Crystal Structure of Human Cytosolic 5′-Nucleotidase II

Abstract

Cytosolic 5-nucleotidase II catalyzes the dephosphorylation of 6-hydroxypurine nucleoside 5-monophosphates and regulates the IMP and GMP pools within the cell. It possesses phosphotransferase activity and thereby also catalyzes the reverse reaction. Both reactions are allosterically activated by adeninebased nucleotides and 2,3-bisphosphoglycerate. We have solved structures of cytosolic 5-nucleotidase II as native protein (2.2 Å ) and in complex with adenosine (1.5 Å ) and beryllium trifluoride (2.15 Å ). The tetrameric enzyme is structurally similar to enzymes of the haloacid dehalogenase (HAD) superfamily, including mitochondrial 5(3)-deoxyribonucleotidase and cytosolic 5-nucleotidase III but possesses additional regulatory regions that contain two allosteric effector sites. At effector site 1 located near a subunit interface we modeled diadenosine tetraphosphate with one adenosine moiety in each subunit. This efficiently glues the tetramer subunits together in pairs. The model shows why diadenosine tetraphosphate but not diadenosine triphosphate activates the enzyme and supports a role for cN-II during apoptosis when the level of diadenosine tetraphosphate increases. We have also modeled 2,3-bisphosphoglycerate in effector site 1 using one phosphate site from each subunit. By comparing the structure of cytosolic 5-nucleotidase II with that of mitochondrial 5(3)-deoxyribonucleotidase in complex with dGMP, we identified residues involved in substrate recognition.Cytosolic 5Ј-nucleotidase II (cN-II), 2 also called purine 5Ј-nucleotidase, IMP-GMP specific nucleotidase or high K m 5Ј-nucleotidase, is one of the seven known mammalian 5Ј-nucleotidases that catalyze the dephosphorylation of ribo-and deoxyribonucleoside monophosphates to nucleoside and inorganic phosphate (1, 2). The seven enzymes differ in substrate specificity, subcellular location, and tissue-specific expression. Intracellular 5Ј-nucleotidases regulate nucleotide levels by counteracting the action of nucleoside kinases and competing with other enzymes that use the nucleoside monophosphates. This complex network of interactions contributes to maintain nucleotide pools in tune with the metabolic needs of the cell (1, 2).The structures of two of the six human intracellular 5Ј-nucleotidases, i.e. mitochondrial 5Ј(3Ј)-deoxyribonucleotidase (mdN) (3) and cytosolic 5Ј-nucleotidase III (cN-III) (PDB entry 2CN1) are known. The latter is almost identical to the published structure of mouse cN-III (4). Both mdN and cN-III belong to the haloacid dehalogenase (HAD) superfamily, which is defined by an ␣/␤-Rossmann-like domain and a smaller 4-helix bundle domain, which, however, is not present in all HAD superfamily enzymes. The intracellular 5Ј-nucleotidases share three conserved motifs that have been found in HAD superfamily enzymes such as phosphoserine phosphatase (5) , are located in the ␣/␤-Rossmann-like domain and build up the catalytic phosphate-binding site in these enzymes. Motif I is directly involved in the reaction mechanism of 5Ј-nucleotidases (3),...

Structural Insights into the Inhibition of Cytosolic 5′-Nucleotidase II (cN-II) by Ribonucleoside 5′-Monophosphate Analogues

“…The potential energy of the phosphonate nucleotides was minimized with a torsion force (set to 1000 kcal/mol) using Discover and the consistent valence force field in two phases, 1000 steps of steepest descent (SD) followed by 5000 steps of conjugate gradient with a tolerance of 0.001 kcal/mol Å (dielectric constant set to 1). The crystal structure of human cN-II [41] solved at high resolution (2JC9, 1.5 Å resolution) was used for docking of the various phosphonate analogues. A few new crystal structures of cN-II were solved since we have started our docking study, our choice to use 2JC9 was based on its better resolution and the absence of mutation in the active site (D52N) that could modify the interaction with the phosphonate group.…”

Crystal Structures of the Novel Cytosolic 5′-Nucleotidase IIIB Explain Its Preference for m7GMP

“…Hs cN-IIIA (originally called pyrimidine-specific 5′-nucleotidase 1) catalyzes the hydrolysis of CMP and UMP [25]. Several crystal structures of cN-IIIA in complex with the substrate UMP [14] or representing different states of the reaction mechanism [10], [13] have been solved showing the molecular basis for substrate binding and catalysis. Superposition of the HAD core domains of cN-IIIA and cN-IIIB shows that the orientation of the cap domains with respect to the HAD core are slightly different ( Figure 6 ).…”

“…Five of them, namely cytosolic 5′-nucleotidase IA (cN-IA), cytosolic 5′-nucleotidase IB (cN-IB), cytosolic 5′-nucleotidase II (cN-II), cytosolic 5′-nucleotidase IIIA (cN-IIIA; previously called cN-III) and cytosolic 5′(3′)-deoxyribonucleotidase (cdN) are located in the cytosol whereas one is mitochondrial (mitochondrial-5′(3′)-deoxyribonucleotidase, mdN) and one is an extracellular enzyme (ecto-5′-nucleotidase, eN). Crystal structures are available for five of the seven nucleotidases including the mitochondrial (mdN) [5]–[7] and the extracellular nucleotidase (eN) [8], [9] as well as the cytosolic nucleotidases cdN [7], cN-II [10]–[12] and cN-IIIA [13], [14]. Although the intracellular 5′-nucleotidases generally share a low sequence similarity, all enzymes are magnesium-dependent and belong to the haloacid dehalogenase (HAD) superfamily [15].…”

ASD v2.0: updated content and novel features focusing on allosteric regulation

“…dual activators/regulators, dual inhibitors/regulators, dual activators/inhibitors and multiple activators/inhibitors/regulators. For example, the endogenous theophylline can function as an allosteric activator for the adenosine receptor A1 (31) and as an allosteric inhibitor for the cAMP-specific 3′,5′-cyclic phosphodiesterase 4D (32); the endogenous ATP is involved in multiple allosteric regulation of targets, including cytosolic 5′-Nucleotidase II as an allosteric activator (33), glycogen phosphorylase α as an allosteric inhibitor (34) and aspartate carbamoyltransferase as an allosteric regulator (35). Due to the multiple targets of such modulators, there are 23 099 allosteric interactions between proteins and modulators recorded in ASD v2.0, larger than the total number of allosteric modulators.…”