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Title

Purification of a recombinant functionally active Streptococcus pneumoniae UDP-glucose pyrophosphorylase and optimization of an assay to screen for inhibitors

AuthorsBonofiglio, L.; Zavala, Agustín; Kovacec, Verónica; Levín, Gustavo; Moglioni, Albertina; Miranda, María Victoria; García, Ernesto ; Mollerach, M.
KeywordsGalU
Antipneumococcal inhibitors
High scale purification
Issue DateMay-2013
PublisherFundación General de la Universidad de Alcalá
Instituto de Salud Carlos III (España)
CSIC - Instituto de Química Física Rocasolano (IQFR)
CSIC - Centro de Investigaciones Biológicas Margarita Salas (CIB)
CitationXI European Meeting on the Molecular Biology of the Pneumococcus (EuroPneumo 2013)
AbstractThe UDP-glucose pyrophosphorylase (GalU) of Streptococcus pneumoniae is absolutely required for the biosynthesis of capsular polysaccharide, the sine qua non virulence factor of pneumococcus. Although GalU is widely distributed, the eukaryotic enzymes are completely unrelated to their prokaryotic counterparts; therefore, we proposed that pneumococcal GalU is an important target to expand our knowledge of the mechanisms underlying capsule formation.This could contribute to the development of new therapeutic strategies to fight the pneumococcus.A recombinant form of the pneumococcal GalU was purified from Escherichia coli and found to be stable and catalytically active. An average of 0.6 g of active rGalU was obtained from 100 ml of culture. We describe a GalU assay that is rapid, sensitive, and easy to perform in 96-well plates.The purified enzyme was tested in the presence of several drugs with structural similarity to natural substrates of the GalU enzyme. Our results document that this colorimetric test is appropriate for screening of chemical libraries for UDP-glucose pyrophosphorylase inhibitors. This work represents a fundamental step in the search of novel antipneumococcal drugs.
Description2 p.
Publisher version (URL)http://europneumo2013.iqfr.csic.es/EP-2013-Program-and-Abstracts-Book.pdf
URIhttp://hdl.handle.net/10261/215143
ISBN978-84-88754-51-6
Appears in Collections:(CIB) Comunicaciones congresos
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