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

BONOFIGLIO, L.; ZAVALA, A.; KOVACEC, V.; LEVIN, G. J.; MOGLIONI, A.; MIRANDA, M. V.; GARCÍA, E.; MOLLERACH, M.

Congreso; XI European Meeting on the Molecular Biology of the Pneumococcus (EuroPneumo 2013); 2013

The UDP-glucose pyrophosphorylase (GalU) is absolutely required for the biosynthesis of capsular polysaccharide, the sine qua non virulence factor of Streptococcus pneumoniae. Although GalU is widely distributed, the eukaryotic are completely unrelated to their prokaryotic counterparts; therefore we proposed that pneumococcal GalU is an important target to expand our knowledge of the underlying mechanism in capsule formation and could contribute to the development of new therapeutic strategies The recombinant GalU (rGalU) was purified in Escherichia coli and found to be stable and catalytically active. An average of 0.6g of active rGalU was obtained from 100 ml of culture broth. Purified enzyme preparation was used to adapt a colorimetric assay that that detects GalU activity. Herein we describe a 96 well assay for UDP-glucose pyrophosphorylase that is rapid, sensitive, and easy to perform. 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 the screening of chemical libraries of UDP-glucose pyrophosphorylase inhibitors. This work represents a fundamental step in the search of putative antipneumococcal drugs base on GalU inhibition.