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Production and characterization of mutants of UDP-glucose pyrophosphorylase
Umeå University, Faculty of Science and Technology, Plant Physiology.
2006 (English)Independent thesis Advanced level (degree of Master (One Year)), 20 credits / 30 HE creditsStudent thesis
Abstract [en]

UDP-glucose pyrophosphorylase (UGPase) is a key component of carbohydrate production in plants, especially with respect to sucrose synthesis/ metabolism, by producing UDP-glucose, a key precursor to sucrose and to many polysaccharides in cell walls. UDP-glucose is also utilized in the synthesis of carbohydrate moiety of glycolipids, glycoproteins and a variety of secondary metabolites, among other functions. The UGPase enzyme may have a rate-limiting function in sugar biosynthesis, and its activity is now known to increase upon variety of abiotic stresses, with possible effects on an overall carbohydrate budget in stressed plants. The enzyme has been proposed to be regulated by (de)oligomerization and it has been estabished that only monomeric form of the enzyme is active. Based on mutant studies, the deoligomerization step (formation of monomers) was found as rate-limiting. A structural model of barley UGPase was recently suggested, based on homology to a human Antigen-X (AGX) protein that has a 40% protein sequence similarity to eukaryotic UGPase. The 3D model shows a bowl-shaped protein with three different domains: (a) N-terminal, (b) central part which includes the nucleotide binding loop (NB-loop) at the active centre and (c) C-terminal which includes an insertion loop (I-loop) that is possibly involved in dimer formation and stabilization. In this study, the model was used as a testable blueprint to verify details of the barley enzyme catalysis and substrate binding, as well as oligomerization process.  In order to test the model, site-directed mutagenesis approaches and heterologous (E. coli) expression system were used to produce several UGPase mutants: Del-NB, lacking 4 amino acids (aa) at the NB region; Del-I-4 and Del-I-8, lacking respectively 4 and 8 aa of the I-loop; and Y192A, by replacing an active-site tyrosine into alanine. The Y192A mutant had about half the apparent activity of the wild-type (wt), whereas Del-I-8 and Del-I-4 had only 0.5 and 0.2 % activity, respectively, of the wt, and Del-NB showed no activity at all. Based on native-PAGE, both Y192A and Del-NB mutants had similar oligomerization status as the wt, i.e. existing as monomer only or a mixture of monomer, dimer and higher order oligomers, depending on incubation conditions. Both Del-I-8 and Del-I-4 were present in all conditions as higher order oligomers. Whereas Y192A mutant had similar Kms with both substrates as the wt protein, significant difference between the Del-I-4 and Del-I-8 mutants and wt could be detected. Both mutants had approximately 16-fold higher Kms for UDP-glucose, and the Kms with PPi were 735- and 1500-fold higher for Del-I-4 and Del-I-8, respectively, when compared to wt.The conclusion of those results: (A) Tyr-192 is not essential for activity and is not involved in substrate binding and/ or oligomerization of the enzyme. (B) The NB-loop is essential for catalysis, as evidenced by a complete lack of activity of the Del-NB mutant, and is not involved in oligomerization. On the other hand, (C) the region corresponding to central part of I-loop is located in the model far from active center, but deletion in this region does affect very strongly both catalysis and substrate binding parameters. This can be explained by the involvement of I-loop in formation of dimers (inactive) from monomers (active), as earlier proposed. Apparently, the Del-I-4 and Del-I-8 mutations lead to an enzyme form with a very high oligomerization ability. This affects both Kms and Vmaxs of the Del-I mutants. Taken together the results verify the essentiality of NB-loop for catalysis support the involvement of I-loop region in oligomerization and, overall, the importance of oligomerization status for enzymatic performance of UGPase.

Place, publisher, year, edition, pages
2006. , 45 p.
URN: urn:nbn:se:umu:diva-26081OAI: diva2:236490
KBF30, UPSC, Umeå universitet, Umeå (English)

Available from: 2009-09-30 Created: 2009-09-23 Last updated: 2009-09-30Bibliographically approved

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