SITE-DIRECTED MUTAGENESIS OF THE CDD-ENCODED CYTIDINE DEAMINASE FROM MYCOBACTERIUM TUBERCULOSIS H37RV AND SOLUBLE EXPRESSION OF THE MUTANT ENZYMES

Valnês da Silva Rodrigues-junior (INCT-TB,CPBMF,PUC-RS); Jacqueline Gonçalves Rehm (INCT-TB,CPBMF,PUC-RS); Christopher Zandoná Schneider (INCT-TB,CPBMF,PUC-RS); Zilpa Adriana Sánchez-quitian (INCT-TB,CPBMF,PUC-RS); Diógenes Santiago Santos (INCT-TB,CPBMF,PUC-RS); Luiz Augusto Basso (INCT-TB,CPBMF,PUC-RS)

Human tuberculosis (TB), caused by Mycobacterium tuberculosis, remains a leading cause of mortality. The emergence of drug resistant strains of M. tuberculosis has exacerbated the treatment and control of TB. There is thus a continuous need to identify promising targets for the development of anti-TB agents and vaccines. Cytidine deaminase (CDA) is a pyrimidine salvage pathway enzyme that recycles cytidine and 2'-deoxycytidine for uridine and 2'-deoxyuridine synthesis, respectively. Our group has previously performed cloning, expression, purification, mass spectrometry, N-terminal amino acid sequencing, gel filtration chromatography and kinetic studies to demonstrate that the cdd gene encodes a protein having CDA activity in M. tuberculosis. In addiction, pH rate profile studies and multiple sequence alignment suggest that the conserved amino acid Glutamate in the position 47 (E47) is involved in catalysis and/or substrate binding to this enzyme. Then, the objective of this study is carrying out site-directed mutagenesis of this invariant residue, E47, replacing it for Alanine, Histidine or Glutamine, and investigating the kinetic properties of the resulting three mutant enzymes. The mutagenic oligonucleotide primers were designed according to the desired mutation, having between 25 and 45 bases in length, and a minimum CG content of 40%. Mutagenesis was performed using a PCR-amplification technique followed by an overlapping-PCR step both using the Pfu DNA polymerase. The gene product was than ligated into the pCR-Blunt cloning vector and subcloned into the pET-23a (+) expression vector. The sequences of the mutant cdd genes have been verified to both confirm the insertion of the desired mutation and to ensure that no unwanted mutations were introduced by the PCR steps. The expression of the mutant proteins in the soluble fraction was observed in Escherichia coli BL21(DE3) grown without isopropyl beta-d-thiogalactoside induction in LB medium at 37 °C for 9 h after the cultures reach an OD₆₀₀ 0.4-0.6. Protein purification experiments are currently underway in our laboratory and will provide enzymes in quantities necessary for kinetic studies, then paving the way for the assignment of the function of this residue in the catalytic mechanism. This work will provide an improved understanding about the enzyme catalytic mechanism and these data may help in the rational production of attenuated mycobacterial samples that could be used as vaccines to prevent TB.