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PDBsum entry 1lv8

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protein ligands metals Protein-protein interface(s) links
Transferase PDB id
1lv8
Jmol
Contents
Protein chains
(+ 0 more) 265 a.a. *
Ligands
9PP ×6
Metals
_CA ×6
Waters ×724
* Residue conservation analysis
PDB id:
1lv8
Name: Transferase
Title: Crystal structure of calf spleen purine nucleoside phosphorylase in a new space group with full trimer in the asymmetric unit
Structure: Purine nucleoside phosphorylase. Chain: a, b, c, d, e, f. Ec: 2.4.2.1
Source: Bos taurus. Cattle. Organism_taxid: 9913. Tissue: spleen
Biol. unit: Trimer (from PQS)
Resolution:
2.30Å     R-factor:   0.189     R-free:   0.258
Authors: A.Bzowska,G.Koellner,B.Wielgus-Kutrowska,A.Stroh, G.Raszewski,A.Holy,T.Steiner,J.Frank
Key ref:
A.Bzowska et al. (2004). Crystal structure of calf spleen purine nucleoside phosphorylase with two full trimers in the asymmetric unit: important implications for the mechanism of catalysis. J Mol Biol, 342, 1015-1032. PubMed id: 15342253 DOI: 10.1016/j.jmb.2004.07.017
Date:
27-May-02     Release date:   02-Sep-03    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chains
Pfam   ArchSchema ?
P55859  (PNPH_BOVIN) -  Purine nucleoside phosphorylase
Seq:
Struc:
289 a.a.
265 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 1 residue position (black cross)

 Enzyme reactions 
   Enzyme class: E.C.2.4.2.1  - Purine-nucleoside phosphorylase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction:
1. Purine nucleoside + phosphate = purine + alpha-D-ribose 1-phosphate
2. Purine deoxynucleoside + phosphate = purine + 2'-deoxy-alpha-D-ribose 1-phosphate
Purine nucleoside
Bound ligand (Het Group name = 9PP)
matches with 52.00% similarity
+ phosphate
= purine
+ alpha-D-ribose 1-phosphate
Purine deoxynucleoside
+ phosphate
= purine
+ 2'-deoxy-alpha-D-ribose 1-phosphate
Molecule diagrams generated from .mol files obtained from the KEGG ftp site
 Gene Ontology (GO) functional annotation 
  GO annot!
  Cellular component     cytoplasm   2 terms 
  Biological process     nucleobase-containing compound metabolic process   2 terms 
  Biochemical function     catalytic activity     5 terms  

 

 
    reference    
 
 
DOI no: 10.1016/j.jmb.2004.07.017 J Mol Biol 342:1015-1032 (2004)
PubMed id: 15342253  
 
 
Crystal structure of calf spleen purine nucleoside phosphorylase with two full trimers in the asymmetric unit: important implications for the mechanism of catalysis.
A.Bzowska, G.Koellner, B.Wielgus-Kutrowska, A.Stroh, G.Raszewski, A.Holý, T.Steiner, J.Frank.
 
  ABSTRACT  
 
The crystal structure of the binary complex of trimeric purine nucleoside phosphorylase (PNP) from calf spleen with the acyclic nucleoside phosphonate inhibitor 2,6-diamino-(S)-9-[2-(phosphonomethoxy)propyl]purine ((S)-PMPDAP) is determined at 2.3A resolution in space group P2(1)2(1)2(1). Crystallization in this space group, which is observed for the first time with a calf spleen PNP crystal structure, is obtained in the presence of calcium atoms. In contrast to the previously described cubic space group P2(1)3, two independent trimers are observed in the asymmetric unit, hence possible differences between monomers forming the biologically active trimer could be detected, if present. Such differences would be expected due to third-of-the-sites binding documented for transition-state events and inhibitors. However, no differences are noted, and binding stoichiometry of three inhibitor molecules per enzyme trimer is observed in the crystal structure, and in the parallel solution studies using isothermal titration calorimetry and spectrofluorimetric titrations. Presence of phosphate was shown to modify binding stoichiometry of hypoxanthine. Therefore, the enzyme was also crystallized in space group P2(1)2(1)2(1) in the presence of (S)-PMPDAP and phosphate, and the resulting structure of the binary PNP/(S)-PMPDAP complex was refined at 2.05A resolution. No qualitative differences between complexes obtained with and without the presence of phosphate were detected, except for the hydrogen bond contact of Arg84 and a phosphonate group, which is observed only in the former complex in three out of six independent monomers. Possible hydrogen bonds observed in the enzyme complexed with (S)-PMPDAP, in particular a putative hydrogen bonding contact N(1)-H cdots, three dots, centered Glu201, indicate that the inhibitor binds in a tautomeric or ionic form in which position N(1) acts as a hydrogen bond donor. This points to a crucial role of this hydrogen bond in defining specificity of trimeric PNPs and is in line with the proposed mechanism of catalysis in which this contact helps to stabilize the negative charge that accumulates on O(6) of the purine base in the transition state. In the present crystal structure the loop between Thr60 and Ala65 was found in a different conformation than that observed in crystal structures of trimeric PNPs up to now. Due to this change a new wide entrance is opened into the active site pocket, which is otherwise buried in the interior of the protein. Hence, our present crystal structure provides no obvious indication for obligatory binding of one of the substrates before binding of a second one; it is rather consistent with random binding of substrates. All these results provide new data for clarifying the mechanism of catalysis and give reasons for the non-Michaelis kinetics of trimeric PNPs.
 
  Selected figure(s)  
 
Figure 2.
Figure 2. Bis-calcium-binding sites linking two biologically active trimers of calf spleen PNP in the crystal. Calcium atoms are coordinated by six water molecules and two residues, Ser51 and Glu58, from each of the two trimers, forming a loosely bound sandwiched hexamer. The example shows the bis-calcium-binding site between monomers A and D of the complex obtained without phosphate. The Figure was drawn with DINO (http://www.bioz.unibas.ch/ xray/dino).
Figure 4.
Figure 4. The binding pocket of calf spleen PNP characterized by the solvent-accessible surface calculated without the ligand (MSMS).47 Upper: Monomer D of the (S)-PMPDAP-PNP complex obtained without phosphate. The biologically active form of the enzyme is a homotrimer, with active sites located at the interfaces of two monomers. The neighbouring subunit contributes the side-chain of Phe159 (here shown in green) to the pocket. The active site is buried in the interior of the protein structure, and the walls of the binding pocket appear to be fairly compact. However, in the present structure, probably due to the presence of calcium ions that form contact with Glu58 located in the flexible loop (here shown in red), a wide path to the active site is opened. This possible entrance is closed in all previously reported structures of calf spleen PNP,3^, 16^, 17^ and 35 because the loop 60-65 adopts a different conformation (compare with the lower panel and Figure 5). Lower: Two possible entrances to the active site pocket observed in the present structure (space group P2[1]2[1]2[1]). The entrances are shown for monomer D of the (S)-PMPDAP-PNP complex obtained without phosphate. The conformational change of the loop 60-65 (red colour for the loop and the ligand (S)-PMPDAP) is shown by superimposition of the ligand and the loop from the present structure with those observed in the previously reported structure of the ternary complex of PNP with an N(7)-acycloguanosine inhibitor and a phosphate anion16 (PDB entry 1FXU; green colour for the loop and the ligands). In both structures, the side-chain of His64 from the flexible loop 60-65 is also shown. In all previously reported structures of the enzyme in the P2[1]3 space group3^, 16^, 17^ and 35 the only possible entrance is located near the six-membered ring of the purine base. The conformational change of loop 60-65 observed in the present structure opens, in addition, a second wide entry into the active site, near the phosphate-binding site, as shown also in the upper panel. The Figure was drawn with DINO (http://www.bioz.unibas.ch/ xray/dino).
 
  The above figures are reprinted by permission from Elsevier: J Mol Biol (2004, 342, 1015-1032) copyright 2004.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
20063024 D.F.Visser, F.Hennessy, K.Rashamuse, M.E.Louw, and D.Brady (2010).
Cloning, purification and characterisation of a recombinant purine nucleoside phosphorylase from Bacillus halodurans Alk36.
  Extremophiles, 14, 185-192.  
20193043 K.Breer, L.Glavas-Obrovac, M.Suver, S.Hikishima, M.Hashimoto, T.Yokomatsu, B.Wielgus-Kutrowska, L.Magnowska, and A.Bzowska (2010).
9-Deazaguanine derivatives connected by a linker to difluoromethylene phosphonic acid are slow-binding picomolar inhibitors of trimeric purine nucleoside phosphorylase.
  FEBS J, 277, 1747-1760.  
18234834 S.Saen-Oon, M.Ghanem, V.L.Schramm, and S.D.Schwartz (2008).
Remote mutations and active site dynamics correlate with catalytic properties of purine nucleoside phosphorylase.
  Biophys J, 94, 4078-4088.  
16220545 A.Ababou, and J.E.Ladbury (2006).
Survey of the year 2004: literature on applications of isothermal titration calorimetry.
  J Mol Recognit, 19, 79-89.  
16239721 A.V.Toms, W.Wang, Y.Li, B.Ganem, and S.E.Ealick (2005).
Novel multisubstrate inhibitors of mammalian purine nucleoside phosphorylase.
  Acta Crystallogr D Biol Crystallogr, 61, 1449-1458.
PDB codes: 2ai1 2ai2 2ai3
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB codes are shown on the right.