Crystallographic structure of PNP from Mycobacterium tuberculosis complexed with 2,3 dideoxyinosine
Cortinóz, J.R.1; Moreno, F.B.1; Souza, M.M.1;Oliveira, J.S.2 ; Basso, L.A.3 ; Santos, D.S.2; Azevedo Jr, W.F.2
1 Departamento de Física, UNESP/IBILCE São José do Rio Preto, SP; 2 Centro de Pesquisa em Biologia Molecular e Funcional PUCRS Porto Alegre, RS; 3 UNESP Rio Claro, SP
Purine nucleoside phosphorylase is a ubiquitous enzyme of purine metabolism that functions in the salvage pathway, enabling the cells to utilize purine bases recovered from metabolized purine ribo- and deoxyribonucleosides to synthesize purine nucleotides, in humans and other organisms. PNP catalyzes the phosphorolysis of purine nucleosides to corresponding bases and sugar 1-phosphate. PNP is specific for purine nucleosides in the b-configuration exhibiting a preference for ribosyl-containing nucleosides relative to the analogs containing the arabinose, xylose, and lyxose stereoisomers. PNP cleaves the glycosidic bond from purine nucleoside with inversion of configuration to produce a-ribose 1-phosphate. In the de novo synthesis of purine ribonucleotides, the formation of AMP and GMP from IMP is irreversible, but purine bases, nucleosides, and nucleotides can be interconverted through the activities of purine nucleoside phosphorylase, adenosine deaminase, and hypoxanthine-guanine phosphoribosyl transferase.
The PNP from Mycobacterium tuberculosis have been identified with approximately 30% of similarity against HsPNP. The specific inhibition of MtPNP could potentially lead to the accumulation of guanine nucleotides since a putative guanylate kinase and nucleoside diphosphate kinase are encoded in the genome.
The ddI is an analogue of the naturally occurring purine nucleoside inosine. This nucleoside is converted within target cells to its active form ddA-triphosphate.
In the experiment, the native MtPNP was concentrated up to 25 mg.ml-1 at 5 mM Tris-HCl, pH 8.0. For crystallization trials the sample was mixed with mother liquor (100 mM Tris, pH 8.0, containing 25% PEG 3350, and 25 mM MgCl2) at a ratio of 1:1. To form the complex, we add on grow crystals the ligant ddI. A small amount of ddI in dust was mixed with 5 mL of de mother liquor and later auditioned in de drop containing the crystals.
The MtPNP.ddI crystals diffracted at 2.35 Å using synchrotron radiation source. The 150 frames were processed in a resolution range of 37,11 – 2.35 Å. Diffraction from the crystals was consistent with the space group P21212 (a = 118.96Å, b = 135.11Å, and c = 44.41Å). The structure of MtPNP.ddI was solved by molecular replacement. The best solution after rigid-body refinement yielded an initial Rfactor of 35.8% and correlation coefficient of 70.8%.
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