Biochemical Characterization of the Pneumococcal Glucose 1-Phosphate Uridylyltransferase (GalU) Essential for Capsule Biosynthesis |
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Authors: | Laura Bonofiglio Ernesto García Marta Mollerach |
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Institution: | (1) Cátedra de Microbiología, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, , Junín 956, , 1113 Buenos Aires, Argentina;(2) Centro de Investigaciones Biólogicas, CSIC, Ramiro de Maetzu 9, 28040 Madrid, Spain |
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Abstract: | The glucose 1-phosphate uridylyltransferase (GalU) is absolutely required for the biosynthesis of capsular polysaccharide,
the sine qua non virulence factor of Streptococcus pneumoniae. The pneumococcal GalU protein was overexpressed in Escherichia coli, and purified. GalU showed a pI of 4.23, and catalyzed the reversible formation of UDP-glucose and pyrophosphate from UTP
and glucose 1-phosphate with Km values of 0.4 mM for UDP-glucose, 0.26 mM for pyrophosphate, 0.19 mM for glucose 1-phosphate, and 0.24 mM for UTP. GalU has an optimum pH of 8–8.5, and requires Mg2+ for activity. Neither ADP-glucose nor TDP-glucose is utilized as substrates in vitro. The purification of GalU represents
a fundamental step to provide insights on drug design to control the biosynthesis of the main pneumococcal virulence factor. |
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