Expression, purification, and characterization of a functionally active Mycobacterium tuberculosis UDP-glucose pyrophosphorylase

• Published in: Protein expression and purification Vol. 61; no. 1; pp. 50 - 56
• Main Authors: Lai, Xuhui, Wu, Jing, Chen, Shudan, Zhang, Xuelian, Wang, Honghai
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.09.2008
• Subjects: Chromatography, Gel; Chromatography, High Pressure Liquid; Escherichia coli; galU; Hydrogen-Ion Concentration; Kinetics; Mass Spectrometry; Mycobacterium tuberculosis; Mycobacterium tuberculosis - enzymology; Mycobacterium tuberculosis - pathogenicity; Rv0993; Subcellular Fractions - enzymology; Temperature; UDP-glucose pyrophosphorylase; UTP-Glucose-1-Phosphate Uridylyltransferase - chemistry; UTP-Glucose-1-Phosphate Uridylyltransferase - genetics; UTP-Glucose-1-Phosphate Uridylyltransferase - isolation & purification; Virulence; Virulence factor; UDP-glucose pyrophosphorylase; galU; Mycobacterium tuberculosis; Rv0993; Virulence factor
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2008.05.015

Tuberculosis, which is caused by Mycobacterium tuberculosis, remains to be a global health problem. The thick and complex cell envelope has been implicated in many aspects of the pathogenicity of M. tuberculosis. M. tuberculosis UDP-glucose pyrophosphorylase (UGP, coded by galU, Rv0993) is involved in cell envelope precursor synthesis. UGP catalyzes the reversible formation of UDP-glucose and inorganic pyrophosphate from UTP and glucose 1-phosphate (Glc-l-P). Bacterial UGPs are completely unrelated to their eukaryotic counterparts. This enzyme is recognized as a virulence factor in several bacterial species and is conserved among mycobacterial species, which makes it a good target for mycobacterial pathogenicity research. The recombinant M. tuberculosis UGP (rMtUGP) was purified in Escherichia coli and found to be stable and catalytically active. The effects of pH, temperature and Mg 2+ on enzyme activity were characterized. In addition, subcellular localization studies revealed that most of M. tuberculosis UGP protein was located in the cell wall. The purification and characterization of M. tuberculosis UGP may help to decipher the pathogenicity of M. tuberculosis.