Expression, purification, and characterization of a functionally active Mycobacterium tuberculosis UDP-glucose pyrophosphorylase.
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 Mg2+ 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.
Lai
Xuhui
X
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Room 301, Handan Road 220, Shanghai 200433, China. xuelianzhang@fudan.edu.cn
Wu
Jing
J
Chen
Shudan
S
Zhang
Xuelian
X
Wang
Honghai
H
D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013058:Mass Spectrometry; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000472:pathogenicity; D013347:Subcellular Fractions / Q000201:enzymology; D013696:Temperature; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D014774:Virulence
D005957:UTP-Glucose-1-Phosphate Uridylyltransferase