THE ADENYLOSSUCINATE LYASE ENZYME FROM MICOBACTERIUM TUBERCULOSIS AS A TARGET FOR THE DEVELOPMENT OF NEW ANTI-TB DRUGS

Candida Deves (INCT-TB); Christopher Schneider (INCT-TB); Luiz Augusto Basso (INCT-TB); Diógenes Santiago Santos (INCT-TB)

Tuberculosis (TB) has become one of the deadliest global emergencies due to the widespread existence of multiple drug-resistant strains of Mycobacterium tuberculosis and the increase of immuno-compromised populations in the world. One of the most promising tools for future drug discovery lies in the elucidation of the molecular structure of potential drug targets from M. tuberculosis. Enzymes of purine nucleotide biosynthesis have been proposed as attractive drug targets for the development of chemotherapeutic agents against infective and parasitic diseases. Adenylosuccinate lyase (ASL; EC 4.3.2.2) is the only enzyme of purine nucleotide biosynthesis to act at two distinct points in the pathway. The first ASL-catalyzed reaction is the conversion of succinylaminoimidazole carboxamide ribotide (SAICAR) to aminoimidazole carboxamide ribotide (AICAR) in the de novo biosynthesis. The second ASL-catalyzed reaction is the conversion of adenylosuccinate (S-AMP) to AMP and fumarate, a step common both to purine nucleotide biosynthesis and the purine salvage pathway. The aim of this work is to study and understand the biochemistry and kinetics of the recombinant ASL enzyme from M. tuberculosis. The full-length purB coding region (1419 bp) was PCR amplified from M. tuberculosis H37Rv genomic DNA. For subsequent cloning, forward and reverse primers were previously designed to contain NdeI and BamHI restriction sites. The amplified PCR fragment was cloned into the pCR-Blunt cloning vector, subcloned into the pET-23a(+) expression vector and its sequence was confirmed by automated DNA sequencing. The resulting pET-23a(+)::purB plasmid was transformed into the Escherichia coli BL21 (DE3) pLys strain, which was incubated at 37ºC in LB medium with and without IPTG induction (1 mM). One ml of each culture was collected throughout the first 48 hours post-induction. Cells were disrupted by sonication and soluble and insoluble fractions were obtained and analyzed by SDS-PAGE. The recombinant M. tuberculosis ASL protein was expressed in the soluble and insoluble fractions of the BL21 (DE3)pLys strain. To obtain a higher expression of soluble protein, other conditions are currently being tested. Purification by FPLC, biochemical assays, crystallization protocols and three-dimensional structure determination of M. tuberculosis ASL will be the next steps of this project.