pubmed.ncbi.nlm.nih.gov

Biochemical characterization of uracil phosphoribosyltransferase from Mycobacterium tuberculosis - PubMed

Biochemical characterization of uracil phosphoribosyltransferase from Mycobacterium tuberculosis

Anne Drumond Villela et al. PLoS One. 2013.

Abstract

Uracil phosphoribosyltransferase (UPRT) catalyzes the conversion of uracil and 5-phosphoribosyl-α-1-pyrophosphate (PRPP) to uridine 5'-monophosphate (UMP) and pyrophosphate (PP(i)). 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 PP(i) 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.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Chemical reaction catalyzed by MtUPRT.
Figure 2
Figure 2. SDS-PAGE analysis of MtUPRT purification steps.

Lane 1, molecular weight protein marker; lane 2, crude extract; lane 3, sample loaded onto DEAE Sepharose CL6B column; lane 4, sample loaded onto Sephacryl S-200 column; lane 5, sample loaded onto Mono Q column; lane 6, homogeneous recombinant MtUPRT eluted from the Mono Q column.

Figure 3
Figure 3. Sedimentation equilibrium experiment.

A model (equation) of absorbance versus cell radius was fitted to the data by applying nonlinear regression. The experimental data for 1.5 mg/mL of protein at 9,000 and 11,000 rpm are shown. The random distribution of the residues (top panel) indicated a good quality fit in agreement with monomer-tetramer equilibrium.

Figure 4
Figure 4. Calibration curve of Superdex 200 HR column with protein standards.

The following standards were employed (solid squares): ribonuclease A (13,700 Da), carbonic anhydrase (29,000 Da), ovalbumin (43,000 Da), conalbumin (75,000 Da), aldolase (158,000 Da), ferritin (440,000 Da) and thyroglobulin (669,000 Da). The K av value was calculated for each standard protein using the equation (V e – V 0)/(V t – V 0), where is V e the elution volume for the protein and V t is the total bed volume, and K av was plotted against the logarithm of standard molecular weights. The experimental K av (open square) suggests a value of 109,650 Da for the molecular mass of recombinant MtUPRT in solution.

Figure 5
Figure 5. Apparent steady-state kinetic parameters.

(A) Initial velocity of MtUPRT (U mg−1) as a function of increasing PRPP concentration in the presence of constant uracil concentration (10 µM). (B) Initial velocity of MtUPRT as a function of increasing uracil concentration in the presence of constant PRPP concentration (100 µM). (C) Initial velocity of MtUPRT as a function of increasing PRPP concentration in the presence of constant concentrations of uracil (10 µM) and GTP (100 µM). (D) Initial velocity of MtUPRT as a function of increasing uracil concentration in the presence of constant concentrations of PRPP (100 µM) and GTP (100 µM).

Figure 6
Figure 6. Multiple sequence alignment of amino acid sequences of UPRTs from M. tuberculosis, B. caldolyticus, T. gondii, E. coli and S. solfataricus.

Amino acids for each polypeptide sequence were independently numbered. Identical conserved residues are indicated by stars below the alignment. Residues proposed to be involved in catalysis (ARg102 and Asp198), PRPP substrate binding (ARg77 and Arg102), and (or not) C-terminal glycine (Gly205) are highlighted (MtUPRT numbering). Multiple sequence alignment was carried out using Clustal W2 software (

http://www.ebi.ac.uk/Tools/msa/clustalw2/

).

Figure 7
Figure 7. Evaluation of nucleotides as allosteric effectors.

All reactions contained 350 µM PRPP and 35 µM uracil. (•) standard reaction, (○) standard reaction containing 500 µM GTP, (□) standard reaction containing 500 µM CTP, (Δ) standard reaction containing 500 µM ATP, (×) standard reaction containing 500 µM UTP, (▪) standard reaction containing 100 µM UMP, (▴) standard reaction containing both 100 µM UMP and 500 µM CTP.

Figure 8
Figure 8. Initial velocity patterns for MtUPRT.

Double-reciprocal plot of enzyme initial velocity−1 (mg U−1) versus [PRPP]−1 (µM−1). Concentrations of uracil were: 6 µM (open circles), 8 µM (filled triangle), and 10 µM (open squares).

Figure 9
Figure 9. Isothermal titration (ITC) curves of binding of ligands to MtUPRT.

(A) Reverse titration of PRPP substrate. (B) Titration of uracil substrate. (C) Reverse titration of UMP product. (D) Titration of PPi product.

Figure 10
Figure 10. Proposed kinetic mechanism for MtUPRT.

This order of substrate binding and product release is suggested on the basis of thermodynamic results.

Figure 11
Figure 11. Dependence of kinetic parameters on pH.

(A) pH dependence of log k cat. (B) pH dependence of log k cat/K PRPP.

Similar articles

Cited by

References

    1. World Health Organization (2011) Global Tuberculosis Control: WHO report 2011.
    1. Jain A, Mondal R (2008) Extensively drug-resistant tuberculosis: current challenges and threats. FEMS Immunol Med Microbiol 53: 145–50. - PubMed
    1. Velayati AA, Farnia P, Masjedi MR, Ibrahim TA, Tabarsi P, et al. (2009) Totally drug-resistant tuberculosis strains: evidence of adaptation at the cellular level. Eur Respir J 34: 1202–1203. - PubMed
    1. Ducati RG, Ruffino-Netto A, Basso LA, Santos DS (2006) The resumption of consumption – a review on tuberculosis. Mem Inst Oswaldo Cruz 101: 697–714. - PubMed
    1. Pieters J (2008) Mycobacterium tuberculosis and the macrophage: maintaining a balance. Cell Host Microbe 3: 399–407. - PubMed

Publication types

MeSH terms

Substances

Grants and funding

This work was supported by funds of Decit/SCTIE/MS-MCT-CNPq-FNDCT-CAPES to National Institute of Science and Technology on Tuberculosis (INCT-TB) to DSS and LAB. LAB and DSS also acknowledge financial support awarded by FAPERGS-CNPq-PRONEX-2009 (Fundacao de Amparoa Pesquisa do estado do Rio Grand do Sul). CB acknowledges financial support from “Embrapa Recursos Genéticos e Biotecnologia”, Brazil. CHIR acknowledges financial support from FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo). LAB (CNPq, 520182/99-5), DSS (CNPq, 304051/1975-06), CBJ (304034/2008-8), and CHIR are Research Career Awardees of the National Research Council of Brazil (CNPq). RGD was a post-doctoral fellow of CNPq (The National Council for Scientific and Technological Development). ADV and LAR acknowledge scholarships awarded by CNPq (The National Council for Scientific and Technological Development). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

LinkOut - more resources