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A luminescence-based assay of UDP-sugar producing pyrophosphorylases
Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).ORCID-id: 0000-0001-8685-9665
2014 (Engelska)Ingår i: Analytical Methods, ISSN 1759-9660, E-ISSN 1759-9679, Vol. 6, nr 1, s. 57-61Artikel i tidskrift (Refereegranskat) 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.

Ort, förlag, år, upplaga, sidor
2014. Vol. 6, nr 1, s. 57-61
Nationell ämneskategori
Kemi Botanik
Identifikatorer
URN: urn:nbn:se:umu:diva-85622DOI: 10.1039/C3AY41811aISI: 000329071500004OAI: oai:DiVA.org:umu-85622DiVA, id: diva2:695139
Forskningsfinansiär
VetenskapsrådetTillgänglig från: 2014-02-10 Skapad: 2014-02-07 Senast uppdaterad: 2018-06-08Bibliografiskt granskad
Ingår i avhandling
1. UDP-sugar metabolizing pyrophosphorylases in plants: formation of precursors for essential glycosylation-reactions
Öppna denna publikation i ny flik eller fönster >>UDP-sugar metabolizing pyrophosphorylases in plants: formation of precursors for essential glycosylation-reactions
2017 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Umeå: Umeå Universitet, 2017. s. 51
Nyckelord
Chemical library screening, Cell wall synthesis, Glycosylation, Nucleotide sugars, Oligomerization, Protein structure, Reverse chemical genetics, Sugar activation, UDP-sugar synthesis
Nationell ämneskategori
Växtbioteknologi Mikrobiologi Farmaceutisk synteskemi
Forskningsämne
biokemisk farmakologi; biokemi; biologi; molekylärbiologi
Identifikatorer
urn:nbn:se:umu:diva-134087 (URN)978-91-7601-713-5 (ISBN)
Disputation
2017-05-22, KB.E3.01, Lilla Hörsalen, KBC-huset, Umeå Universitet, Umeå, 10:15 (Engelska)
Opponent
Handledare
Tillgänglig från: 2017-04-28 Skapad: 2017-04-26 Senast uppdaterad: 2018-06-09Bibliografiskt granskad

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