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Benini, Stefano, Toccafondi, Mirco, Rejzek, Martin, Musiani, Francesco, Wagstaff, Ben A., Wuerges, Jochen, Cianci, Michele and Field, Robert A. 
ORCID: https://orcid.org/0000-0001-8574-0275
(2017)
Glucose-1-phosphate uridylyltransferase from Erwinia amylovora:Activity, structure and substrate specificity.
Biochimica et Biophysica Acta - Proteins and Proteomics, 1865 (11).
pp. 1348-1357.
ISSN 1570-9639
Abstract
Erwinia amylovora, a Gram-negative plant pathogen, is the causal agent of Fire Blight, a contagious necrotic disease affecting plants belonging to the Rosaceae family, including apple and pear. E. amylovora is highly virulent and capable of rapid dissemination in orchards; effective control methods are still lacking. One of its most important pathogenicity factors is the exopolysaccharide amylovoran. Amylovoran is a branched polymer made by the repetition of units mainly composed of galactose, with some residues of glucose, glucuronic acid and pyruvate. E. amylovora glucose-1-phosphate uridylyltransferase (UDP-glucose pyrophosphorylase, EC 2.7.7.9) has a key role in amylovoran biosynthesis. This enzyme catalyses the production of UDP-glucose from glucose-1-phosphate and UTP, which the epimerase GalE converts into UDP-galactose, the main building block of amylovoran. We determined EaGalU kinetic parameters and substrate specificity with a range of sugar 1-phosphates. At time point 120 min the enzyme catalysed conversion of the sugar 1-phosphate into the corresponding UDP-sugar reached 74% for N-acetyl-α-D-glucosamine 1-phosphate, 28% for α-D-galactose 1-phosphate, 0% for α-D-galactosamine 1-phosphate, 100% for α-D-xylose 1-phosphate, 100% for α-D-glucosamine 1-phosphate, 70% for α-D-mannose 1-phosphate, and 0% for α-D-galacturonic acid 1-phosphate. To explain our results we obtained the crystal structure of EaGalU and augmented our study by docking the different sugar 1-phosphates into EaGalU active site, providing both reliable models for substrate binding and enzyme specificity, and a rationale that explains the different activity of EaGalU on the sugar 1-phosphates used. These data demonstrate EaGalU potential as a biocatalyst for biotechnological purposes, as an alternative to the enzyme from Escherichia coli, besides playing an important role in E. amylovora pathogenicity.
| Item Type: | Article |
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| Additional Information: | Funding Information: This work was supported by the Autonomous Province of Bolzano (project 98 : “A structural genomics approach for the study of the virulence and pathogenesis of Erwinia amylovora ”), by the Free University of Bolzano (project 103 GAMEs: Galactose and glucuronic acid metabolism in Erwinia spp ), by the Faculty of Science and Technology (project 1466 , MACSIMA I: MAthematics, Chemistry, Statistics: Innovative Methods for Applications I), by the UK BBSRC Institute Strategic Programme Grant on Understanding and Exploiting Metabolism (MET) [ BB/J004561/1 ] and by the John Innes Foundation . F.M. was supported by CIRMMP (Consorzio Interuniversitario di Risonanze Magnetiche di Metallo-Proteine). Publisher Copyright: © 2017 Elsevier B.V. |
| Uncontrolled Keywords: | amylovoran,biotechnology,erwinia amylovora,galu,molecular docking,udp-glucose pyrophosphorylase,analytical chemistry,biophysics,biochemistry,molecular biology ,/dk/atira/pure/subjectarea/asjc/1600/1602 |
| Faculty \ School: | Faculty of Science > School of Chemistry, Pharmacy and Pharmacology |
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| Depositing User: | LivePure Connector |
| Date Deposited: | 04 Sep 2024 09:35 |
| Last Modified: | 25 Sep 2024 18:06 |
| URI: | https://ueaeprints.uea.ac.uk/id/eprint/96512 |
| DOI: | 10.1016/j.bbapap.2017.08.015 |
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