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Substrate kinetics and substrate effects on the quaternary structure of barley UDP-glucose pyrophosphorylase
Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
Vise andre og tillknytning
2012 (engelsk)Inngår i: Phytochemistry, ISSN 0031-9422, E-ISSN 1873-3700, Vol. 79, s. 39-45Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

UDP-Glc pyrophosphorylase (UGPase) is an essential enzyme responsible for production of UDP-Glc, which is used in hundreds of glycosylation reactions involving addition of Glc to a variety of compounds. In this study, barley UGPase was characterized with respect to effects of its substrates on activity and quaternary structure of the protein. Its K(m) values with Glc-1-P and UTP were 0.33 and 0.25 mM, respectively. Besides using Glc-1-P as a substrate, the enzyme had also considerable activity with Gal-1-P; however, the K(m) for Gal-1-P was very high (>10 mM), rendering this reaction unlikely under physiological conditions. UGPase had a relatively broad pH optimum of 6.5-8.5, regardless of the direction of reaction. The enzyme equilibrium constant was 0.4, suggesting slight preference for the Glc-1-P synthesis direction of the reaction. The quaternary structure of the enzyme, studied by Gas-phase Electrophoretic Mobility Macromolecule Analysis (GEMMA), was affected by addition of either single or both substrates in either direction of the reaction, resulting in a shift from UGPase dimers toward monomers, the active form of the enzyme. The substrate-induced changes in quaternary structure of the enzyme may have a regulatory role to assure maximal activity. Kinetics and factors affecting the oligomerization status of UGPase are discussed.

sted, utgiver, år, opplag, sider
2012. Vol. 79, s. 39-45
Emneord [en]
Cell wall synthesis, Oligomerization, Protein structure, Sucrose metabolism, Sucrose synthase, Sugar activation, UDP-sugar synthesis
HSV kategori
Identifikatorer
URN: urn:nbn:se:umu:diva-58610DOI: 10.1016/j.phytochem.2012.04.002ISI: 000307031800003PubMedID: 22552276OAI: oai:DiVA.org:umu-58610DiVA, id: diva2:549270
Tilgjengelig fra: 2012-09-04 Laget: 2012-09-04 Sist oppdatert: 2018-06-08bibliografisk kontrollert
Inngår i avhandling
1. UDP-sugar metabolizing pyrophosphorylases in plants: formation of precursors for essential glycosylation-reactions
Åpne denne publikasjonen i ny fane eller vindu >>UDP-sugar metabolizing pyrophosphorylases in plants: formation of precursors for essential glycosylation-reactions
2017 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

UDP-sugar metabolizing pyrophosphorylases provide the primary mechanism for de novo synthesis of UDP-sugars, which can then be used for myriads of glycosyltranferase reactions, producing cell wall carbohydrates, sucrose, glycoproteins and glycolipids, as well as many other glycosylated compounds. The pyrophosphorylases can be divided into three families: UDP-Glc pyrophosphorylase (UGPase), UDP-sugar pyrophosphorylase (USPase) and UDP-N-acety lglucosamine pyrophosphorylase (UAGPase), which can be discriminated both by differences in accepted substrate range and amino acid sequences.

This thesis focuses both on experimental examination (and re-examination) of some enzymatic/ biochemical properties of selected members of the UGPases and USPases and UAGPase families and on the design and implementation of a strategy to study in vivo roles of these pyrophosphorylases using specific inhibitors. In the first part, substrate specificities of members of the Arabidopsis UGPase, USPase and UAGPase families were comprehensively surveyed and kinetically analyzed, with barley UGPase also further studied with regard to itspH dependency, regulation by oligomerization, etc. Whereas all the enzymes preferentially used UTP as nucleotide donor, they differed in their specificity for sugar-1-P. UGPases had high activity with D-Glc-1-P, but could also react with Frc-1-P, whereas USPase reacted with arange of sugar-1-phosphates, including D-Glc-1-P, D-Gal-1-P, D-GalA-1-P, β-L-Ara-1-P and α-D-Fuc-1-P. In contrast, UAGPase2 reacted only with D-GlcNAc-1-P, D-GalNAc-1-P and, to some extent, with D-Glc-1-P. A structure activity relationship was established to connect enzyme activity, the examined sugar-1-phosphates and the three pyrophosphorylases. The UGPase/USPase/UAGPase active sites were subsequently compared in an attempt to identify amino acids which may contribute to the experimentally determined differences in substrate specificities.

The second part of the thesis deals with identification and characterization of inhibitors of the pyrophosphorylases and with studies on in vivo effects of those inhibitors in Arabidopsis-based systems. A novel luminescence-based high-throughput assay system was designed, which allowed for quantitative measurement of UGPase and USPase activities, down to a pmol per min level. The assay was then used to screen a chemical library (which contained 17,500 potential inhibitors) to identify several compounds affecting UGPase and USPase. Hit-optimization on one of the compounds revealed even stronger inhibitors of UGPase and USPase which also strongly inhibited Arabidopsis pollen germination, by disturbing UDP-sugar metabolism. The inhibitors may represent useful tools to study in vivo roles of the pyrophosphorylases, as a complement to previous genetics-based studies.

The thesis also includes two review papers on mechanisms of synthesis of NDP-sugars. The first review covered the characterization of USPase from both prokaryotic and eukaryotic organisms, whereas the second review was a comprehensive survey of NDP-sugar producing enzymes (not only UDP-sugar producing and not only pyrophosphorylases). All these enzymes were discussed with respect to their substrate specificities and structural features (if known) and their proposed in vivo functions.

sted, utgiver, år, opplag, sider
Umeå: Umeå Universitet, 2017. s. 51
Emneord
Chemical library screening, Cell wall synthesis, Glycosylation, Nucleotide sugars, Oligomerization, Protein structure, Reverse chemical genetics, Sugar activation, UDP-sugar synthesis
HSV kategori
Forskningsprogram
biokemisk farmakologi; biokemi; biologi; molekylärbiologi
Identifikatorer
urn:nbn:se:umu:diva-134087 (URN)978-91-7601-713-5 (ISBN)
Disputas
2017-05-22, KB.E3.01, Lilla Hörsalen, KBC-huset, Umeå Universitet, Umeå, 10:15 (engelsk)
Opponent
Veileder
Tilgjengelig fra: 2017-04-28 Laget: 2017-04-26 Sist oppdatert: 2018-06-09bibliografisk kontrollert

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