XI Salão de
Iniciação Científica
PUCRS
Cytidine 5’-triphosphate synthase from Mycobacterium
tuberculosis: expression, purification and characterization for
novel antitubercular drug development
Jacqueline Gonçalves Rehm1,2, Ardala Breda1, Diógenes Santiago Santos1, Luiz Augusto Basso1
1
Centro de Pesquisas em Biologia Molecular e Funcional - Instituto Nacional de Ciência e Tecnologia em
Tuberculose - PUCRS; 2Faculdade de Farmácia. Universidade Federal do Rio Grande do Sul - UFRGS
Abstract
Human tuberculosis (TB) is an infectious bacterial disease mainly caused by
Mycobacterium tuberculosis (MTB). There were a global estimative of 9.4 million incident
cases and 1.3 million deaths in 2008 [1]. The easily transmission, high rates of mortality and
HIV co-infection, along with the emergence of MTB drug-resistant strains, highlight the need
for development of novel, effective (against drug-resistant and latent MTB) and less toxic
anti-TB drugs, capable of shortening current therapy duration. The cytidine 5’-triphosphate
synthase enzyme (CTPS, E.C. 6.3.4.2) from MTB, encoded by pyrG gene, catalyzes the CTP
formation from UTP in presence of ATP, using L-glutamine or ammonia as the nitrogen
source; a critical step of the de novo pyrimidine pathway [2]. Human CTPS activity is
augmented in some forms of leukemia and some solid tumors. Since it plays a central role in
nucleic acid and membrane phospholipid biosynthesis, CTPS is a recognized target for
antineoplastic, antiviral and antiprotozoal agents’ development [3]. The objective of this study
is the molecular, kinetic and structural characterization of the CTPS enzyme from MTB
aiming future development of novel anti-TB agents.
Synthetic primers were designed and MTB genomic DNA was used as template for
pyrG PCR amplification. The amplicon was cloned into the pCR-Blunt® vector and
subcloned into the pET-23a(+) expression vector. Protein expression tests were performed in
different conditions using Escherichia coli as a host. Best CTPS soluble expression was
observed in Escherichia coli BL21(DE3) cells with Terrific Broth medium at 30°C,
transformed with pET-23a(+)::pyrG recombinant plasmids. Tests of enzyme purification
using High Performance Liquid Chromatography are currently underway to obtain
homogeneous CTPS for kinetics parameters determinations and structural studies. CTPS
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activity will monitored directly by the increase in the absorbance at 291nm, due to conversion
of UTP to CTP [4], for kinetic parameters determination. For the structural studies, initial
crystallization trials will be performed using screening kits from Hampton Research (Crystal
screens I/II) [5]. Results from this work will establish the biological role of the pyrG gene
from M. tuberculosis and these data might assist the rational design of CTPS specific
inhibitors, aiming the development of novel and more effective anti-TB agents.
[1] World Health Organization. Global Tuberculosis Control: a short update to the 2009 report. Available at:
http://whqlibdoc.who.int/publications/2009/9789241598866_eng.pdf . Acessed: 21 maio 2010.
[2] LONG C., KOSHLAND Jr D. E., Cytidine triphosphate synthetase, Methods Enzymol. 51 (1978) 79-83.
[3] TRAVIS J. et al., The role of lysine residues 297 and 306 in nucleoside triphosphate regulation of E. coli
CTP synthase: Inactivation by 2’,3’-dialdehyde ATP and mutational analyses, Biochimica et Biophysica Acta.
1764 (2006) 199-210.
[4] ANDERSON P. M., CTP Synthetase from E. coli: An Improved Purification Procedure and Characterization
of Hysteretic and Enzyme Concentration Effects on Kinetic Properties, Biochemistry, 22 (1983) 3285-3292.
[5] JANCARIK J., KIM S.-H., Sparse matrix sampling: a screening method for crystallization of proteins, J.
Appl. Cryst. 24 (1991) 409-411.
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