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Molecular and kinetic characterization of two UDP-glucose pyrophosphorylases, products of distinct genes, from Arabidopsis.
Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).ORCID iD: 0000-0001-8685-9665
2008 (English)In: Biochimica et Biophysica Acta - Proteins and Proteomics, ISSN 1570-9639, E-ISSN 1878-1454, Vol. 1784, no 6, 967-972 p.Article in journal (Refereed) Published
Abstract [en]

UDP-glucose pyrophosphorylase (UGPase) is an important enzyme in the production (and conversions) of UDP-glucose, a key precursor for carbohydrate biosynthesis. cDNAs corresponding to two UGPase isozymes in Arabidopsis were overexpressed in Escherichia coli and, subsequently, the recombinant proteins were purified and characterized. Both proteins were highly conserved, sharing 93% identity. Based on crystal structure-derived images, the main amino acid differences mapped to N- and C-termini domains, but not to central active site region. The two proteins existed mainly as monomers, and they had similar molecular masses of ca. 53 kDa. However, comparison of molecular masses of UGPases from Arabidopsis root and leaf extracts revealed that the root protein was slightly larger, suggesting a post-translational modification. Specific activity of the purified UGPase-1 was ca. 10–30% lower than that of UGPase-2, depending on direction of the reaction, whereas its Km values with all substrates in both directions of the reaction were consistently ca. twice lower than those of UGPase-2 (0.03–0.14 mM vs. 0.07–0.36 mM, respectively). Both proteins were “true” UGPases, and had no activity with ADP-glucose/ATP or galactose-1-P. Equilibrium constant for both proteins was ca. 0.3, suggesting preference for the pyrophosphorolysis direction of the reaction. The data are discussed with respect to potential roles of UGPase in carbohydrate synthesis/metabolism in Arabidopsis.

Place, publisher, year, edition, pages
2008. Vol. 1784, no 6, 967-972 p.
URN: urn:nbn:se:umu:diva-9502DOI: doi:10.1016/j.bbapap.2008.02.021PubMedID: 18395530OAI: diva2:149173
Available from: 2008-05-27 Created: 2008-05-27 Last updated: 2015-04-29Bibliographically approved
In thesis
1. Plant UDP-glucose Pyrophosphorylase: Function and Regulation
Open this publication in new window or tab >>Plant UDP-glucose Pyrophosphorylase: Function and Regulation
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

UDP-glucose pyrophosphorylase (UGPase) is an important enzyme of carbohydrate metabolism in all living organisms. The main aim of this thesis was to investigate the function and regulation of plant UGP genes as well as the UGPase proteins. Both in vivo and in vitro approaches were used, including the use of transgenic plants deficient in UGPase activity, and using purified proteins and their mutants to elucidate the structure/ function properties of UGPase.

In both Arabidopsis and aspen, there are two highly similar UGP genes being actively transcribed, but not to the same extent. For both species, the UGP genes could be classified into two categories: a “house-keeping” gene and a subsidiary gene, with the former functioning universally in all the tissues to support the normal growth, whereas the latter usually expressed at a lower level in most of the organs/tissues tested. Besides, the two UGP genes were also found being differentially regulated under abiotic stress conditions, e.g. low temperature. By investigating the Arabidopsis T-DNA insertion mutants, which respectively have one or both of the UGP genes knocked out, we noticed that as little as 10% of the remaining UGPase activity could still support normal growth and development under controlled conditions, with little or no changes in carbohydrate contents, including soluble sugars (e.g. sucrose), starch and cell wall polysaccharides. Those plants, however, had a significantly decreased fitness under field conditions, i.e. the plants most deficient in UGPase activity produced up to 50% less seeds than in wt. Therefore, we concluded that UGPase is not a rate-limiting enzyme in carbohydrate synthesis pathways, but still is essential in viability of Arabidopsis plants.

In order to characterize two Arabidopsis UGPase isozymes, both proteins were heterologously overexpressed in prokaryotic cells and purified by affinity chromatography. The two isozymes showed little differences in physical and biochemical properties, including substrate specificity, Km values with substrates in both directions of the reaction, molecular masses, isoelectric point (pI), and equilibrium constant. On the other hand, possibilities of distinct post-translational regulatory mechanisms were indicated, based on amino acid (aa) motif analyses, and on 3D analyses of derived crystal structures of the two proteins.

We used the heterologous bacterial system also to overexpress barley UGPase and several of its mutants, both single mutants and those with whole domains/ exons deleted. As a result, we have identified several aa residues/ protein domains that may be essential for structural integrity and catalytic/ substrate-binding properties of the protein. For instance, we found that the last exon of UGPase (8 aa at the end of C-terminus) was important for the protein ability to oligomerize and that Lys-260 and the second-to-last exon were essential for pyrophosphate (but not UDP-glucose) binding. The data emphasized the critical role of central part of the active site (so called NB-loop) in catalysis, but also pointed out to the role of N-terminus in catalysis and oligomerization, but not substrate binding, and that of C-terminus in both catalysis/substrate binding and oligomerization.

Place, publisher, year, edition, pages
Umeå: Fysiologisk botanik, 2008. 38 p.
UDP-glucose pyrophosphorylase, carbohydrate metabolism, in vivo regulation, T-DNA knockout, heterologous expression, isozyme, mutagenesis, kinetic property, oligomerization
National Category
urn:nbn:se:umu:diva-1796 (URN)978-91-7264-604-9 (ISBN)
Public defence
2008-09-12, KB3B1, KBC, Umeå, 10:00 (English)
Available from: 2008-09-01 Created: 2008-09-01 Last updated: 2009-06-25Bibliographically approved

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Meng, MengWilczynska, MalgorzataKleczkowski, Leszek A
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Department of Plant PhysiologyUmeå Plant Science Centre (UPSC)Department of Medical Biochemistry and Biophysics
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