Cytosolic 5′‐nucleotidase (cN‐II), which acts preferentially on 6‐hydroxypurine nucleotides, is essential for the survival of several cell types. cN‐II catalyses both the hydrolysis of nucleotides and transfer of their phosphate moiety to a nucleoside acceptor through formation of a covalent phospho‐intermediate. Both activities are regulated by a number of phosphorylated compounds, such as diadenosine tetraphosphate (Ap4A), ADP, ATP, 2,3‐bisphosphoglycerate (BPG) and phosphate. On the basis of a partial crystal structure of cN‐II, we mutated two residues located in the active site, Y55 and T56. We ascertained that the ability to catalyse the transfer of phosphate depends on the presence of a bulky residue in the active site very close to the aspartate residue that forms the covalent phospho‐intermediate. The molecular model indicates two possible sites at which adenylic compounds may interact. We mutated three residues that mediate interaction in the first activation site (R144, N154, I152) and three in the second (F127, M436 and H428), and found that Ap4A and ADP interact with the same site, but the sites for ATP and BPG remain uncertain. The structural model indicates that cN‐II is a homotetrameric protein that results from interaction through a specific interface B of two identical dimers that have arisen from interaction of two identical subunits through interface A. Point mutations in the two interfaces and gel‐filtration experiments indicated that the dimer is the smallest active oligomerization state. Finally, gel‐filtration and light‐scattering experiments demonstrated that the native enzyme exists as a tetramer, and no further oligomerization is required for enzyme activation. Structured digital abstract http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-8011572: cN‐II (uniprotkb:http://www.uniprot.org/uniprot/O46411) and cN‐II (uniprotkb:http://www.uniprot.org/uniprot/O46411) bind (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407) by dynamic light scattering (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0038) http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-8011493, http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-8011481: cN‐II (uniprotkb:http://www.uniprot.org/uniprot/O46411) and cN‐II (uniprotkb:http://www.uniprot.org/uniprot/O46411) bind (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407) by molecular sieving (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0071)
Cytosolic 5′-nucleotidase II is a widespread IMP hydrolyzing enzyme, essential for cell vitality, whose role in nucleotide metabolism and cell function is still to be exactly determined. Cytosolic 5′-nucleotidase overexpression and silencing have both been demonstrated to be toxic for mammalian cultured cells. In order to ascertain the effect of enzyme expression on a well-known eukaryote simple model, we expressed cytosolic 5′-nucleotidase II in Saccharomyces cerevisiae, which normally hydrolyzes IMP through the action of a nucleotidase with distinct functional and structural features. Heterologous expression was successful. The yeast cells harbouring cytosolic 5′-nucleotidase II displayed a shorter duplication time and a significant modification of purine and pyrimidine derivatives concentration as compared with the control strain. Furthermore the capacity of homologous recombination in the presence of mutagenic compounds of yeast expressing cytosolic 5′-nucleotidase II was markedly impaired.
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