediated by NO production induced by the activation of endothelial α2-adrenoceptor and nitric oxide synthase. PMID:27610030

  9. Resistance Phenotypes Mediated by Aminoacyl-Phosphatidylglycerol Synthases

    PubMed Central

    Arendt, Wiebke; Hebecker, Stefanie; Jäger, Sonja; Nimtz, Manfred

    2012-01-01

    The specific aminoacylation of the phospholipid phosphatidylglycerol (PG) with alanine or with lysine catalyzed by aminoacyl-phosphatidylglycerol synthases (aaPGS) was shown to render various organisms less susceptible to antibacterial agents. This study makes use of Pseudomonas aeruginosa chimeric mutant strains producing lysyl-phosphatidylglycerol (L-PG) instead of the naturally occurring alanyl-phosphatidylglycerol (A-PG) to study the resulting impact on bacterial resistance. Consequences of such artificial phospholipid composition were studied in the presence of an overall of seven antimicrobials (β-lactams, a lipopeptide antibiotic, cationic antimicrobial peptides [CAMPs]) to quantitatively assess the effect of A-PG substitution (with L-PG, L-PG and A-PG, increased A-PG levels). For the employed Gram-negative P. aeruginosa model system, an exclusive charge repulsion mechanism does not explain the attenuated antimicrobial susceptibility due to PG modification. Additionally, the specificity of nine orthologous aaPGS enzymes was experimentally determined. The newly characterized protein sequences allowed for the establishment of a significant group of A-PG synthase sequences which were bioinformatically compared to the related group of L-PG synthesizing enzymes. The analysis revealed a diverse origin for the evolution of A-PG and L-PG synthases, as the specificity of an individual enzyme is not reflected in terms of a characteristic sequence motif. This finding is relevant for future development of potential aaPGS inhibitors. PMID:22267511

  10. Mechanism of Action and Inhibition of dehydrosqualene Synthase

    SciTech Connect

    F Lin; C Liu; Y Liu; Y Zhang; K Wang; W Jeng; T Ko; R Cao; A Wang; E Oldfield

    2011-12-31

    'Head-to-head' terpene synthases catalyze the first committed steps in sterol and carotenoid biosynthesis: the condensation of two isoprenoid diphosphates to form cyclopropylcarbinyl diphosphates, followed by ring opening. Here, we report the structures of Staphylococcus aureus dehydrosqualene synthase (CrtM) complexed with its reaction intermediate, presqualene diphosphate (PSPP), the dehydrosqualene (DHS) product, as well as a series of inhibitors. The results indicate that, on initial diphosphate loss, the primary carbocation so formed bends down into the interior of the protein to react with C2,3 double bond in the prenyl acceptor to form PSPP, with the lower two-thirds of both PSPP chains occupying essentially the same positions as found in the two farnesyl chains in the substrates. The second-half reaction is then initiated by the PSPP diphosphate returning back to the Mg{sup 2+} cluster for ionization, with the resultant DHS so formed being trapped in a surface pocket. This mechanism is supported by the observation that cationic inhibitors (of interest as antiinfectives) bind with their positive charge located in the same region as the cyclopropyl carbinyl group; that S-thiolo-diphosphates only inhibit when in the allylic site; activity results on 11 mutants show that both DXXXD conserved domains are essential for PSPP ionization; and the observation that head-to-tail isoprenoid synthases as well as terpene cyclases have ionization and alkene-donor sites which spatially overlap those found in CrtM.

  11. From bacterial to human dihydrouridine synthase: automated structure determination

    SciTech Connect

    Whelan, Fiona Jenkins, Huw T.; Griffiths, Samuel C.; Byrne, Robert T.; Dodson, Eleanor J.; Antson, Alfred A.

    2015-06-30

    The crystal structure of a human dihydrouridine synthase, an enzyme associated with lung cancer, with 18% sequence identity to a T. maritima enzyme, has been determined at 1.9 Å resolution by molecular replacement after extensive molecular remodelling of the template. The reduction of uridine to dihydrouridine at specific positions in tRNA is catalysed by dihydrouridine synthase (Dus) enzymes. Increased expression of human dihydrouridine synthase 2 (hDus2) has been linked to pulmonary carcinogenesis, while its knockdown decreased cancer cell line viability, suggesting that it may serve as a valuable target for therapeutic intervention. Here, the X-ray crystal structure of a construct of hDus2 encompassing the catalytic and tRNA-recognition domains (residues 1–340) determined at 1.9 Å resolution is presented. It is shown that the structure can be determined automatically by phenix.mr-rosetta starting from a bacterial Dus enzyme with only 18% sequence identity and a significantly divergent structure. The overall fold of the human Dus2 is similar to that of bacterial enzymes, but has a larger recognition domain and a unique three-stranded antiparallel β-sheet insertion into the catalytic domain that packs next to the recognition domain, contributing to domain–domain interactions. The structure may inform the development of novel therapeutic approaches in the fight against lung cancer.

  12. Cellulose Microfibril Formation by Surface-Tethered Cellulose Synthase Enzymes.

    PubMed

    Basu, Snehasish; Omadjela, Okako; Gaddes, David; Tadigadapa, Srinivas; Zimmer, Jochen; Catchmark, Jeffrey M

    2016-02-23

    Cellulose microfibrils are pseudocrystalline arrays of cellulose chains that are synthesized by cellulose synthases. The enzymes are organized into large membrane-embedded complexes in which each enzyme likely synthesizes and secretes a β-(1→4) glucan. The relationship between the organization of the enzymes in these complexes and cellulose crystallization has not been explored. To better understand this relationship, we used atomic force microscopy to visualize cellulose microfibril formation from nickel-film-immobilized bacterial cellulose synthase enzymes (BcsA-Bs), which in standard solution only form amorphous cellulose from monomeric BcsA-B complexes. Fourier transform infrared spectroscopy and X-ray diffraction techniques show that surface-tethered BcsA-Bs synthesize highly crystalline cellulose II in the presence of UDP-Glc, the allosteric activator cyclic-di-GMP, as well as magnesium. The cellulose II cross section/diameter and the crystal size and crystallinity depend on the surface density of tethered enzymes as well as the overall concentration of substrates. Our results provide the correlation between cellulose microfibril formation and the spatial organization of cellulose synthases. PMID:26799780

  13. Rotation and structure of FoF1-ATP synthase.

    PubMed

    Okuno, Daichi; Iino, Ryota; Noji, Hiroyuki

    2011-06-01

    F(o)F(1)-ATP synthase is one of the most ubiquitous enzymes; it is found widely in the biological world, including the plasma membrane of bacteria, inner membrane of mitochondria and thylakoid membrane of chloroplasts. However, this enzyme has a unique mechanism of action: it is composed of two mechanical rotary motors, each driven by ATP hydrolysis or proton flux down the membrane potential of protons. The two molecular motors interconvert the chemical energy of ATP hydrolysis and proton electrochemical potential via the mechanical rotation of the rotary shaft. This unique energy transmission mechanism is not found in other biological systems. Although there are other similar man-made systems like hydroelectric generators, F(o)F(1)-ATP synthase operates on the nanometre scale and works with extremely high efficiency. Therefore, this enzyme has attracted significant attention in a wide variety of fields from bioenergetics and biophysics to chemistry, physics and nanoscience. This review summarizes the latest findings about the two motors of F(o)F(1)-ATP synthase as well as a brief historical background. PMID:21524994

  14. Multi-Substrate Terpene Synthases: Their Occurrence and Physiological Significance

    PubMed Central

    Pazouki, Leila; Niinemets, Ülo

    2016-01-01

    Terpene synthases are responsible for synthesis of a large number of terpenes in plants using substrates provided by two distinct metabolic pathways, the mevalonate-dependent pathway that is located in cytosol and has been suggested to be responsible for synthesis of sesquiterpenes (C15), and 2-C-methyl-D-erythritol-4-phosphate pathway located in plastids and suggested to be responsible for the synthesis of hemi- (C5), mono- (C10), and diterpenes (C20). Recent advances in characterization of genes and enzymes responsible for substrate and end product biosynthesis as well as efforts in metabolic engineering have demonstrated existence of a number of multi-substrate terpene synthases. This review summarizes the progress in the characterization of such multi-substrate terpene synthases and suggests that the presence of multi-substrate use might have been significantly underestimated. Multi-substrate use could lead to important changes in terpene product profiles upon substrate profile changes under perturbation of metabolism in stressed plants as well as under certain developmental stages. We therefore argue that multi-substrate use can be significant under physiological conditions and can result in complicate modifications in terpene profiles. PMID:27462341

  15. Characterization and expression of human bifunctional 3'-phosphoadenosine 5'-phosphosulphate synthase isoforms.

    PubMed Central

    Fuda, Hirotoshi; Shimizu, Chikara; Lee, Young C; Akita, Harukuni; Strott, Charles A

    2002-01-01

    Sulphonation, a fundamental process essential for normal growth and development, requires the sulphonate donor molecule 3'-phosphoadenosine 5'-phosphosulphate (PAPS), which is produced from ATP and inorganic sulphate by the bifunctional enzyme PAPS synthase. In humans, two genes encode isoenzymes that are 77% identical at the amino acid level, and alternative splicing creates two subtypes of PAPS synthase 2. The question as to whether distinctions in amino acid composition are reflected in differences in activity has been examined. The specific activity of the PAPS synthase 2 subtypes is 10- to 15-fold higher than that for PAPS synthase 1. The greater catalytic efficiency of the PAPS synthase 2 subtypes is demonstrated further by the 3- to 6-fold higher k(cat)/K(m) ratios for ATP and inorganic sulphate as compared with the ratios for PAPS synthase 1. In humans, PAPS synthase 1 is expressed ubiquitously, and is the dominant isoform in most tissues, whereas expression of the PAPS synthase 2 subtypes is variable and tissue-specific. It is noteworthy that, similar to other human tissues, PAPS synthase 1 also appears to be the dominant isoform expressed in cartilage. The latter finding initially created a conundrum, since there is a specific human dwarfing disorder that is known to be caused by a mutation in the PAPS synthase 2 gene. This apparent enigma would seem to be resolved by examination of cartilage from guinea-pigs as an animal model. Similar to humans, cartilage from mature animals predominantly expresses PAPS synthase 1. In contrast, expression of PAPS synthase 1 is relatively low in the cartilage of immature guinea-pigs, including the growth plate of long bones, whereas PAPS synthase 2 is the highly expressed isoenzyme. PMID:11931637

  16. Chloropropionyl-CoA: a mechanism-based inhibitor of HMG-CoA synthase and fatty acid synthase

    SciTech Connect

    Miziorko, H.M.; Ahmad, F.; Behnke, C.E.

    1986-05-01

    Recent work on the mechanisms of inactivation of HMG-CoA synthase and fatty acid synthase by chloropropionyl-CoA (Cl-prop-CoA) suggests that this analog is a mechanism-based (suicide) inhibitor; the acyl group is enzymatically converted to an acrylyl derivative prior to alkylation of the target proteins. When Cl-(/sup 3/H)prop-CoA is incubated with the target enzymes, /sup 3/H/sub 2/O is produced concomitantly with enzyme inactivation; this suggests that deprotonation and chloride elimination to form an acrylyl moiety occurs. Difficulty in cleanly synthesizing acrylyl-CoA complicates direct demonstration of the intermediacy of this species. However, synthesis of a functionally equivalent reactive substrate analog, S-acrylyl-N-acetylcysteamine has been accomplished. This analog irreversibly inhibits both HMG-CoA synthase and fatty acid synthase in a site directed fashion. Concentrations required for effective inhibition (K/sub i/ values of 1.9 mM and 3.6 mM, respectively) are much higher than observed with Cl-prop-CoA. Maximal rates of inactivation (as vertical bar ..-->.. infinity) are comparable to those measured with Cl-prop-CoA, indicating that an acrylyl derivative is kinetically competent to function as an intermediate, as required if Cl-prop-CoA is a mechanism-based inhibitor. S-acrylyl-N-acetylcysteamine also inactivates HMG-CoA lyase. In this case, kinetic studies indicate that a bimolecular process is involved (k/sub 2/ = 86.7M/sup -1/min/sup -1/ at 30/sup 0/, pH 7.0).

  17. Identification of core 1 O-glycan T-synthase from Caenorhabditis elegans.

    PubMed

    Ju, Tongzhong; Zheng, Qinlong; Cummings, Richard D

    2006-10-01

    The common O-glycan core structure in animal glycoproteins is the core 1 disaccharide Galbeta1-3GalNAcalpha1-Ser/Thr, which is generated by the addition of Gal to GalNAcalpha1-Ser/Thr by core 1 UDP-alpha-galactose (UDP-Gal):GalNAcalpha1-Ser/Thr beta1,3-galactosyltransferase (core 1 beta3-Gal-T or T-synthase, EC2.4.1.122). Although O-glycans play important roles in vertebrates, much remains to be learned from model organisms such as the free-living nematode Caenorhabditis elegans, which offer many advantages in exploring O-glycan structure/function. Here, we report the cloning and enzymatic characterization of T-synthase from C. elegans (Ce-T-synthase). A putative C. elegans gene for T-synthase, C38H2.2, was identified in GenBank by a BlastP search using the human T-synthase protein sequence. The full-length cDNA for Ce-T-synthase, which was generated by polymerase chain reaction using a C. elegans cDNA library as the template, contains 1170 bp including the stop TAA. The cDNA encodes a protein of 389 amino acids with typical type II membrane topology and a remarkable 42.7% identity to the human T-synthase. Ce-T-synthase has seven Cys residues in the lumenal domain including six conserved Cys residues in all orthologs. The Ce-T-synthase has four potential N-glycosylation sequons, whereas the mammalian orthologs lack N-glycosylation sequons. Only one gene for Ce-T-synthase was identified in the genome-wide search, and it contains eight exons. Promoter analysis of the Ce-T-synthase using green fluorescent protein (GFP) constructs shows that the gene is expressed at all developmental stages and appears to be in all cells. Unexpectedly, only minimal activity was recovered in the recombinant, soluble Ce-T-synthase secreted from a wide variety of mammalian cell lines, whereas robust enzyme activity was recovered in the soluble Ce-T-synthase expressed in Hi-5 insect cells. Vertebrate T-synthase requires the molecular chaperone Cosmc, but our results show that Ce-T-synthase

  18. Molecular and biochemical characterization of benzalacetone synthase and chalcone synthase genes and their proteins from raspberry (Rubus idaeus L.).

    PubMed

    Zheng, Desen; Hrazdina, Geza

    2008-02-15

    Two new members of the polyketide synthase (PKS) gene family (RiPKS4 and RiPKS5) were cloned from raspberry fruits (Rubus idaeus L., cv Royalty) and expressed in Escherichia coli. Characterization of the recombinant enzyme products indicated that RiPKS4 is a bifunctional polyketide synthase producing both 4-hydroxybenzalacetone and naringenin chalcone. The recombinant RiPKS4 protein, like the native protein from raspberry fruits [W. Borejsza-Wysocki, G. Hrazdina, Plant Physiol. 1996;110: 791-799] accepted p-coumaryl-CoA and ferulyl-CoA as starter substrates and catalyzed the formation of both naringenin chalcone, 4-hydroxy-benzalacetone and 3-methoxy-4-hydroxy-benzalacetone. Although activity of RiPKS4 was higher with ferulyl-CoA than with p-coumaryl-CoA, the corresponding product, 3-methoxy-4-hydroxy phenylbutanone could not be detected in raspberries to date. Sequence analysis of the genes and proteins suggested that this feature of RiPKS4 was created by variation in the C-terminus due to DNA recombination at the 3' region of its coding sequence. RiPKS5 is a typical chalcone synthase (CHS) that uses p-coumaryl-CoA only as starter substrate and produces naringenin chalcone exclusively as the reaction product. PMID:18068110

  19. Vanadate and selenium inhibit the triiodothyronine induced enzyme activity and mRNA level for both fatty acid synthase and malic enzyme

    SciTech Connect

    Zhu, Y.; Mirmiran, R.; Goodridge, A.G.; Stapleton, S.R. Western Michigan Univ., Kalamazoo )

    1991-03-15

    In chick-embryo hepatocytes in culture, triiodothyronine stimulates enzyme activity, mRNA level and transcription rate for both fatty acid synthase (FAS) and malic enzyme (ME). Insulin alone has no effect but amplifies the induction by T3. Recent evidence has demonstrated the insulin-mimicking action of vanadate and selenium on various physiological processes. Little information, however, is available on the affects of vanadate and selenium on the expression of genes that are regulated by insulin. These studies were initiated to test the potential of vanadate and selenium to mimic the amplification affect of insulin on the T3 induction of FAS and ME. In chick-embryo hepatocytes incubated in a chemically defined medium, addition of T3 for 48h causes an increase in the enzyme activity and mRNA level for both FAS and ME. Addition of sodium vanadate or sodium selenate (20 {mu}M) coincident with the T3 almost completely inhibited the stimulation of FAS and ME activity and accumulation of their respective mRNA's. Fifty percent maximal inhibition occurred at about 3-40{mu}M vanadate or 5-10{mu}M selenium. Vanadate and selenium similarity inhibited FAS and ME enzyme activity and mRNA level when the cells were incubated in the presence of insulin and T3. The effect of these metals was selective; isocitrate dehydrogenase activity as well as the level of glyceraldehyde 3-phosphate mRNA were not affected by any of the additions made to the cells in culture. This effect by vanadate and selenium also does not appear to be a generalized effect of metals on lipogenic enzymes as molydate under similar experimental conditions has no effect on either the enzyme activity or mRNA level of FAS or ME. Studies are continuing to determine the mechanism of action of these agents on the regulation of lipogenic enzymes.

  20. Myo-inositol phosphate synthase expression in the European eel (Anguilla anguilla) and Nile tilapia (Oreochromis niloticus): effect of seawater acclimation

    PubMed Central

    Kalujnaia, Svetlana; Hazon, Neil

    2016-01-01

    A single MIPS gene (Isyna1/Ino1) exists in eel and tilapia genomes with a single myo-d-inositol 3-phosphate synthase (MIPS) transcript identified in all eel tissues, although two MIPS spliced variants [termed MIPS(s) and MIPS(l)] are found in all tilapia tissues. The larger tilapia transcript [MIPS(l)] results from the inclusion of the 87-nucleotide intron between exons 5 and 6 in the genomic sequence. In most tilapia tissues, the MIPS(s) transcript exhibits much higher abundance (generally >10-fold) with the exception of white skeletal muscle and oocytes, in which the MIPS(l) transcript predominates. SW acclimation resulted in large (6- to 32-fold) increases in mRNA expression for both MIPS(s) and MIPS(l) in all tilapia tissues tested, whereas in the eel, changes in expression were limited to a more modest 2.5-fold increase and only in the kidney. Western blots identified a number of species- and tissue-specific immunoreactive MIPS proteins ranging from 40 to 67 kDa molecular weight. SW acclimation failed to affect the abundance of any immunoreactive protein in any tissue tested from the eel. However, a major 67-kDa immunoreactive protein (presumed to be MIPS) found in tilapia tissues exhibited 11- and 54-fold increases in expression in gill and fin samples from SW-acclimated fish. Immunohistochemical investigations revealed specific immunoreactivity in the gill, fin, skin, and intestine taken from only SW-acclimated tilapia. Immunofluorescence indicated that MIPS was expressed within gill chondrocytes and epithelial cells of the primary filaments, basal epithelial cell layers of the skin and fin, the cytosol of columnar intestinal epithelial and mucous cells, as well as unknown entero-endocrine-like cells. PMID:27252471

  1. Myo-inositol phosphate synthase expression in the European eel (Anguilla anguilla) and Nile tilapia (Oreochromis niloticus): effect of seawater acclimation.

    PubMed

    Kalujnaia, Svetlana; Hazon, Neil; Cramb, Gordon

    2016-08-01

    A single MIPS gene (Isyna1/Ino1) exists in eel and tilapia genomes with a single myo-d-inositol 3-phosphate synthase (MIPS) transcript identified in all eel tissues, although two MIPS spliced variants [termed MIPS(s) and MIPS(l)] are found in all tilapia tissues. The larger tilapia transcript [MIPS(l)] results from the inclusion of the 87-nucleotide intron between exons 5 and 6 in the genomic sequence. In most tilapia tissues, the MIPS(s) transcript exhibits much higher abundance (generally >10-fold) with the exception of white skeletal muscle and oocytes, in which the MIPS(l) transcript predominates. SW acclimation resulted in large (6- to 32-fold) increases in mRNA expression for both MIPS(s) and MIPS(l) in all tilapia tissues tested, whereas in the eel, changes in expression were limited to a more modest 2.5-fold increase and only in the kidney. Western blots identified a number of species- and tissue-specific immunoreactive MIPS proteins ranging from 40 to 67 kDa molecular weight. SW acclimation failed to affect the abundance of any immunoreactive protein in any tissue tested from the eel. However, a major 67-kDa immunoreactive protein (presumed to be MIPS) found in tilapia tissues exhibited 11- and 54-fold increases in expression in gill and fin samples from SW-acclimated fish. Immunohistochemical investigations revealed specific immunoreactivity in the gill, fin, skin, and intestine taken from only SW-acclimated tilapia. Immunofluorescence indicated that MIPS was expressed within gill chondrocytes and epithelial cells of the primary filaments, basal epithelial cell layers of the skin and fin, the cytosol of columnar intestinal epithelial and mucous cells, as well as unknown entero-endocrine-like cells. PMID:27252471

  2. Surrogate Splicing for Functional Analysis of Sesquiterpene Synthase Genes1[w

    PubMed Central

    Wu, Shuiqin; Schoenbeck, Mark A.; Greenhagen, Bryan T.; Takahashi, Shunji; Lee, Sungbeom; Coates, Robert M.; Chappell, Joseph

    2005-01-01

    A method for the recovery of full-length cDNAs from predicted terpene synthase genes containing introns is described. The approach utilizes Agrobacterium-mediated transient expression coupled with a reverse transcription-polydeoxyribonucleotide chain reaction assay to facilitate expression cloning of processed transcripts. Subsequent expression of intronless cDNAs in a suitable prokaryotic host provides for direct functional testing of the encoded gene product. The method was optimized by examining the expression of an intron-containing β-glucuronidase gene agroinfiltrated into petunia (Petunia hybrida) leaves, and its utility was demonstrated by defining the function of two previously uncharacterized terpene synthases. A tobacco (Nicotiana tabacum) terpene synthase-like gene containing six predicted introns was characterized as having 5-epi-aristolochene synthase activity, while an Arabidopsis (Arabidopsis thaliana) gene previously annotated as a terpene synthase was shown to possess a novel sesquiterpene synthase activity for α-barbatene, thujopsene, and β-chamigrene biosynthesis. PMID:15965019

  3. Ectopic ATP synthase in endothelial cells: a novel cardiovascular therapeutic target.

    PubMed

    Fu, Yi; Zhu, Yi

    2010-01-01

    Adenosine triphosphate (ATP) synthase produces ATP in cells and is found on the inner membrane of mitochondria or the cell plasma membrane (ectopic ATP synthase). Here, we summarize the functions of ectopic ATP synthase in vascular endothelial cells (ECs). Ectopic ATP synthase is involved in adenosine metabolism on the cell surface through its ATP generation or hydrolysis activity. The ATP/ADP generated by the enzyme on the plasma membrane can bind to P2X/P2Y receptors and activate the related signalling pathways to regulate endothelial function. The β-chain of ectopic ATP synthase on the EC surface can recruit inflammatory cells and activate cytotoxic activity to damage ECs and induce vascular inflammation. Angiostatin and other angiogenesis inhibitors can have anti-angiogenic functions by inhibiting ectopic ATP synthase on ECs. Moreover, ectopic ATP synthase on ECs is a receptor for apoA-I, the acceptor of cholesterol efflux, which implies that endothelial ectopic ATP synthase is involved in cholesterol metabolism. Coupling factor 6 (CF6), a part of ectopic ATP synthase, is released from ECs and can inhibit prostacyclin synthesis and promote nitric oxide (NO) degradation to enhance NO bioactivity. Because ATP/ADP generated by ectopic ATP synthase can induce NO production, substances such as CF6 can inhibit NO generation by inhibiting surface ATP/ADP production. Thus, the components of ectopic ATP synthase are associated with regulation of vascular tone. Through these functions, ectopic ATP synthase on ECs is considered a potential and novel therapeutic target for atherosclerosis, hypertension and lipid disorders. PMID:21247400

  4. Potential therapeutic target for malignant paragangliomas: ATP synthase on the surface of paraganglioma cells

    PubMed Central

    Fliedner, Stephanie MJ; Yang, Chunzhang; Thompson, Eli; Abu-Asab, Mones; Hsu, Chang-Mei; Lampert, Gary; Eiden, Lee; Tischler, Arthur S; Wesley, Robert; Zhuang, Zhengping; Lehnert, Hendrik; Pacak, Karel

    2015-01-01

    F1FoATP synthase (ATP synthase) is a ubiquitous enzyme complex in eukaryotes. In general it is localized to the mitochondrial inner membrane and serves as the last step in the mitochondrial oxidative phosphorylation of ADP to ATP, utilizing a proton gradient across the inner mitochondrial membrane built by the complexes of the electron transfer chain. However some cell types, including tumors, carry ATP synthase on the cell surface. It was suggested that cell surface ATP synthase helps tumor cells thriving on glycolysis to survive their high acid generation. Angiostatin, aurovertin, resveratrol, and antibodies against the α and β subunits of ATP synthase were shown to bind and selectively inhibit cell surface ATP synthase, promoting tumor cell death. Here we show that ATP synthase β (ATP5B) is present on the cell surface of mouse pheochromocytoma cells as well as tumor cells of human SDHB-derived paragangliomas (PGLs), while being virtually absent on chromaffin primary cells from bovine adrenal medulla by confocal microscopy. The cell surface location of ATP5B was verified in the tissue of an SDHB-derived PGL by immunoelectron microscopy. Treatment of mouse pheochromocytoma cells with resveratrol as well as ATP5B antibody led to statistically significant proliferation inhibition. Our data suggest that PGLs carry ATP synthase on their surface that promotes cell survival or proliferation. Thus, cell surface ATP synthase may present a novel therapeutic target in treating metastatic or inoperable PGLs. PMID:26101719

  5. The type I fatty acid and polyketide synthases: a tale of two megasynthases

    PubMed Central

    Tsai, Shiou-Chuan

    2008-01-01

    This review chronicles the synergistic growth of the fields of fatty acid and polyketide synthesis over the last century. In both animal fatty acid synthases and modular polyketide synthases, similar catalytic elements are covalently linked in the same order in megasynthases. Whereas in fatty acid synthases the basic elements of the design remain immutable, guaranteeing the faithful production of saturated fatty acids, in the modular polyketide synthases, the potential of the basic design has been exploited to the full for the elaboration of a wide range of secondary metabolites of extraordinary structural diversity. PMID:17898897

  6. Salmonella typhimurium mutants defective in acetohydroxy acid synthases I and II.

    PubMed

    Shaw, K J; Berg, C M; Sobol, T J

    1980-03-01

    An analysis of transposon-induced mutants shows that Salmonella typhimurium possesses two major isozymes of acetohydroxy acid synthase, the enzymes which mediate the first common step in isoleucine and valine biosynthesis. A third (minor) acetohydroxy acid synthase is present, but its significance in isoleucine and valine synthesis may be negligible. Mutants defective in acetohydroxy acid synthase II (ilvG::Tn10) require isoleucine, alpha-ketobutyrate, or threonine for growth, a mutant defective in acetohydroxy acid synthase I (ilvB::Tn5) is a prototroph, and a double mutant (ilvG::Tn10 ilvB::Tn5) requires isoleucine plus valine for growth. PMID:6245063

  7. Detection of the Messenger RNA Encoding for the Ferredoxin-Dependent Glutamate Synthase in Maize Leaf

    PubMed Central

    Commere, Bernard; Vidal, Jean; Suzuki, Akira; Gadal, Pierre; Caboche, Michel

    1986-01-01

    Ferredoxin-dependent glutamate synthase (EC 1.4.7.1), glutamate oxoglutarate aminotransferase (glutamate synthase) (GOGAT) messenger RNA was extracted from maize (Zea mays L.) leaves and partially purified through oligo(dT)-cellulose chromatography and ultracentrifugation in a sucrose gradient. mRNA were translated in vitro using a reticulocyte system. The glutamate synthase subunit was characterized by immunoprecipitation with antibodies raised against the rice (Oryza sativa L.) ferredoxin-glutamate synthase. The in vitro synthesized protein and the 145 kilodaltons genuine maize leaf subunit of GOGAT were found to comigrate in sodium dodecyl sulfate-polyacrylamide gel electrophoresis experiments. Images Fig. 1 Fig. 2 Fig. 3 PMID:16664732

  8. LAP6/POLYKETIDE SYNTHASE A and LAP5/POLYKETIDE SYNTHASE B encode hydroxyalkyl α-pyrone synthases required for pollen development and sporopollenin biosynthesis in Arabidopsis thaliana.

    PubMed

    Kim, Sung Soo; Grienenberger, Etienne; Lallemand, Benjamin; Colpitts, Che C; Kim, Sun Young; Souza, Clarice de Azevedo; Geoffroy, Pierrette; Heintz, Dimitri; Krahn, Daniel; Kaiser, Markus; Kombrink, Erich; Heitz, Thierry; Suh, Dae-Yeon; Legrand, Michel; Douglas, Carl J

    2010-12-01

    Plant type III polyketide synthases (PKSs) catalyze the condensation of malonyl-CoA units with various CoA ester starter molecules to generate a diverse array of natural products. The fatty acyl-CoA esters synthesized by Arabidopsis thaliana ACYL-COA SYNTHETASE5 (ACOS5) are key intermediates in the biosynthesis of sporopollenin, the major constituent of exine in the outer pollen wall. By coexpression analysis, we identified two Arabidopsis PKS genes, POLYKETIDE SYNTHASE A (PKSA) and PKSB (also known as LAP6 and LAP5, respectively) that are tightly coexpressed with ACOS5. Recombinant PKSA and PKSB proteins generated tri-and tetraketide α-pyrone compounds in vitro from a broad range of potential ACOS5-generated fatty acyl-CoA starter substrates by condensation with malonyl-CoA. Furthermore, substrate preference profile and kinetic analyses strongly suggested that in planta substrates for both enzymes are midchain- and ω-hydroxylated fatty acyl-CoAs (e.g., 12-hydroxyoctadecanoyl-CoA and 16-hydroxyhexadecanoyl-CoA), which are the products of sequential actions of anther-specific fatty acid hydroxylases and acyl-CoA synthetase. PKSA and PKSB are specifically and transiently expressed in tapetal cells during microspore development in Arabidopsis anthers. Mutants compromised in expression of the PKS genes displayed pollen exine layer defects, and a double pksa pksb mutant was completely male sterile, with no apparent exine. These results show that hydroxylated α-pyrone polyketide compounds generated by the sequential action of ACOS5 and PKSA/B are potential and previously unknown sporopollenin precursors. PMID:21193570

  9. Nitric oxide synthase in macula densa regulates glomerular capillary pressure.

    PubMed Central

    Wilcox, C S; Welch, W J; Murad, F; Gross, S S; Taylor, G; Levi, R; Schmidt, H H

    1992-01-01

    Tubular-fluid reabsorption by specialized cells of the nephron at the junction of the ascending limb of the loop of Henle and the distal convoluted tubule, termed the macula densa, releases compounds causing vasoconstriction of the adjacent afferent arteriole. Activation of this tubuloglomerular feedback response reduces glomerular capillary pressure of the nephron and, hence, the glomerular filtration rate. The tubuloglomerular feedback response functions in a negative-feedback mode to relate glomerular capillary pressure to tubular-fluid delivery and reabsorption. This system has been implicated in renal autoregulation, renin release, and longterm body fluid and blood-pressure homeostasis. Here we report that arginine-derived nitric oxide, generated in the macula densa, is an additional intercellular signaling molecule that is released during tubular-fluid reabsorption and counters the vasoconstriction of the afferent arteriole. Antibody to rat cerebellar constitutive nitric oxide synthase stained rat macula densa cells specifically. Microperfusion of the macula densa segment of single nephrons with N omega-methyl-L-arginine (an inhibitor of nitric oxide synthase) or with pyocyanin (a lipid-soluble inhibitor of endothelium-derived relaxation factor) showed that generation of nitric oxide can vasodilate the afferent arteriole and increase glomerular capillary pressure; this effect was blocked by drugs that prevent tubular-fluid reabsorption. We conclude that nitric oxide synthase in macula densa cells is activated by tubular-fluid reabsorption and mediates a vasodilating component to the tubuloglomerular feedback response. These findings imply a role for arginine-derived nitric oxide in body fluid-volume and blood-pressure homeostasis, in addition to its established roles in modulation of vascular tone by the endothelium and in neurotransmission. Images PMID:1281548

  10. Chondroitin sulfate synthase-3. Molecular cloning and characterization.

    PubMed

    Yada, Toshikazu; Sato, Takashi; Kaseyama, Hiromi; Gotoh, Masanori; Iwasaki, Hiroko; Kikuchi, Norihiro; Kwon, Yeon-Dae; Togayachi, Akira; Kudo, Takashi; Watanabe, Hideto; Narimatsu, Hisashi; Kimata, Koji

    2003-10-10

    Recently, it has become evident that chondroitin sulfate (CS) glycosyltransferases, which transfer glucuronic acid and/or N-acetylgalactosamine residues from each UDP-sugar to the nonreducing terminus of the CS chain, form a gene family. We report here a novel human gene (GenBank trade mark accession number AB086062) that possesses a sequence homologous with the human chondroitin sulfate synthase-1 (CSS1) gene, formerly known as chondroitin synthase. The full-length open reading frame consists of 882 amino acids and encodes a typical type II membrane protein. This enzyme contains a beta 3-glycosyltransferase motif and a beta 4-glycosyltransferase motif similar to that found in CSS1. Both the enzymes were expressed in COS-7 cells as soluble proteins, and their enzymatic natures were characterized. Both glucuronyltransferase and N-acetylgalactosaminyltransferase activities were observed when chondroitin, CS polymer, and their corresponding oligosaccharides were used as the acceptor substrates, but no polymerization reaction was observed as in the case of CSS1. The new enzyme was thus designated chondroitin sulfate synthase-3 (CSS3). However, the specific activity of CSS3 was much lower than that of CSS1. The reaction products were shown to have a GlcUA beta 1-3GalNAc linkage and a GalNAc beta 1-4GlcUA linkage in the nonreducing terminus of chondroitin resulting from glucuronyltransferase activity and N-acetylgalactosaminyltransferase activity, respectively. Quantitative real time PCR analysis revealed that the transcript level of CSS3 was much lower than that of CSS1, although it was ubiquitously expressed in various human tissues. These results indicate that CSS3 is a glycosyltransferase having both glucuronyltransferase and N-acetylgalactosaminyltransferase activities. It may make a contribution to CS biosynthesis that differs from that of CSS1. PMID:12907687

  11. Evolutionary Dynamics of the Cellulose Synthase Gene Superfamily in Grasses.

    PubMed

    Schwerdt, Julian G; MacKenzie, Katrin; Wright, Frank; Oehme, Daniel; Wagner, John M; Harvey, Andrew J; Shirley, Neil J; Burton, Rachel A; Schreiber, Miriam; Halpin, Claire; Zimmer, Jochen; Marshall, David F; Waugh, Robbie; Fincher, Geoffrey B

    2015-07-01

    Phylogenetic analyses of cellulose synthase (CesA) and cellulose synthase-like (Csl) families from the cellulose synthase gene superfamily were used to reconstruct their evolutionary origins and selection histories. Counterintuitively, genes encoding primary cell wall CesAs have undergone extensive expansion and diversification following an ancestral duplication from a secondary cell wall-associated CesA. Selection pressure across entire CesA and Csl clades appears to be low, but this conceals considerable variation within individual clades. Genes in the CslF clade are of particular interest because some mediate the synthesis of (1,3;1,4)-β-glucan, a polysaccharide characteristic of the evolutionarily successful grasses that is not widely distributed elsewhere in the plant kingdom. The phylogeny suggests that duplication of either CslF6 and/or CslF7 produced the ancestor of a highly conserved cluster of CslF genes that remain located in syntenic regions of all the grass genomes examined. A CslF6-specific insert encoding approximately 55 amino acid residues has subsequently been incorporated into the gene, or possibly lost from other CslFs, and the CslF7 clade has undergone a significant long-term shift in selection pressure. Homology modeling and molecular dynamics of the CslF6 protein were used to define the three-dimensional dispositions of individual amino acids that are subject to strong ongoing selection, together with the position of the conserved 55-amino acid insert that is known to influence the amounts and fine structures of (1,3;1,4)-β-glucans synthesized. These wall polysaccharides are attracting renewed interest because of their central roles as sources of dietary fiber in human health and for the generation of renewable liquid biofuels. PMID:25999407

  12. Assembly Line Polyketide Synthases: Mechanistic Insights and Unsolved Problems

    PubMed Central

    2015-01-01

    Two hallmarks of assembly line polyketide synthases have motivated an interest in these unusual multienzyme systems, their stereospecificity and their capacity for directional biosynthesis. In this review, we summarize the state of knowledge regarding the mechanistic origins of these two remarkable features, using the 6-deoxyerythronolide B synthase as a prototype. Of the 10 stereocenters in 6-deoxyerythronolide B, the stereochemistry of nine carbon atoms is directly set by ketoreductase domains, which catalyze epimerization and/or diastereospecific reduction reactions. The 10th stereocenter is established by the sequential action of three enzymatic domains. Thus, the problem has been reduced to a challenge in mainstream enzymology, where fundamental gaps remain in our understanding of the structural basis for this exquisite stereochemical control by relatively well-defined active sites. In contrast, testable mechanistic hypotheses for the phenomenon of vectorial biosynthesis are only just beginning to emerge. Starting from an elegant theoretical framework for understanding coupled vectorial processes in biology [Jencks, W. P. (1980) Adv. Enzymol. Relat. Areas Mol. Biol. 51, 75–106], we present a simple model that can explain assembly line polyketide biosynthesis as a coupled vectorial process. Our model, which highlights the important role of domain–domain interactions, not only is consistent with recent observations but also is amenable to further experimental verification and refinement. Ultimately, a definitive view of the coordinated motions within and between polyketide synthase modules will require a combination of structural, kinetic, spectroscopic, and computational tools and could be one of the most exciting frontiers in 21st Century enzymology. PMID:24779441

  13. Preliminary crystallographic analysis of a polyadenylate synthase from Megavirus

    PubMed Central

    Lartigue, Audrey; Jeudy, Sandra; Bertaux, Lionel; Abergel, Chantal

    2013-01-01

    Megavirus chilensis, a close relative of the Mimivirus giant virus, is also the most complex virus sequenced to date, with a 1.26 Mb double-stranded DNA genome encoding 1120 genes. The two viruses share common regulatory elements such as a peculiar palindrome governing the termination/polyadenylation of viral transcripts. They also share a predicted polyadenylate synthase that presents a higher than average percentage of residue conservation. The Megavirus enzyme Mg561 was overexpressed in Escherichia coli, purified and crystallized. A 2.24 Å resolution MAD data set was recorded from a single crystal on the ID29 beamline at the ESRF. PMID:23295487

  14. Nitroaromatic amino acids as inhibitors of neuronal nitric oxide synthase.

    PubMed

    Cowart, M; Kowaluk, E A; Daanen, J F; Kohlhaas, K L; Alexander, K M; Wagenaar, F L; Kerwin, J F

    1998-07-01

    Nitric oxide (NO.) is an important biomodulator of many physiological processes. The inhibition of inappropriate production of NO. by the isoforms of nitric oxide synthase (NOS) has been proposed as a therapeutic approach for the treatment of stroke, inflammation, and other processes. In this study, certain 2-nitroaryl-substituted amino acid analogues were discovered to inhibit NOS. Analogues bearing a 5-methyl substituent on the aromatic ring demonstrated maximal inhibitory potency. For two selected inhibitors, investigation of the kinetics of the enzyme showed the inhibition to be competitive with l-arginine. Additionally, functional NOS inhibition in tissue preparations was demonstrated. PMID:9651169

  15. Preliminary crystallographic analysis of a polyadenylate synthase from Megavirus.

    PubMed

    Lartigue, Audrey; Jeudy, Sandra; Bertaux, Lionel; Abergel, Chantal

    2013-01-01

    Megavirus chilensis, a close relative of the Mimivirus giant virus, is also the most complex virus sequenced to date, with a 1.26 Mb double-stranded DNA genome encoding 1120 genes. The two viruses share common regulatory elements such as a peculiar palindrome governing the termination/polyadenylation of viral transcripts. They also share a predicted polyadenylate synthase that presents a higher than average percentage of residue conservation. The Megavirus enzyme Mg561 was overexpressed in Escherichia coli, purified and crystallized. A 2.24 Å resolution MAD data set was recorded from a single crystal on the ID29 beamline at the ESRF. PMID:23295487

  16. Producing a trimethylpentanoic acid using hybrid polyketide synthases

    SciTech Connect

    Katz, Leonard; Fortman, Jeffrey L; Keasling, Jay D

    2014-10-07

    The present invention provides for a polyketide synthase (PKS) capable of synthesizing trimethylpentanoic acid. The present invention also provides for a host cell comprising the PKS and when cultured produces the trimethylpentanoic acid. The present invention also provides for a method of producing the trimethylpentanoic acid, comprising: providing a host cell of the present invention, and culturing said host cell in a suitable culture medium such that the trimethylpentanoic acid is produced, optionally isolating the trimethylpentanoic acid, and optionally, reducing the isolated trimethylpentanoic acid into a trimethylpentanol or an iso-octane.

  17. Structure of a 6-pyruvoyltetrahydropterin synthase homolog from Streptomyces coelicolor

    PubMed Central

    Spoonamore, James E.; Roberts, Sue A.; Heroux, Annie; Bandarian, Vahe

    2008-01-01

    The X-ray crystal structure of the 6-pyruvoyltetrahydropterin synthase (PTPS) homolog from Streptomyces coelicolor, SCO 6650, was solved at 1.5 Å resolution. SCO 6650 forms a hexameric T-fold that closely resembles other PTPS proteins. The biological activity of SCO 6650 is unknown, but it lacks both a required active-site zinc metal ion and the essential catalytic triad and does not catalyze the PTPS reaction. However, SCO 6650 maintains active-site residues consistent with binding a pterin-like substrate. PMID:18931427

  18. Inhibitors of nitric oxide synthase in inflammatory arthritis.

    PubMed

    Boughton-Smith, N K; Tinker, A C

    1998-07-01

    There is considerable evidence that excessive nitric oxide (NO) synthesized from L-arginine by inducible nitric oxide synthase (iNOS) plays an important pathological role in inflammatory arthritis. Since NO synthesized by constitutive isoforms of NOS has a physiological role, a great deal of activity has been directed at identifying inhibitors of NOS that are selective for the induced isoform. The major chemical areas that have been described so far in the search for such selective iNOS inhibitors and the activity of some of these compounds in animal models of arthritis are reviewed. PMID:18465556

  19. Topography of prostaglandin H synthase. Antiinflammatory agents and the protease-sensitive arginine 253 region.

    PubMed

    Kulmacz, R J

    1989-08-25

    Prostaglandin H synthase catalyzes two reactions: the bis-dioxygenation of arachidonic acid to form prostaglandin G2 (cyclooxygenase activity), and the reduction of hydroperoxides to the corresponding alcohols (peroxidase activity). The cyclooxygenase activity can be selectively inhibited by many nonsteroidal antiinflammatory agents including indomethacin. In the native synthase, there is a single prominent protease-sensitive region, located near Arg253; binding of the heme prosthetic group makes the synthase resistant to proteases. To investigate the spatial relationship between the area of the synthase which interacts with indomethacin and the protease-sensitive region, the effects of indomethacin and similar agents on the protease sensitivity of the two enzymatic activities and of the synthase polypeptide were examined. Incubation of the synthase apoenzyme with trypsin (3.6% w/w) resulted in the time-dependent coordinate loss (75% at 1 h) of both enzymatic activities and the cleavage (85% at 1 h) of the 70-kDa subunit into 38- and 33-kDa fragments, indicating that proteolytic cleavage of the polypeptide at Arg253, destroyed both activities of the synthase simultaneously. Indomethacin, (S)-flurbiprofen, or meclofenamate (each at 20 microM) rendered both activities and the synthase polypeptide (at 5 microM subunit) resistant to attack by trypsin or proteinase K; these agents also inhibited the cyclooxygenase activity of the intact synthase. Two reversible cyclooxygenase inhibitors, ibuprofen and flufenamate, also made both of the activities and the synthase polypeptide more resistant to trypsin. Titration of the apoenzyme with indomethacin (0-3 mol/mol of synthase dimer) resulted in proportional increases in the inhibition of the cyclooxygenase and in the resistance to attack by trypsin. (R)-Flurbiprofen did not increase the resistance to protease or appreciably inhibit the cyclooxygenase. These results suggest that the same stereospecific interaction of these

  20. Human uroporphyrinogen III synthase: NMR-based mapping of the active site.

    PubMed

    Cunha, Luis; Kuti, Miklos; Bishop, David F; Mezei, Mihaly; Zeng, Lei; Zhou, Ming-Ming; Desnick, Robert J

    2008-05-01

    Uroporphyrinogen III synthase (URO-synthase) catalyzes the cyclization and D-ring isomerization of hydroxymethylbilane (HMB) to uroporphyrinogen (URO'gen) III, the cyclic tetrapyrrole and physiologic precursor of heme, chlorophyl, and corrin. The deficient activity of human URO-synthase results in the autosomal recessive cutaneous disorder, congenital erythropoietic porphyria. Mapping of the structural determinants that specify catalysis and, potentially, protein-protein interactions is lacking. To map the active site and assess the enzyme's possible interaction in a complex with hydroxymethylbilane-synthase (HMB-synthase) and/or uroporphyrinogen-decarboxylase (URO-decarboxylase) by NMR, an efficient expression and purification procedure was developed for these cytosolic enzymes of heme biosynthesis that enabled preparation of special isotopically-labeled protein samples for NMR characterization. Using an 800 MHz instrument, assignment of the URO-synthase backbone (13)C(alpha) (100%), (1)H(alpha) (99.6%), and nonproline (1)H(N) and (15)N resonances (94%) was achieved as well as 85% of the side-chain (13)C and (1)H resonances. NMR analyses of URO-synthase titrated with competitive inhibitors N(D)-methyl-1-formylbilane (NMF-bilane) or URO'gen III, revealed resonance perturbations of specific residues lining the cleft between the two major domains of URO synthase that mapped the enzyme's active site. In silico docking of the URO-synthase crystal structure with NMF-bilane and URO'gen III was consistent with the perturbation results and provided a 3D model of the enzyme-inhibitor complex. The absence of chemical shift changes in the (15)N spectrum of URO-synthase mixed with the homogeneous HMB-synthase holoenzyme or URO-decarboxylase precluded occurrence of a stable cytosolic enzyme complex. PMID:18004775

  1. Cloning and Molecular Characterization of β-1,3-Glucan Synthase from Sparassis crispa

    PubMed Central

    Yang, Yun Hui; Kang, Hyeon-Woo

    2014-01-01

    A β-glucan synthase gene was isolated from the genomic DNA of polypore mushroom Sparassis crispa, which reportedly produces unusually high amount of soluble β-1,3-glucan (β-glucan). Sequencing and subsequent open reading frame analysis of the isolated gene revealed that the gene (5,502 bp) consisted of 10 exons separated by nine introns. The predicted mRNA encoded a β-glucan synthase protein, consisting of 1,576 amino acid residues. Comparison of the predicted protein sequence with multiple fungal β-glucan synthases estimated that the isolated gene contained a complete N-terminus but was lacking approximately 70 amino acid residues in the C-terminus. Fungal β-glucan synthases are integral membrane proteins, containing the two catalytic and two transmembrane domains. The lacking C-terminal part of S. crispa β-glucan synthase was estimated to include catalytically insignificant transmembrane α-helices and loops. Sequence analysis of 101 fungal β-glucan synthases, obtained from public databases, revealed that the β-glucan synthases with various fungal origins were categorized into corresponding fungal groups in the classification system. Interestingly, mushrooms belonging to the class Agaricomycetes were found to contain two distinct types (Type I and II) of β-glucan synthases with the type-specific sequence signatures in the loop regions. S. crispa β-glucan synthase in this study belonged to Type II family, meaning Type I β-glucan synthase is expected to be discovered in S. crispa. The high productivity of soluble β-glucan was not explained but detailed biochemical studies on the catalytic loop domain in the S. crispa β-glucan synthase will provide better explanations. PMID:25071386

  2. Conversion of aminodeoxychorismate synthase into anthranilate synthase with Janus mutations: mechanism of pyruvate elimination catalyzed by chorismate enzymes.

    PubMed

    Culbertson, Justin E; Chung, Dong hee; Ziebart, Kristin T; Espiritu, Eduardo; Toney, Michael D

    2015-04-14

    The central importance of chorismate enzymes in bacteria, fungi, parasites, and plants combined with their absence in mammals makes them attractive targets for antimicrobials and herbicides. Two of these enzymes, anthranilate synthase (AS) and aminodeoxychorismate synthase (ADCS), are structurally and mechanistically similar. The first catalytic step, amination at C2, is common between them, but AS additionally catalyzes pyruvate elimination, aromatizing the aminated intermediate to anthranilate. Despite prior attempts, the conversion of a pyruvate elimination-deficient enzyme into an elimination-proficient one has not been reported. Janus, a bioinformatics method for predicting mutations required to functionally interconvert homologous enzymes, was employed to predict mutations to convert ADCS into AS. A genetic selection on a library of Janus-predicted mutations was performed. Complementation of an AS-deficient strain of Escherichia coli grown on minimal medium led to several ADCS mutants that allow growth in 6 days compared to 2 days for wild-type AS. The purified mutant enzymes catalyze the conversion of chorismate to anthranilate at rates that are ∼50% of the rate of wild-type ADCS-catalyzed conversion of chorismate to aminodeoxychorismate. The residues mutated do not contact the substrate. Molecular dynamics studies suggest that pyruvate elimination is controlled by the conformation of the C2-aminated intermediate. Enzymes that catalyze elimination favor the equatorial conformation, which presents the C2-H to a conserved active site lysine (Lys424) for deprotonation and maximizes stereoelectronic activation. Acid/base catalysis of pyruvate elimination was confirmed in AS and salicylate synthase by showing incorporation of a solvent-derived proton into the pyruvate methyl group and by solvent kinetic isotope effects on pyruvate elimination catalyzed by AS. PMID:25710100

  3. Direct transfer of starter substrates from type I fatty acid synthase to type III polyketide synthases in phenolic lipid synthesis.

    PubMed

    Miyanaga, Akimasa; Funa, Nobutaka; Awakawa, Takayoshi; Horinouchi, Sueharu

    2008-01-22

    Alkylresorcinols and alkylpyrones, which have a polar aromatic ring and a hydrophobic alkyl chain, are phenolic lipids found in plants, fungi, and bacteria. In the Gram-negative bacterium Azotobacter vinelandii, phenolic lipids in the membrane of dormant cysts are essential for encystment. The aromatic moieties of the phenolic lipids in A. vinelandii are synthesized by two type III polyketide synthases (PKSs), ArsB and ArsC, which are encoded by the ars operon. However, details of the synthesis of hydrophobic acyl chains, which might serve as starter substrates for the type III polyketide synthases (PKSs), were unknown. Here, we show that two type I fatty acid synthases (FASs), ArsA and ArsD, which are members of the ars operon, are responsible for the biosynthesis of C(22)-C(26) fatty acids from malonyl-CoA. In vivo and in vitro reconstitution of phenolic lipid synthesis systems with the Ars enzymes suggested that the C(22)-C(26) fatty acids produced by ArsA and ArsD remained attached to the ACP domain of ArsA and were transferred hand-to-hand to the active-site cysteine residues of ArsB and ArsC. The type III PKSs then used the fatty acids as starter substrates and carried out two or three extensions with malonyl-Co