Structure of Pyrimidine 5′-Nucleotidase Type 1

Abstract

Eukaryotic pyrimidine 5-nucleotidase type 1 (P5N-1) catalyzes dephosphorylation of pyrimidine 5-mononucleotides. Deficiency of P5N-1 activity in red blood cells results in nonspherocytic hemolytic anemia. The enzyme deficiency is either familial or can be acquired through lead poisoning. We present the crystal structure of mouse P5N-1 refined to 2.35 Å resolution. The mouse P5N-1 has a 92% sequence identity to its human counterpart. The structure revealed that P5N-1 adopts a fold similar to enzymes of the haloacid dehydrogenase superfamily. The active site of this enzyme is structurally highly similar to those of phosphoserine phosphatases. We propose a catalytic mechanism for P5N-1 that is also similar to that of phosphoserine phosphatases and provide experimental evidence for the mechanism in the form of structures of several reaction cycle states, including: 1) P5N-1 with bound Mg(II) at 2.25 Å , 2) phosphoenzyme intermediate analog at 2.30 Å , 3) product-transition complex analog at 2.35 Å , and 4) product complex at 2.1 Å resolution with phosphate bound in the active site. Furthermore the structure of Pb(II)-inhibited P5N-1 (at 2.35 Å ) revealed that Pb(II) binds within the active site in a way that compromises function of the cationic cavity, which is required for the recognition and binding of the phosphate group of nucleotides.Eukaryotic pyrimidine nucleotidases are enzymes involved in catabolism of RNA and in the pyrimidine salvage pathway (1, 2). These enzymes catalyze dephosphorylation of pyrimidine mononucleotides to their respective nucleosides. In addition, pyrimidine nucleotidases also possess phosphotransferase activity between pyrimidine mononucleotides and pyrimidine nucleosides (3, 4). Two different subtypes, hP5N-1 2 (UniProt code Q9H0P0) and hPN-2, which differ in their substrate specificities, were identified in human erythrocytes (5-7). Subtype hP5N-1 preferentially dephosphorylates 5Ј-UMP, 5Ј-CMP, and at a lower rate also 5Ј-dCMP, 5Ј-dTMP, and 5Ј-dUMP, whereas hPN-2 preferentially dephosphorylates 3Ј-dTMP, 3Ј-dUMP, and 3Ј-UMP but shows no activity against 5Ј-CMP and 5Ј-dCMP (3,4,8). Both enzymes are essentially inactive against purine nucleotides. This specificity of hPNs is unique among 5Ј-nucleotidases (EC 3.1.3.5), which are typically not discriminatory to the identity of the nucleotide base (9). The specificity of hPNs is crucial in erythrocytes for preservation of the internal pool of ATP/GTP (6). Two types of hP5N-1 deficiency are known: hereditary and acquired. Hereditary deficiency is associated with nonspherocytic hemolytic anemia (NHA) (2, 7, 10). Acquired deficiency results from inhibition of hP5N-1 by Pb(II) during lead poisoning, and its severity is related to the blood levels of Pb(II) (11)(12)(13)(14). Both NHA and severe lead poisoning (Ͼ80 g/dl Pb(II) in blood) have the same clinical manifestation: erythrocytes in blood smears of affected patients show a distinct basophilic stippling upon Wright's staining (7,14,15). At the molecular level, NHA is characterized by signifi...

Functional Role for a Conserved Aspartate in the Spo0E Signature Motif Involved in the Dephosphorylation of the Bacillus subtilis Sporulation Regulator Spo0A

“…These residues, Asn 12 32 of BA1655 (dimer) project outward from the face of the ␣2 helix; this face of the helix is distal to helix ␣1 in the monomer and also from the interface with the other molecule in the dimer, arguing against a structural role for this residue. Furthermore, the Spo0E I36A mutant protein did not interact with Spo0AϳP in the FRET assay, suggesting a possible role for Ile 36 in the interaction with Spo0AϳP. In support of this idea, the corresponding Ile 28 residue of BA5174 makes van der Waals contacts with residues 106 -108 of Spo0AϳP in the modeled complex.…”

“…The results shown in Table 1 indicated that whereas alanine substitution at residues Thr 35 , Glu 41 , and Cys 44 did not affect the activity of the Spo0E protein, the remaining mutant proteins were significantly affected in their ability to inhibit sporulation. These observations suggested a significant role in the structure and/or function of Spo0E for Ile 36 , Ser 39 Fig. 3.…”

“…The capacity of the Ser 35 hydroxyl to hydrogen bond to the main chain carbonyl oxygen of Val 31 could compensate for loss of intrahelical main chain hydrogen bonding were the helix to bend (29). Such an influence on the helix conformation may facilitate the extension of the Gln 36 and Asp 39 side chains into the active site cleft of Spo0A.…”

“…8). In the HADDOCK generated model, Gln 36 of BA5174 forms a hydrogen bond with Lys 106 of Spo0A, which in turn forms an ionic interaction with the Asp 56 phosphoryl group. Flanking the active sites of the respective molecules in the complex are interactions between the His 29 imidazole of BA5174 and the main chain carbonyl of Phe 108 of Spo0A and between the side chains of Asn 43 of BA5174 and Asp 10 of Spo0A (Fig.…”

The use of biophysical methods increases success in obtaining liganded crystal structures

“…These can show unexpected opportunities in recognition or selectivity that cannot be readily predicted by bioinformatics or computational analysis. Intricacies of enzyme mechanism only surmised and guessed at by detailed kinetic experiments can be greatly facilitated by the ability to trap complexes along the reaction pathway (Bitto et al, 2006). Whilst the site of ligand binding is often known, sometimes only a crystal structure of the liganded protein can truly confirm the nature of the interactions involved and the extent of the binding site.…”