- Structures of trehalose-6-phosphate phosphatase from pathogenic fungi reveal the mechanisms of substrate recognition and catalysis
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Trehalose is a disaccharide essential for the survival and virulence of pathogenic fungi. The biosynthesis of trehalose requires trehalose-6-phosphate synthase, Tps1, and trehalose-6-phosphate phosphatase, Tps2. Here, we report the structures of the N-terminal domain of Tps2 (Tps2NTD) from Candida albicans, a transition-state complex of the Tps2 C-terminal trehalose-6-phosphate phosphatase domain (Tps2PD) bound to BeF3 and trehalose, and catalytically dead Tps2PD(D24N) from Cryptococcus neoformans bound to trehalose-6-phosphate (T6P). The Tps2NTD closely resembles the structure of Tps1 but lacks any catalytic activity. The Tps2PD-BeF3 -trehalose and Tps2PD(D24N)-T6P complex structures reveal a "closed" conformation that is effected by extensive interactions between each trehalose hydroxyl group and residues of the cap and core domains of the protein, thereby providing exquisite substrate specificity. Disruption of any of the direct substrate-protein residue interactions leads to significant or complete loss of phosphatase activity. Notably, the Tps2PD-BeF3 -trehalose complex structure captures an aspartyl-BeF3 covalent adduct, which closely mimics the proposed aspartyl-phosphate intermediate of the phosphatase catalytic cycle. Structures of substrate-free Tps2PD reveal an "open" conformation whereby the cap and core domains separate and visualize the striking conformational changes effected by substrate binding and product release and the role of two hinge regions centered at approximately residues 102-103 and 184-188. Significantly, tps2Δ, tps2NTDAΔ, and tps2D705N strains are unable to grow at elevated temperatures. Combined, these studies provide a deeper understanding of the substrate recognition and catalytic mechanism of Tps2 and provide a structural basis for the future design of novel antifungal compounds against a target found in three major fungal pathogens.
- Miaoa, Yi,Tenorb, Jennifer L.,Toffalettib, Dena L.,Washingtona, Erica J.,Liuc, Jiuyu,Shadrickc, William R.,Schumachera, Maria A.,Leec, Richard E.,Perfectb, John R.,Brennana, Richard G.
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p. 7148 - 7153
(2016/07/07)
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- Control of phosphoryl migratory transesterifications allows regioselecive access to sugar phosphates
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Phosphate esters in polyhydroxylated systems are normally blighted by uncontrolled migration under a variety of reaction conditions. Cesium fluoride is demonstrated as a reagent to control migration of primary phosphates during transesterifications. This allows easy exchange of phosphoryl protecting groups enabling enhanced synthetic strategic flexibility and regioselective phosphate installation. Mechanistic analysis suggests that a fluoride-induced extended solvent sphere modulates steric bulk at phosphorus to favor the primary position.
- Patel, Mitul K.,Davis, Benjamin G.
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p. 346 - 349
(2013/03/14)
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- MODIFICATION OF TREHALOSE-6-PHOSPHATE LEVELS IN PLANTS
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Compounds which are trehalose-6-phosphate or trehalose-6-phosphonate precursors of formula (I) or agriculturally acceptable salts thereof are provided: (I) The compounds are useful in increasing starch production in plants.
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- DETECTION OF MYCOBACTERIA
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A method for determining the presence of mycobacteria species in an organism or biological sample, the method comprising adding to the organism or biological sample a probe molecule comprising a substrate and a label, which probe molecule can be incorporated into mycobacteria, the presence of mycobacteria being determined by a detector responsive to the presence of the label, optionally after applying a stimulus; suitable probe molecules include compounds comprising a label and a substrate, which label is can be detected by a detector responsive to the presence of the label, optionally after applying a stimulus, characterised by compound being able to engage with the active site of Antigen 85B (Ag85B) such that it can form simultaneous hydrogen bonds with two or more amino acids in the active site selected from Arg 43, Trp 264, Ser126, His 262 and Leu 42, or the corresponding amino acids in Antigen 85A (Ag85A) or Antigen 85C (Ag85C), at least one of which is with Ser126.
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Page/Page column 75-76
(2011/04/18)
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- Flow chemistry kinetic studies reveal reaction conditions for ready access to unsymmetrical trehalose analogues
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Monofunctionalization of trehalose, a widely-found symmetric plant disaccharide, was studied in a microreactor to give valuable kinetic insights that have allowed improvements in desymmetrization yields and the development of a reaction sequence for large scale monofunctionalizations that allow access to probes of trehalose's biological function.
- Patel, Mitul K.,Davis, Benjamin G.
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supporting information; experimental part
p. 4232 - 4235
(2010/11/18)
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- Saturation transfer difference NMR reveals functionally essential kinetic differences for a sugar-binding repressor protein
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The binding kinetics of disaccharides trehalose and trehalose-6-phosphate to repressor protein TreR have been determined using STD NMR and shed light on the contrasting biological roles of these two sugars.
- Perez-Victoria, Ignacio,Kemper, Sebastian,Patel, Mitul K.,Edwards, John M.,Errey, James C.,Primavesi, Lucia F.,Paul, Matthew J.,Claridge, Timothy D. W.,Davis, Benjamin G.
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supporting information; experimental part
p. 5862 - 5864
(2010/01/31)
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- Trehalose 6-Phosphate Production with Energy Coupling Fermentation by Yeast Cells
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We tried a method for the production of trehalose 6-phosphate (T6P) with energy-coupling fermentation by baker's yeast. T6P was produced in a reaction mixture containing glucose, 5′-UMP, MgSO4, inorganic phosphate, and dried cells of baker's yeast as the enzyme preparation. T6P was isolated from the reaction mixture and identified by TLC, HPLC, GC-MS, and enzymatic methods. The reaction conditions suitable for T6P production were investigated. The formation of T6P and its precursors, glucose 6-phosphate and UDPglucose, at various pHs and concentrations of substrates was examined. Accumulation of T6P was maximum with a reaction mixture containing 1 M glucose, 20 mM 5′-UMP, 20 mM MgSO4, 400 mM sodium phosphate buffer (pH 6.2), and 100 mg/ml dried cells of baker's yeast shaken at 37°C for 6 h. The yield of T6P as a percentage of glucose was 11% (mol/mol) under these reaction conditions.
- Doi, Junko,Yokoigawa, Kumio,Isobe, Yuka,Kawai, Hiroyasu
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p. 735 - 739
(2007/10/03)
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- Gram-scale synthesis of α,α-trehalose 6-monophosphate and α,α- trehalose 6,6'-diphosphate
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α,α-Trehalose derivatives α,α-trehalose 6-monophosphate and α,α-trehalose 6,6′-diphosphate were synthesized to investigate their biological properties of the nonreducing disaccharide. These compounds were significant to the biosynthetic pathway since α,α-trehalose 6-phosphate serves as an intermediate of α,α-trehalose. α,α-Trehalose 6-phosphate was also found to be significant in regulating the first steps of yeast glycolysis. In this paper, a number of methods was described to derive the α,α-trehalose phosphates. Trimethylsilylation and selective methanolysis of primary trimethylsilyloxy group were used.
- Ronnow,Meldal,Bock
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p. 323 - 328
(2007/10/02)
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