Abstract
Purine nucleoside phosphorylases (PNPs) play an important role in the blood fluke parasite Schistosoma mansoni as a key enzyme of the purine salvage pathway. Here we present the structural and kinetic characterization of a new PNP isoform from S. mansoni, SmPNP2. Thermofluorescence screening of different ligands suggested cytidine and cytosine are potential ligands. The binding of cytosine and cytidine were confirmed by isothermal titration calorimetry, with a KD of 27 μM for cytosine, and a KM of 76.3 μM for cytidine. SmPNP2 also displays catalytic activity against inosine and adenosine, making it the first described PNP with robust catalytic activity towards both pyrimidines and purines. Crystal structures of SmPNP2 with different ligands were obtained and comparison of these structures with the previously described S. mansoni PNP (SmPNP1) provided clues for the unique capacity of SmPNP2 to bind pyrimidines. When compared with the structure of SmPNP1, substitutions in the vicinity of SmPNP2 active site alter the architecture of the nucleoside base binding site thus permitting an alternative binding mode for nucleosides, with a 180° rotation from the canonical binding mode. The remarkable plasticity of this binding site enhances our understanding of the correlation between structure and nucleotide selectivity, thus suggesting new ways to analyse PNP activity.
Highlights
Schistosomiasis is a tropical neglected disease that affects almost 206 million people worldwide according to World Health Organization (WHO), causing approximately 250,000 deaths annually [1]
Comparison of expression profiles derived from previous RNA-Seq experiments [26] available at GeneDB showed that SmPNP1 and SmPNP2 present a different expression pattern: SmPNP1 was highly expressed in adults while SmPNP2 is more abundant in cercariae
The unique properties of SmPNP2 were discovered using a combination of techniques such as Differential scanning fluorimetry (DSF), kinetics and X-ray crystallography
Summary
Schistosomiasis is a tropical neglected disease that affects almost 206 million people worldwide according to World Health Organization (WHO), causing approximately 250,000 deaths annually [1]. Zhou et al [14] have characterized three HMW NPs from the thermophiles Deinococcus geothermalis, Geobacilus thermoglucosidasius and Aeropyrum pernix All these NPs catalyze both 6-oxo and 6-amino purine substrates and some analogs, the natural substrates (inosine and adenosine) are better substrates than their 2-amino modified substrates. Structural information of SmPNP2 in both the apo form and in complexes with cytidine, cytosine, ribose-1-phosphate, adenine, hypoxanthine, and tubercidin was obtained. These data allow us to describe, for the first time, the kinetics and structure of a LMW PNP well suited for inosine, adenosine and unexpectedly cytidine phosphorolysis. We describe the structural basis for binding and catalysis of both purine and cytidine nucleosides
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