Biochemical Characterization of Uracil Phosphoribosyltransferase from Mycobacterium tuberculosis

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.

Enzymatic Production of Non‐Natural Nucleoside‐5′‐Monophosphates by a Thermostable Uracil Phosphoribosyltransferase

“…Notably, Tt UPRT shows higher specific activities than most other reported UPRTs under similar enzymatic assay conditions. By way of example, for the biosynthesis of UMP, the specific activity showed by Tt UPRT (3.7 μmol min −1 mg −1 ) was 1.2, 1.82, 2.4, 6.2, and 26.4 times higher than activity values of UPRTs from Giardia intestinalis , Gi UPRT (0.6 μmol min −1 mg −1 ); Toxoplasma gondii , Tg UPRT (2.03 μmol min −1 mg −1 ); Mycobacterium tuberculosis , Mt UPRT (1.7 μmol min −1 mg −1 ); Crithidia luciliae , Cl UPRT (0.6 μmol min −1 mg −1 ; adapted from) and Sulfolobus shibatae , Ssh UPRT, (0.14 μmol min −1 mg −1 , adapted from), respectively. UPRTase activity of UPRT from E. coli , Ec UPRT, and B. caldolyticus , Bc UPRT, is higher than specific activity showed by Tt UPRT.…”

“…Tt UPRT shows lower K M values for uracil than for PRPP. These results agree with kinetic data described for other UPRTs, In this sense, it is reasonable to think that similar to other UPRTs, catalysis could follow a sequential mechanism, in which PRPP binding is followed by uracil, and PPi product is released first, followed by UMP…”

“…Moreover, multiple sequence alignment of Tt UPRT with other homologous UPRTs reveals a high similarity between different UPRTs and clearly suggests that their three‐dimensional structures will be very similar, with a conserved core structure encompassing the PRPP/PPi and pyrimidine nucleobase binding sites and sharing similar substrate binding contacts. In this regard, four typical short regions (I–IV) of the UPRTs family, involved in substrate recognition, catalysis, and stability of the protein, can easily be recognized in Tt UPRT (Figure ), a finding which supports a similar active site (Figure ) . All described UPRTs contain a conserved PRPP binding domain (region III), thirteen‐residue PRPP‐binding motif region, which typically comprises four hydrophobic residues followed by two acidic residues, two hydrophobic residues, and four small residues .…”

Phosphoribosyl Diphosphate (PRPP): Biosynthesis, Enzymology, Utilization, and Metabolic Significance

“…Alternatively, GTP functions as an allosteric activator of the oligomeric uracil phosphoribosyltransferase of Sulfolobus species (234)(235)(236)(237). The enzyme from M. tuberculosis differs from the aforementioned in being unaffected by GTP (238,239), and the enzyme from Giardia intestinalis dimerizes in the presence of GTP (240).…”

Proteomic profiling of Botrytis cinerea conidial germination

“…This enzyme synthesizes Uridine 5′-monophosphate, a common precursor tuberculosis (Villela et al 2013). In Candida albicans, it has been related to primary resistance to flucytosine, an antifungal reagent (Hope et al 2004), which may indicate a role of the pyrimidine "de novo" synthesis as a therapeutic target for fungicide design and the development of fungal virulence.…”