Abstract
Uracil phosphoribosyltransferase (UPRT) catalyzes the conversion of uracil and 5-phosphoribosyl-α-1-pyrophosphate (PRPP) to uridine 5′-monophosphate (UMP) and pyrophosphate (PPi). UPRT plays an important role in the pyrimidine salvage pathway since UMP is a common precursor of all pyrimidine nucleotides. Here we describe cloning, expression and purification to homogeneity of upp-encoded UPRT from Mycobacterium tuberculosis (MtUPRT). Mass spectrometry and N-terminal amino acid sequencing unambiguously identified the homogeneous protein as MtUPRT. Analytical ultracentrifugation showed that native MtUPRT follows a monomer-tetramer association model. MtUPRT is specific for uracil. GTP is not a modulator of MtUPRT ativity. MtUPRT was not significantly activated or inhibited by ATP, UTP, and CTP. Initial velocity and isothermal titration calorimetry studies suggest that catalysis follows a sequential ordered mechanism, in which PRPP binding is followed by uracil, and PPi product is released first followed by UMP. The pH-rate profiles indicated that groups with pK values of 5.7 and 8.1 are important for catalysis, and a group with a pK value of 9.5 is involved in PRPP binding. The results here described provide a solid foundation on which to base upp gene knockout aiming at the development of strategies to prevent tuberculosis.
Highlights
The major etiological agent of human tuberculosis (TB), Mycobacterium tuberculosis, currently infects one-third of the world’s population
Results on initial velocity measurements and isothermal titration calorimetry (ITC) data on substrate(s)/product(s) binding suggest that M. tuberculosis UPRT (MtUPRT) follows a sequential ordered mechanism, in which PRPP binding is followed by uracil, and PPi dissociation is followed by Uridine 59-monophosphate (UMP) release into solution
The 624 bp upp gene was amplified from M. tuberculosis H37Rv genomic DNA, cloned into the pCR-Blunt cloning vector, and subcloned into the pET-23a(+) expression vector between the NdeI and BamHI restriction sites
Summary
The major etiological agent of human tuberculosis (TB), Mycobacterium tuberculosis, currently infects one-third of the world’s population. Results on initial velocity measurements and isothermal titration calorimetry (ITC) data on substrate(s)/product(s) binding suggest that MtUPRT follows a sequential ordered mechanism, in which PRPP binding is followed by uracil, and PPi dissociation is followed by UMP release into solution.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
