Mechanistic studies on bovine cytosolic 5′‐nucleotidase II, an enzyme belonging to the HAD superfamily

Abstract

Cytosolic 5¢-nucleotidase/phosphotransferase specific for 6-hydroxypurine monophosphate derivatives (cN-II), belongs to a class of phosphohydrolases that act through the formation of an enzyme-phosphate intermediate. Sequence alignment with members of the P-type ATPases/L-2-haloacid dehalogenase superfamily identified three highly conserved motifs in cN-II and other cytosolic nucleotidases. Mutagenesis studies at specific amino acids occurring in cN-II conserved motifs were performed. The modification of the measured kinetic parameters, caused by conservative and nonconservative substitutions, suggested that motif I is involved in the formation and stabilization of the covalent enzyme-phosphate intermediate. Similarly, T249 in motif II as well as K292 in motif III also contribute to stabilize the phospho-enzyme adduct. Finally, D351 and D356 in motif III coordinate magnesium ion, which is required for catalysis. These findings were consistent with data already determined for P-type ATPases, haloacid dehalogenases and phosphotransferases, thus suggesting that cN-II and other mammalian 5¢-nucleotidases are characterized by a 3D arrangement related to the 2-haloacid dehalogenase superfold. Structural determinants involved in differential regulation by nonprotein ligands and redox reagents of the two naturally occurring cN-II forms generated by proteolysis were ascertained by combined biochemical and mass spectrometric investigations. These experiments indicated that the C-terminal region of cN-II contains a cysteine prone to form a disulfide bond, thereby inactivating the enzyme. Proteolysis events that generate the observed cN-II forms, eliminating this C-terminal portion, may prevent loss of enzymic activity and can be regarded as regulatory phenomena.Keywords: catalytic residues; HAD; nucleotidase; regulation; site-directed mutagenesis.Mammalian 5¢-nucleotidases (eN, cN-Ia, cN-Ib, cN-II, cN-III, cdN and mdN) make up a family of proteins with different subcellular locations and remarkably low sequence similarities [1]. Besides ectosolic 5¢-nucleotidase, one mitochondrial and five cytosolic enzymes have been described to date. According to its substrate specificity and tissue distribution, each protein seems to play a specific role within the cell. In fact, cN-Is, which is highly expressed in skeletal muscle, heart and testis, is specific for AMP and seems to be involved in adenosine production during hypoxia or ischemia, because it mediates the cell response to low energy charges [2]. On the other hand, cN-II is more specific for inosine monophosphate (IMP) and GMP, and is a ubiquitous enzyme involved in the regulation of intracellular IMP and GMP concentrations [3]. Furthermore, cN-III, which is expressed in red blood cells and is specific for pyrimidines, seems to participate in RNA degradation during erythrocyte maturation [4]. Likewise, cytosolic and mitochondrial deoxynucleotidases (cdN and mdN) regulate nucleotide pools in their respective compartments [1].cN-II was the first member of the cytosolic 5¢-nucl...

Identification of Drosophila and Human 7-Methyl GMP-specific Nucleotidases

“…The protein encoded by CG3362 has a predicted molecular mass of 36.3 kDa and a similar extent of sequence identity (ϳ 35%) both with the human cytosolic 5Ј nucleotidase III (SwissProt Q9H0P0.3) (47) and the human cNIII-like protein (SwissProt Q969T7.3). The catalytic motifs for nucleotidase activity (30,48) are well conserved in CG3362 (Fig. 4) (see "Discussion").…”

Cell proliferation and drug sensitivity of human glioblastoma cells are altered by the stable modulation of cytosolic 5′-nucleotidase II

“…During the last decades cN-II has been intensively studied and shown to be an allosterically regulated bifunctional enzyme (Pesi et al, 1994(Pesi et al, , 2010Allegrini et al, 2001) belonging to the haloacids dehalogenase superfamily (Allegrini et al, 2004;Bretonnet et al, 2005). Its activity relies on the formation of a phospho-enzyme intermediate where the phosphate is either transferred to a nucleoside (phosphotransferase activity) or released by hydrolysis (Allegrini et al, 2001).…”

Activating mutations in the NT5C2 nucleotidase gene drive chemotherapy resistance in relapsed ALL

“…3). An additional structurally interesting allele is the NT5C2 Q523* nonsense mutation, which removes an inhibitory region located in the C-terminal segment of the NT5C2 protein 26 . In addition, and despite the absence of clear structural cues suggesting a role of other mutations in NT5C2 activation, nucleotidase activity analysis of NT5C2 R367Q and NT5C2 D407A mutant proteins revealed an 18 fold and a 16 fold increase in their 5'-IMP nucleotidase activity compared with wild type NT5C2, respectively (Fig.…”