Change search
ReferencesLink to record
Permanent link

Direct link
Domain-specific determinants of catalysis/ substrate binding and the oligomerization status of barley UDP-glucose pyrophosphorylase
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 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.
Show others and affiliations
2009 (English)In: Biochimica et Biophysica Acta - Proteins and Proteomics, ISSN 1570-9639, E-ISSN 1878-1454, Vol. 1794, 1734-1742 p.Article in journal (Refereed) Published
Abstract [en]

UDP-glucose (UDPG) pyrophosphorylase (UGPase) produces UDPG for sucrose and polysaccharide synthesis and glycosylation reactions. In this study, several barley UGPase mutants were produced, either single amino acid mutants or involving deletions of N- and C-terminal domains (Ncut and Ccut mutants, respectively) and of active site region ("NB loop"). The Del-NB mutant yielded no activity, whereas Ncut deletions and most of Ccut mutants, including short deletions at the so called "I-loop" region of C-terminal domain, as well as a single K260A mutant resulted in very low activity. For wt and the mutants, kinetics with UDPG were linear on reciprocal plots, whereas PPi at concentrations above 1mM exerted strong substrate inhibition. Both K260A and most of the Ccut mutants had very high K(m) with PPi (up to 33mM), whereas Ncut deletions had greatly increased K(m) with UDPG (up to 57mM). Surprisingly, an 8 amino acid deletion from end of the C-terminus resulted in an enzyme (Ccut-8 mutant) with 44% higher activity when compared to wt, but with similar K(m) values. Whereas Ccut-8 existed solely as a monomer, other deletion mutants had a more oligomerized status, e.g. Ncut mutants existing primarily as dimers. Overall, the data confirmed the essential role of NB loop in catalysis, but also pointed out to the role of both N- and C-termini for activity, substrate binding and oligomerization. The importance of oligomerization status for enzymatic activity of UGPase is discussed.

Place, publisher, year, edition, pages
2009. Vol. 1794, 1734-1742 p.
URN: urn:nbn:se:umu:diva-23574DOI: 10.1016/j.bbapap.2009.08.009OAI: diva2:225254
Available from: 2009-06-25 Created: 2009-06-25 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

Open Access in DiVA

No full text

Other links

Publisher's full text

Search in DiVA

By author/editor
Meng, MengWilczynska, MalgorzataKleczkowski, Leszek A
By organisation
Department of Plant PhysiologyUmeå Plant Science Centre (UPSC)Department of Medical Biochemistry and Biophysics
In the same journal
Biochimica et Biophysica Acta - Proteins and Proteomics

Search outside of DiVA

GoogleGoogle Scholar
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

Altmetric score

Total: 107 hits
ReferencesLink to record
Permanent link

Direct link