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Identification and characterization of inhibitors of UDP-glucose and UDP-sugar pyrophosphorylases for in vivo studies
Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC). (Leszek A. Kleczkowski)
Umeå University, Faculty of Science and Technology, Department of Chemistry.
Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
2017 (English)In: The Plant Journal, ISSN 0960-7412, E-ISSN 1365-313X, Vol. 90, no 6, 1093-1107 p.Article in journal (Other academic) Published
Abstract [en]

UDP-sugars serve as ultimate precursors in hundreds of glycosylation reactions (e.g. for protein and lipid glycosylation, synthesis of sucrose, cell wall polysaccharides, etc.), underlying an important role of UDP-sugar-producing enzymes in cellular metabolism. However, genetic studies on mechanisms of UDP-sugar formation were frequently hampered by reproductive impairment of the resulting mutants, making it difficult to assess an in vivo role of a given enzyme. Here, a chemical library containing 17 500 compounds was separately screened against purified UDP-glucose pyrophosphorylase (UGPase) and UDP-sugar pyrophosphorylase (USPase), both enzymes representing the primary mechanisms of UDP-sugar formation. Several compounds have been identified which, at 50 μm, exerted at least 50% inhibition of the pyrophosphorylase activity. In all cases, both UGPase and USPase activities were inhibited, probably reflecting common structural features of active sites of these enzymes. One of these compounds (cmp #6), a salicylamide derivative, was found as effective inhibitor of Arabidopsis pollen germination and Arabidopsis cell culture growth. Hit optimization on cmp #6 yielded two analogs (cmp #6D and cmp #6D2), which acted as uncompetitive inhibitors against both UGPase and USPase, and were strong inhibitors in the pollen test, with apparent inhibition constants of less than 1 μm. Their effects on pollen germination were relieved by addition of UDP-glucose and UDP-galactose, suggesting that the inhibitors targeted UDP-sugar formation. The results suggest that cmp #6 and its analogs may represent useful tools to study in vivo roles of the pyrophosphorylases, helping to overcome the limitations of genetic approaches.

Place, publisher, year, edition, pages
John Wiley & Sons, 2017. Vol. 90, no 6, 1093-1107 p.
Keyword [en]
chemical library screening, inhibitors, UDP-sugar synthesis, pyrophosphorylases, pollen germination, Arabidopsis cell culture, enzyme kinetics, Arabidopsis thaliana, reverse chemical genetics
National Category
Biochemistry and Molecular Biology Pharmaceutical Sciences Plant Biotechnology
Identifiers
URN: urn:nbn:se:umu:diva-134159DOI: 10.1111/tpj.13531ISI: 000403881500006PubMedID: 28273406OAI: oai:DiVA.org:umu-134159DiVA: diva2:1091670
Note

First published in thesis in manuscript form.

Available from: 2017-04-27 Created: 2017-04-27 Last updated: 2017-07-19Bibliographically approved
In thesis
1. UDP-sugar metabolizing pyrophosphorylases in plants: formation of precursors for essential glycosylation-reactions
Open this publication in new window or tab >>UDP-sugar metabolizing pyrophosphorylases in plants: formation of precursors for essential glycosylation-reactions
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
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.

Place, publisher, year, edition, pages
Umeå: Umeå Universitet, 2017. 51 p.
Keyword
Chemical library screening, Cell wall synthesis, Glycosylation, Nucleotide sugars, Oligomerization, Protein structure, Reverse chemical genetics, Sugar activation, UDP-sugar synthesis
National Category
Plant Biotechnology Microbiology Pharmaceutical Chemistry
Research subject
biokemisk farmakologi; Biochemistry; biology; Molecular Biology
Identifiers
urn:nbn:se:umu:diva-134087 (URN)978-91-7601-713-5 (ISBN)
Public defence
2017-05-22, KB.E3.01, Lilla Hörsalen, KBC-huset, Umeå Universitet, Umeå, 10:15 (English)
Opponent
Supervisors
Available from: 2017-04-28 Created: 2017-04-26 Last updated: 2017-05-11Bibliographically approved

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