PDBsum entry 1v48

Transferase

PDB id: 1v48

Contents

Protein chain

261 a.a. *

Ligands

HA1

Metals

_ZN

_MG

Waters ×179

* Residue conservation analysis

PDB id:

1v48

Name:

Transferase

Title:

Calf spleen purine nucleoside phosphorylase (pnp) binary complex with 9-(5,5-difluoro-5-phosphonopenthyl)guanine

Structure:

Purine nucleoside phosphorylase. Chain: a. Synonym: inosine phosphorylase, pnp. Ec: 2.4.2.1

Source:

Bos taurus. Cattle. Organism_taxid: 9913

Biol. unit:

Trimer (from PDB file)

Resolution:

2.20Å R-factor: 0.191 R-free: 0.242

Authors:

M.Luic,G.Koellner,T.Yokomatsu,S.Shibuya,A.Bzowska

Key ref:

M.Luić et al. (2004). Calf spleen purine-nucleoside phosphorylase: crystal structure of the binary complex with a potent multisubstrate analogue inhibitor. Acta Crystallogr D Biol Crystallogr, 60, 1417-1424. PubMed id: 15272165 DOI: 10.1107/S0907444904013861

Date:

11-Nov-03 Release date: 03-Aug-04

Protein chain

P55859  (PNPH_BOVIN) -  Purine nucleoside phosphorylase from Bos taurus

Seq: 289 a.a.

Struc: 261 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.2.4.2.1 - purine-nucleoside phosphorylase.
• Reaction:
  1. a purine D-ribonucleoside + phosphate = a purine nucleobase + alpha- D-ribose 1-phosphate
  2. a purine 2'-deoxy-D-ribonucleoside + phosphate = a purine nucleobase + 2-deoxy-alpha-D-ribose 1-phosphate

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1107/S0907444904013861

Acta Crystallogr D Biol Crystallogr 60:1417-1424 (2004)

PubMed id: 15272165

Calf spleen purine-nucleoside phosphorylase: crystal structure of the binary complex with a potent multisubstrate analogue inhibitor.

M.Luić, G.Koellner, T.Yokomatsu, S.Shibuya, A.Bzowska.

ABSTRACT

Purine-nucleoside phosphorylase (PNP) deficiency in humans leads to inhibition of the T-cell response. Potent membrane-permeable inhibitors of this enzyme are therefore considered to be potential immunosuppressive agents. The binary complex of the trimeric calf spleen phosphorylase, which is highly homologous to human PNP, with the potent ground-state analogue inhibitor 9-(5,5-difluoro-5-phosphonopentyl)guanine (DFPP-G) was crystallized in the cubic space group P2(1)3, with unit-cell parameter a = 93.183 A and one monomer per asymmetric unit. High-resolution X-ray diffraction data were collected using synchrotron radiation (EMBL Outstation, DESY, Hamburg, station X13). The crystal structure was refined to a resolution of 2.2 A and R and Rfree values of 19.1 and 24.2%, respectively. The crystal structure confirms that DFPP-G acts as a multisubstrate analogue inhibitor as it binds to both nucleoside- and phosphate-binding sites. The structure also provides the answers to some questions regarding the substrate specificity and molecular mechanism of trimeric PNPs. The wide access to the active-site pocket that was observed in the reported structure as a result of the flexibility or disorder of two loops (residues 60-65 and 251-266) strongly supports the random binding of substrates. The putative hydrogen bonds identified in the base-binding site indicate that N1-H and not O6 of the purine base defines the specificity of trimeric PNPs. This is confirmed by the fact that the contact of guanine O6 with Asn243 Odelta1 is not a direct contact but is mediated by a water molecule. Participation of Arg84 in the binding of the phosphonate group experimentally verifies the previous suggestion [Blackburn & Kent (1986), J. Chem. Soc. Perkin Trans. I, pp. 913-917; Halazy et al. (1991), J. Am. Chem. Soc. 113, 315-317] that fluorination of alkylphosphonates yields compounds with properties that suitably resemble those of phosphate esters and in turn leads to optimized interactions of such analogues with the phosphate-binding site residues. DFPP-G shows a Ki(app) in the nanomolar range towards calf and human PNPs. To date, no high-resolution X-ray structures of these enzymes with such potent ground-state analogue inhibitors have been available in the Protein Data Bank. The present structure may thus be used in the rational structure-based design of new PNP inhibitors with potential medical applications.

Selected figure(s)

Figure 1.
Figure 1 Structures of the multisubstrate analogue inhibitor DFPP-G and some of its precursors: non-fluorinated (DPP-G), non-fluorinated and without terminal phosphonyl group (HPG) and orthophosphate (P[i]).

Figure 4.
Figure 4 Superposition of the active sites of three complexes of calf spleen PNP with various inhibitors: (i) the binary complex with the multisubstrate analogue inhibitor DFPP-G (cpk colours, present structure; PDB code [254]1v48 ), (ii) the binary complex with the multisubstrate analogue inhibitor (S)-PMPDAP (magenta; PDB code [255]1lv8 , monomer A; Bzowska et al., 2004[256] [Bzowska, A., Koellner, G., Wielgus-Kutrowska, B., Stroh, A., Raszewski, G., Holý, A., Steiner, T. & Frank, J. (2004). Submitted.]-[257][bluearr.gif] ) and (iii) the ternary complex with the transition-state inhibitor immucillin G and phosphate (green; PDB code [258]1b8n ; Kicska et al., 2002[259] [Kicska, G. A., Tyler, P. C., Evans, G. B., Furneaux, R. H., Schramm, V. L. & Kim, K. (2002). J. Biol. Chem. 277, 3226-3231.]-[260][bluearr.gif] ). Active-site amino acids that form putative hydrogen bonds with the inhibitors are included (see also Fig. 2[261] [link]-[262][turqarr.gif] ). In addition, the location of Phe200 involved in [263][pi] -stacking interaction with the inhibitor base is also shown (thin lines). Phosphate and phosphonate groups of the multisubstrate analogue inhibitors [DFPP-G and (S)-PMPDAP] and orthophosphate in the ternary complex with immucillin G occupy the same position in all three structures. The side chain of Arg84 in the complex with (S)-PMPDAP (drawn in light magenta) is directed away from the phosphate-binding site and does not form hydrogen bonds with the phosphonate group of the inhibitor. Tyr88, Met219 and His257 only form direct hydrogen-bonding contacts in the complex with immucillin G. The inhibition potency of the three inhibitors shown in the figure spans almost six orders of magnitude: from 30 pM for immucillin G to 6 µM for (S)-PMPDAP (see text for details). The number of direct hydrogen-bonding contacts formed by these three ligands correlates well with their inhibition potency as observed in solution.

The above figures are reprinted by permission from the IUCr: Acta Crystallogr D Biol Crystallogr (2004, 60, 1417-1424) copyright 2004.

Figures were selected by an automated process.