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UDP-sugar metabolizing pyrophosphorylases in plants: formation of precursors for essential glycosylation-reactions
Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. (Leszek A. Kleczkowski)
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 [en]
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: urn:nbn:se:umu:diva-134087ISBN: 978-91-7601-713-5 (tryckt)OAI: oai:DiVA.org:umu-134087DiVA, id: diva2:1091316
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
Delarbeid
1. Substrate kinetics and substrate effects on the quaternary structure of barley UDP-glucose pyrophosphorylase
Åpne denne publikasjonen i ny fane eller vindu >>Substrate kinetics and substrate effects on the quaternary structure of barley UDP-glucose pyrophosphorylase
Vise andre…
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.

Emneord
Cell wall synthesis, Oligomerization, Protein structure, Sucrose metabolism, Sucrose synthase, Sugar activation, UDP-sugar synthesis
HSV kategori
Identifikatorer
urn:nbn:se:umu:diva-58610 (URN)10.1016/j.phytochem.2012.04.002 (DOI)000307031800003 ()22552276 (PubMedID)
Tilgjengelig fra: 2012-09-04 Laget: 2012-09-04 Sist oppdatert: 2018-06-08bibliografisk kontrollert
2. A luminescence-based assay of UDP-sugar producing pyrophosphorylases
Åpne denne publikasjonen i ny fane eller vindu >>A luminescence-based assay of UDP-sugar producing pyrophosphorylases
2014 (engelsk)Inngår i: Analytical Methods, ISSN 1759-9660, E-ISSN 1759-9679, Vol. 6, nr 1, s. 57-61Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

A coupled luminescence assay was applied to monitor pyrophosphate (PPi) production by either purified barley UDP-glucose pyrophosphorylase (UGPase) or purified Leishmania UDP-sugar pyrophosphorylase (USPase). In the assay, the PPi produced by the pyrophosphorylases was converted to ATP by ATP-sulfurylase, and the ATP produced was linked to luminescent light formation through the action of firefly luciferase. The assay allowed for a quantitative measurement of UGPase and USPase activities, down to a pmol per min level. The activities were linear with time and proportional to the amount of the enzyme added, and were neither affected by Pi nor by DTT. For UGPase, K-m values with UTP and Glc-1-P were 0.14 and 0.26 mM, respectively, whereas for USPase the respective K-m values with UTP, Glc-1-P and Gal-1-P were 0.4, 2.9 and 3.9 mM. Possible applications of the luminescence-based assay for not only UDP-sugar producing pyrophosphorylases, but also other types of pyrophosphorylases are discussed.

HSV kategori
Identifikatorer
urn:nbn:se:umu:diva-85622 (URN)10.1039/C3AY41811a (DOI)000329071500004 ()
Forskningsfinansiär
Swedish Research Council
Tilgjengelig fra: 2014-02-10 Laget: 2014-02-07 Sist oppdatert: 2018-06-08bibliografisk kontrollert
3. Identification and characterization of inhibitors of UDP-glucose and UDP-sugar pyrophosphorylases for in vivo studies
Åpne denne publikasjonen i ny fane eller vindu >>Identification and characterization of inhibitors of UDP-glucose and UDP-sugar pyrophosphorylases for in vivo studies
2017 (engelsk)Inngår i: The Plant Journal, ISSN 0960-7412, E-ISSN 1365-313X, Vol. 90, nr 6, s. 1093-1107Artikkel i tidsskrift (Annet vitenskapelig) 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.

sted, utgiver, år, opplag, sider
John Wiley & Sons, 2017
Emneord
chemical library screening, inhibitors, UDP-sugar synthesis, pyrophosphorylases, pollen germination, Arabidopsis cell culture, enzyme kinetics, Arabidopsis thaliana, reverse chemical genetics
HSV kategori
Identifikatorer
urn:nbn:se:umu:diva-134159 (URN)10.1111/tpj.13531 (DOI)000403881500006 ()28273406 (PubMedID)
Merknad

Originally included in thesis in manuscript form.

Tilgjengelig fra: 2017-04-27 Laget: 2017-04-27 Sist oppdatert: 2018-06-25bibliografisk kontrollert
4. Substrate specificity and inhibitor sensitivity of plant UDP-sugar producing pyrophosphorylases
Åpne denne publikasjonen i ny fane eller vindu >>Substrate specificity and inhibitor sensitivity of plant UDP-sugar producing pyrophosphorylases
(engelsk)Manuskript (preprint) (Annet vitenskapelig)
HSV kategori
Identifikatorer
urn:nbn:se:umu:diva-134158 (URN)
Tilgjengelig fra: 2017-04-27 Laget: 2017-04-27 Sist oppdatert: 2018-06-09
5. UDP-sugar pyrophosphorylase: a new old mechanism for sugar activation
Åpne denne publikasjonen i ny fane eller vindu >>UDP-sugar pyrophosphorylase: a new old mechanism for sugar activation
2011 (engelsk)Inngår i: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 156, nr 1, s. 3-10Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Recent developments in studies on properties and functions of UDP-sugar pyrophosphorylase (USPase) in metabolism are presented. The protein was characterized from plants and protozoans (Leishmania, Trypanosoma), but apparently it is also present in bacteria. In plants, USPase deficiency leads to male-sterility. USPase produces a variety of UDP-sugars and their analogs required for cell wall biosynthesis as well as for protein and lipid glycosylation, among other functions. Substrate specificity of USPases from different sources is reviewed, and their function/ structure properties are discussed, based on recent crystallization of the protein, with emphasis on common structural blueprint with some other pyrophosphorylases. Some strategies for future research on USPase are discussed.

HSV kategori
Identifikatorer
urn:nbn:se:umu:diva-41618 (URN)10.1104/pp.111.174706 (DOI)000290207100001 ()21444645 (PubMedID)
Tilgjengelig fra: 2011-03-30 Laget: 2011-03-30 Sist oppdatert: 2018-06-08bibliografisk kontrollert
6. Sugar activation for production of nucleotide sugars as aubstrates for glycosyltransferases in plants
Åpne denne publikasjonen i ny fane eller vindu >>Sugar activation for production of nucleotide sugars as aubstrates for glycosyltransferases in plants
2015 (engelsk)Inngår i: Journal of Applied Glycoscience, ISSN 1344-7882, Vol. 62, nr 2, s. 25-36Artikkel, forskningsoversikt (Fagfellevurdert) Published
Abstract [en]

In order to serve as a glycosyl donor, a sugar or a sugar derivative (e.g. GlcA) needs to be “activated” to a highly energetic state of a nucleotide-sugar. This activation requires the involvement of specific enzymes which produce NDP-sugars (or, in one case, NMP-sugar), using NTP or NDP as substrate. The present review provides concise survey of distinct plant nucleotide-sugar pyrophosphorylases (all using NTP as one of the substrates and differing in sugar specificity) as well as nucleotide-sugar phosphorylases and sucrose synthase (all using NDP as one of substrates). The pyrophosphorylases discussed include UGPase, USPase, UAGPase, AGPase, GMPase (VTC1), and FKGP, whereas phosphorylases include ADP-Glc phosphorylase and GDP-Gal phosphorylase (VTC2/VTC5). We also discuss the activation mechanism of 3-deoxy-D-manno-octulosonic acid (Kdo) by CKS, leading to the formation of a unique NMP-linked sugar (CMP-Kdo).

sted, utgiver, år, opplag, sider
Tokyo: The Japanese Society of Applied Glycoscience, 2015
Emneord
Cell wall formation, glycosyltransferases, nucleotide sugars, sugar activation
HSV kategori
Forskningsprogram
biokemi
Identifikatorer
urn:nbn:se:umu:diva-118718 (URN)10.5458/jag.jag.JAG-2015_003 (DOI)
Forskningsfinansiär
Swedish Research Council
Tilgjengelig fra: 2016-03-30 Laget: 2016-03-30 Sist oppdatert: 2018-06-07bibliografisk kontrollert

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