- Inhibition of Trypanosoma brucei glucose-6-phosphate dehydrogenase by human steroids and their effects on the viability of cultured parasites
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Dehydroepiandrosterone (DHEA) is known as an intermediate in the synthesis of mammalian steroids and a potent uncompetitive inhibitor of mammalian glucose-6-phosphate dehydrogenase (G6PDH), but not the enzyme from plants and lower eukaryotes. G6PDH cataly
- Cordeiro, Artur T.,Thiemann, Otavio H.,Michels, Paul A.M.
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- Mutations in the tetramer interface of human glucose-6-phosphate dehydrogenase reveals kinetic differences between oligomeric states
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Glucose-6-phosphate dehydrogenase (G6PDH) catalyzes the oxidation of glucose-6-phoshate to 6-phospho-gluconolactone with the concomitant reduction of NADP+ to NADPH. In solution, the recombinant human G6PDH is known to be active as dimers and tetramers. To distinguish between the kinetic properties of dimers and tetramers of the G6PDH is not trivial. Steady-state kinetic experiments are often performed at low enzyme concentrations, which favor the dimeric state. The present work describes two novel human G6PDH mutants, one that creates four disulfide bonds among apposing dimers, resulting in a ‘cross-linked’ tetramer, and another that prevents the dimer to dimer association. The functional and structural characterizations of such mutants indicate the tetramer as the most active form of human G6PDH.
- Ranzani, Americo Tavares,Cordeiro, Artur Torres
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- Non-thermal effect of a ceramics radiation on a yeast glucose-6-phosphate dehydrogenase.
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Non-thermal effect of a ceramics radiation on glucose-6-phosphate dehydrogenase has been investigated using the enzyme, glucose-6-phosphate and NADP+ separately irradiated at 10 degrees C by a ceracompo R plate and a ceramics un-sewed cloth (sheet). The Km for glucose-6-phosphate was increased 20% after 6 h of irradiation by the plate, but the Vmax/Km was decreased 24%. After 3 h of irradiation by the sheet, the Km was increased 17%, but after 6 h of irradiation it was decreased 11%. The 3 h of irradiation by the sheet slightly increased both enthalpy and entropy changes of the reaction, but the 6 h of irradiation significantly decreased them. Both thermodynamic parameters in the activated state were increased by the sheet irradiation. The promotion energy for both formations of the enzyme-substrate and their activated complex depended on enthalpy. The different effects of two ceramics radiators on G6PDH activity were discussed.
- Kohashi,Ohta,Watanabe
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- Structural and functional characterization of the phosphoglucomutase from Xanthomonas citri subsp. citri
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Citrus canker, caused by bacteria Xanthomonas citri subsp. citri, can affect all economically important varieties of citrus. Studying Xanthomonas genes related to the invasive capacity may improve the knowledge on how this works and ultimately use the information to avoid the disease. Some annotated genes from Xanthomonas citri subsp. citri published genome are addressed to an interesting class of genes named “pathogenicity, virulence and adaptation”. One of them is xanA, which encodes a predicted phosphoglucomutase. Phosphoglucomutases are ubiquitous enzymes among the living kingdoms that play roles in carbohydrate metabolism, catalyzing the reversible conversion of 1- to 6-phosphoglucose. In Xanthomonas, phosphoglucomutase activity is required to synthesize precursors of the pathogenesis-related polysaccharide xanthan. In this work, a characterization of this gene product is presented by structural and functional studies. Molecular cloning was used for heterologous expression and deletion of xanA. A Michaelis-Menten kinetics model was obtained using the recombinant protein. The protein structure was also determined by X-ray diffraction on the recombinant enzyme substrate-free, bound to glucose-1,6-biphosphate and to glucose-1-phosphate. Deletion of xanA was done with a suicide plasmid construct and the obtained mutant was tested for pathogenic capacity. This study is the first describing the properties of the Xanthomonas citri subsp. citri phosphoglucomutase.
- Goto, Leandro Seiji,Vessoni Alexandrino, André,Malvessi Pereira, Camila,Silva Martins, Carla,D'Muniz Pereira, Humberto,Brand?o-Neto, José,Marques Novo-Mansur, Maria Teresa
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- Genome-wide screening reveals the genetic determinants of an antibiotic insecticide in Bacillus thuringiensis
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Thuringiensin is a thermostable secondary metabolite in Bacillus thuringiensis and has insecticidal activity against a wide range of insects. Until now, the regulatory mechanisms and genetic determinants involved in thuringiensin production have remained unclear. Here, we successfully used heterologous expression-guided screening in an Escherichia coli-Bacillus thuringiensis shuttle bacterial artificial chromosome library, to clone the intact thuringiensin synthesis (thu) cluster. Then the thu cluster was located on a 110-kb endogenous plasmid bearing insecticide crystal protein gene cry1Ba in strain CT-43. Furthermore, the plasmid, named pBMB0558, was indirectly cloned and sequenced. The gene functions on pBMB0558 were annotated by BLAST based on the GenBank and KEGG databases. The genes on pBMB0558 could be classified into three functional modules: a thuringiensin synthesis cluster, a type IV secretion system-like module, and mobile genetic elements. By HPLC coupling mass spectrometer, atmospheric pressure ionization with ion trap, and TOF technologies, biosynthetic intermediates of thuringiensin were detected. The thuE gene is proved to be responsible for the phosphorylation of thuringiensin at the last step by vivo and vitro activity assays. The thuringiensin biosynthesis pathway was deduced and clarified. We propose that thuringiensin is an adenine nucleoside oligosaccharide rather than an adenine nucleotide analog, as is traditionally believed, based on the predicted functions of the key enzymes, glycosyltransferase (ThuF) and exopolysaccharide polymerization protein (Thu1).
- Liu, Xiao-Yan,Ruan, Li-Fang,Hu, Zhen-Fei,Peng, Dong-Hai,Cao, Shi-Yun,Yu, Zi-Niu,Liu, Yao,Zheng, Jin-Shui,Sun, Ming
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- Glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase: A unique bifunctional enzyme from Plasmodium falciparum
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The survival of malaria parasites in human RBCs (red blood cells) depends on the pentose phosphate pathway, both in Plasmodium falciparum and its human host. G6PD (glucose-6-phosphate dehydrogenase) deficiency, the most common human enzyme deficiency, leads to a lack of NADPH in erythrocytes, and protects from malaria. In P. falciparum, G6PD is combined with the second enzyme of the pentose phosphate pathway to create a unique bifunctional enzyme named GluPho (glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase). In the present paper,we report for the first time the cloning, heterologous overexpression, purification and kinetic characterization of both enzymatic activities of full-length PfGluPho (P. falciparum GluPho), and demonstrate striking structural and functional differences with the human enzymes. Detailed kinetic analyses indicate that PfGluPho functions on the basis of a rapid equilibrium random Bi Bi mechanism, where the binding of the second substrate depends on the first substrate. We furthermore show that PfGluPho is inhibited by S-glutathionylation. The availability of recombinant PfGluPho and the major differences to hG6PD (human G6PD) facilitate studies on PfGluPho as an excellent drug target candidate in the search for new antimalarial drugs. The Authors Journal compilation
- Jortzik, Esther,Mailu, Boniface M.,Preuss, Janina,Fischer, Marina,Bode, Lars,Rahlfs, Stefan,Becker, Katja
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- Functional Replacement of Histidine in Proteins to Generate Noncanonical Amino Acid Dependent Organisms
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Simple strategies to produce organisms whose growth is strictly dependent on the presence of a noncanonical amino acid are useful for the generation of live vaccines and the biological containment of recombinant organisms. To this end, we report an approach based on genetically replacing key histidine (His) residues in essential proteins with functional His analogs. We demonstrate that 3-methyl-l-histidine (MeH) functionally substitutes for a key metal binding ligand, H264, in the zinc-containing metalloenzyme mannose-6-phosphate isomerase (ManA). An evolved variant, Opt5, harboring both N262S and H264MeH substitutions exhibited comparable activities to wild type ManA. An engineered Escherichia coli strain containing the ManA variant Opt5 was strictly dependent on MeH for growth with an extremely low reversion rate. This straightforward strategy should be applicable to other metallo- or nonmetalloproteins that contain essential His residues.
- Gan, Fei,Liu, Renhe,Wang, Feng,Schultz, Peter G.
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- Sequential Reactions of Surface- Tethered Glycolytic Enzymes
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The development of complex hybrid organic-inorganic devices faces several challenges, including how they can generate energy. Cells face similar challenges regarding local energy production. Mammalian sperm solve this problem by generating ATP down the fl
- Mukai, Chinatsu,Bergkvist, Magnus,Nelson, Jacquelyn L.,Travis, Alexander J.
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- carba Nicotinamide Adenine Dinucleotide Phosphate: Robust Cofactor for Redox Biocatalysis
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Here we report a new robust nicotinamide dinucleotide phosphate cofactor analog (carba-NADP+) and its acceptance by many enzymes in the class of oxidoreductases. Replacing one ribose oxygen with a methylene group of the natural NADP+ was found to enhance stability dramatically. Decomposition experiments at moderate and high temperatures with the cofactors showed a drastic increase in half-life time at elevated temperatures since it significantly disfavors hydrolysis of the pyridinium-N?glycoside bond. Overall, more than 27 different oxidoreductases were successfully tested, and a thorough analytical characterization and comparison is given. The cofactor carba-NADP+ opens up the field of redox-biocatalysis under harsh conditions.
- D?ring, Manuel,Sieber, Volker,Simon, Robert C.,Tafertshofer, Georg,Zachos, Ioannis
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supporting information
p. 14701 - 14706
(2021/05/13)
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- Enzyme aggregation and fragmentation induced by catalysis relevant species
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It is usually assumed that enzymes retain their native structure during catalysis. However, the aggregation and fragmentation of proteins can be difficult to detect and sometimes conclusions are drawn based on the assumption that the protein is in its native form. We have examined three model enzymes, alkaline phosphatase (AkP), hexokinase (HK) and glucose oxidase (GOx). We find that these enzymes aggregate or fragment after addition of chemical species directly related to their catalysis. We used several independent techniques to study this behavior. Specifically, we found that glucose oxidase and hexokinase fragment in the presence ofd-glucose but notl-glucose, while hexokinase aggregates in the presence of Mg2+ion and either ATP or ADP at low pH. Alkaline phosphatase aggregates in the presence of Zn2+ion and inorganic phosphate. The aggregation of hexokinase and alkaline phosphatase does not appear to attenuate their catalytic activity. Our study indicates that specific multimeric structures of native enzymes may not be retained during catalysis and suggests pathways for different enzymes to associate or separate over the course of substrate turnover.
- Gentile, Kayla,Bhide, Ashlesha,Kauffman, Joshua,Ghosh, Subhadip,Maiti, Subhabrata,Adair, James,Lee, Tae-Hee,Sen, Ayusman
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p. 20709 - 20717
(2021/10/02)
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- Enzymatic glycosylation of indoxyglycosides catalyzed by a novel maltose phosphorylase from Emticicia oligotrophica
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Maltose phosphorylases (EC 2.4.1.8) catalyze the reversible conversion of maltose to glucose and glucose-1-phosphate in the presence of inorganic phosphate. Herein, we describe for the first time the use of a maltose phosphorylase for the synthesis of various anomerically modified diglycosides. The maltose phosphorylase used was isolated from the bacterium Emticicia oligotrophica and showed a high selectivity towards the phosphorolysis of maltose, whereas no phosphorolysis was observed using other glucose-containing disaccharides such as cellobiose, melibiose, sucrose and trehalose. The addition of glucose to various 5-bromo-4-chloro-3-indolyl-glycosides (X-sugars) was used to evaluate the promiscuity of the maltose phosphorylase, and product formation was verified by LC-ESI-MS and MALDI-TOF-MS. The simple expression and purification protocol and the use of maltose as an inexpensive starting material make this maltose phosphorylase from Emticicia oligotrophica a valuable novel biocatalyst for the synthesis of glucose-containing glycosides.
- Awad, Faisal Nureldin,Kulinich, Anna,Yao, Ming Jun,Duan, Xu Chu,Cai, Zhi Peng,Gu, Bin,Liu, Li,Voglmeir, Josef
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p. 301 - 314
(2016/12/07)
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- The metabolic and biochemical impact of glucose 6-sulfonate (sulfoquinovose), a dietary sugar, on carbohydrate metabolism
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Increased activity of the main carbohydrate pathways (glycolysis, pentose phosphate, and hexosamine biosynthetic pathways) is one of the hallmarks of metabolic diseases such as cancer. Sulfoquinovosyl diacylglycerol is a sulfoglycolipid found in the human
- Sacoman, Juliana L.,Badish, Lauren N.,Sharkey, Thomas D.,Hollingsworth, Rawle I.
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p. 21 - 29,9
(2012/12/12)
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- The metabolic and biochemical impact of glucose 6-sulfonate (sulfoquinovose), a dietary sugar, on carbohydrate metabolism
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Increased activity of the main carbohydrate pathways (glycolysis, pentose phosphate, and hexosamine biosynthetic pathways) is one of the hallmarks of metabolic diseases such as cancer. Sulfoquinovosyl diacylglycerol is a sulfoglycolipid found in the human
- Sacoman, Juliana L.,Badish, Lauren N.,Sharkey, Thomas D.,Hollingsworth, Rawle I.
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- Kinetics and Mechanism of Oxidation of D-Glucopyranose 1-Phosphate by Chromium(VI) and Vanadium(V) in Perchloric Acid Medium
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The kinetics of oxidation D-glucopyranose 1-phosphate by Cr(VI) and V(V) have been studied in perchloric acid medium spectrophotometrically in the UV and visible region respectively.Comparison of the present results with those observed in the oxidation of glucose and glucose 6-phosphate by these metal ion oxidants reveals that the present substrate undergoes oxidation by Cr(VI) or V(V) by mechanisms which are different from those suggested for the oxidation of glucose or glucose 6-phosphate.
- Sen Gupta, Kalyan Kali,Sen Gupta, Shipra,Mandal, Subrata Kumar,Basu, Samarendra Nath
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