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Overexpression of UDP-glucose dehydrogenase in Escherichia coli results in decreased biosynthesis of K5 polysaccharide
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
2003 (English)In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 374, no 3, 767-772 p.Article in journal (Refereed) Published
Abstract [en]

The Escherichia coli K5 capsular polysaccharide (glycosaminoglycan) chains are composed of the repeated disaccharide structure: -GlcAbeta1,4-GlcNAcalpha1,4-(where GlcA is glucuronic acid and GlcNAc is N-acetyl-D-glucosamine). The GlcA, present in most glycosaminoglycans, is donated from UDP-GlcA, which, in turn, is generated from UDP-glucose by the enzyme UDP-glucose dehydrogenase (UDPGDH). The formation of UDP-GlcA is critical for the biosynthesis of glycosaminoglycans. To investigate the role of UDPGDH in glycosaminoglycan biosynthesis, we used K5 polysaccharide biosynthesis as a model. E. coli was transformed with the complete gene cluster for K5 polysaccharide production. Additional transformation with an extra copy of UDPGDH resulted in an approx. 15-fold increase in the in vitro UDPGDH enzyme activity compared with the strain lacking extra UDPGDH. UDP-GlcA levels were increased 3-fold in overexpressing strains. However, metabolic labelling with [14C]glucose showed, unexpectedly, that overexpression of UDPGDH lead to decreased formation of K5 polysaccharide. No significant difference in the K5 polysaccharide chain length was observed between control and overexpressing strains, indicating that the decrease in K5-polysaccharide production most probably was due to synthesis of fewer chains. Our results suggest that K5-polysaccharide biosynthesis is strictly regulated such that increasing the amount of available UDP-GlcA results in diminished K5-polysaccharide production.

Place, publisher, year, edition, pages
2003. Vol. 374, no 3, 767-772 p.
Keyword [en]
bacterial capsule, Escherichia coli, glycosaminoglycan, K5 polysaccharide, UDP-glucose dehydrogenase, UDP-glucuronic acid
National Category
Medical and Health Sciences
URN: urn:nbn:se:uu:diva-92360DOI: 10.1042/BJ20030365ISI: 000185546800021PubMedID: 12775214OAI: oai:DiVA.org:uu-92360DiVA: diva2:165404
Available from: 2004-11-17 Created: 2004-11-17 Last updated: 2011-06-27Bibliographically approved
In thesis
1. Studies on the Role of UDP-Glucose Dehydrogenase in Polysaccharide Biosynthesis
Open this publication in new window or tab >>Studies on the Role of UDP-Glucose Dehydrogenase in Polysaccharide Biosynthesis
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Polysaccharides are found in all forms of life and serve diverse purposes. They are enzymatically synthesised from activated monosaccharide precursors, nucleotide sugars. One such nucleotide sugar is UDP-glucuronic acid, which is formed from UDP-glucose by the UDP-glucose dehydrogenase (UGDH) enzyme. UGDH has been proposed to have a regulatory role in the biosynthesis of polysaccharides. The aim of the studies presented in this thesis was to investigate the role of UGDH in the polysaccharide biosynthesis in three different systems: human cell culture, bacterial cultures and growing plants. The effects of UGDH-overexpression on polysaccharide biosyntheses and, when achievable, on UDP-sugar levels, were investigated.

A mammalian UGDH was cloned from a kidney cDNA library. Transient expression of the cloned enzyme in mammalian cells led to an increased UGDH-activity. Northern blotting analyses revealed a single transcript of 2.6 kb in adult mouse tissues whereas human tissues expressed a predominant transcript of 3.2 kb and a minor transcript of 2.6 kb.

Overexpression of the bovine UGDH in mammalian cells induced increased synthesis of the glycosaminoglycans; heparan sulphate, chondroitin sulphate and hyaluronan, without changing their relative proportions. The effects on glycosaminoglycan synthesis caused by an increased demand of UDP-glucuronic acid were studied by overexpression of hyaluronan synthase (Has3), which requires UDP-glucuronic acid as substrate. Overexpression of Has3 and coexpression of Has3 and UGDH resulted in highly augmented production of hyaluronan without noticeably affecting heparan sulfate and chondroitin sulfate synthesis.

Expression of the bacterial UGDH in E. coli resulted in increased formation of UDP-glucuronic acid, but, unexpectedly, also to synthesis of fewer K5 polysaccharide chains.

Overexpression of UGD1, one of four A. thaliana UGDH genes, in A. thaliana, resulted in dwarfism. Analysis of the cell wall polysaccharides showed alteration in saccharide composition. Paradoxically, the UDP-sugars derived from UDP-glucuronic acid decreased in amount.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2004. 58 p.
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 0282-7476 ; 1388
Biochemistry, UDP-glucose dehydrogenase, polysaccharide biosynthesis, glycosaminoglycan, A. thaliana, E. coli K5, Biokemi
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Medical and Physiological Chemistry
urn:nbn:se:uu:diva-4657 (URN)91-554-6088-7 (ISBN)
Public defence
2004-12-10, B22, BMC, Husargatan 3, Uppsala, 13:15
Available from: 2004-11-17 Created: 2004-11-17Bibliographically approved

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