Adenylosuccinate Lyase from Leishmania major Friedlin and its role in the purine nucleotides salvage pathway / Adenylosuccinato lyase (ADSL) de leishmania major friedlin: sua relevância na via de recuperação de purino-nucleotídeos

AUTOR(ES)
DATA DE PUBLICAÇÃO

2006

RESUMO

Many species of Leishmania are responsible for serious visceral or skin diseases that exhibit high incidence in tropical and subtropical regions. The drugs currently employed in the treatment of parasitic diseases are potentially carcinogenic, often require prolonged treatment and patient hospitalization. An effective program of drug design requires the validation of the potential target. In this context, one of the most striking metabolic discrepancies between Trypanosomatidae and their human hosts is the purine nucleotide biosynthesis pathway. Adenylosuccinate lyase (ADSL) is a bifunctional enzyme that catalyses two non-sequential steps in this cycle (one in the de novo purine pathway and another in the salvage pathway). This particular ADSL feature motivated us to investigate if ADSL could give us information about purine biosynthesis evolution in Kinetoplastida. Hence, the present work is aimed to validate ADSL as a potential target using the RNAi (RNAi) technique, as well as to characterize the adsl gene and the recombinant enzyme from Leishmania major Friedlin (ADSL-Lm). The RNAi results proved that ADSL can be considered a potential target, because it is shown to be essential for parasite viability. Regarding the molecular characterization of the adsl-Lm gene, the mature mRNA transcript containing 2060 nucleotides was defined by 5and 3RT-PCR. Restriction analysis and Pulse Field Gel Electrophoresis (PFGE) followed by Southern Blot hybridizations showed that adsl-Lm is a single copy gene and is located in chromosome 4 of this parasite. The adsl-Lm gene was cloned into an expression vector and a purification protocol of the recombinant enzyme was established. The tetrameric form of the recombinant ADSL-Lm enzyme was confirmed by native gel electrophoresis and Dynamic Light Scattering. ADSL-Lm has an experimental pI of 6.07 and exhibited maximum enzymatic activity at pH 8.5. The kinetic parameters of Km, Vmax , Kcat and kinetic efficiency (Kcat/Vm) were obtained for adenylosuccinate substrate. Functional complementation experiments showed that the adsl-Lm gene can effectively complement the E. coli JK268 purB58 mutation. However, this complementation must be in the salvage pathway, because enzymatic assays were performed using SAICAR (de novo pathway substrate) and ADSL-Lm did not convert this compound into product. This result indicates that probably Trypanosomatidae is not an example of de novo purine nucleotide cycle lost. In addition, ADSL-Lm was crystallized in the tetragonal I4122 space group, with unit cell parameters a= b= 130,023 angstron, c= 316,826 angstron, = = =90° and diffracted beyond 2.2angstron. ADSL-Lm crystal structure has been determined by SIRAS (Single Isomorphous Replacement with Anomalous Dispersion), using both native and Gadolinium derivative crystals. The ADSL-Lm monomer is composed of three domains arranged in the elongated manner typical of enzymes in the p-elimination superfamily, and is constituted almost exclusively by helices. Three subunits are necessary to form the active site cleft and the residues His 153, His 231 (general bases), Gln 308, Asn 364 and Glu 369 are involved. Without a bound inhibitor or substrate, the specific contacts made by the enzyme to its two substrates cannot be analyzed in detail. Hence, co-crystallization and docking can help in this question.

ASSUNTO(S)

purino-nucleotídeos protein crystalography rna interference purine nucleotides adsl adsl rna de interferência cristalografia de proteínas

ACESSO AO ARTIGO

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