PDBsum entry 1mn7

Transferase

PDB id: 1mn7

Contents

Protein chains

150 a.a. *

Ligands

ABT ×2

Metals

_MG ×2

Waters ×307

* Residue conservation analysis

PDB id:

1mn7

Name:

Transferase

Title:

Ndp kinase mutant (h122g;n119s;f64w) in complex with abazttp

Structure:

Ndp kinase. Chain: a, b. Synonym: nucleoside diphosphate kinase, cytosolic. Engineered: yes. Mutation: yes

Source:

Dictyostelium discoideum. Organism_taxid: 44689. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Hexamer (from PDB file)

Resolution:

2.15Å R-factor: 0.232 R-free: 0.295

Authors:

S.Gallois-Montbrun,B.Schneider,Y.Chen,V.Giacomoni-Fernandes,L.Mulard, S.Morera,J.Janin,D.Deville-Bonne,M.Veron

Key ref:

S.Gallois-Montbrun et al. (2002). Improving nucleoside diphosphate kinase for antiviral nucleotide analogs activation. J Biol Chem, 277, 39953-39959. PubMed id: 12171931 DOI: 10.1074/jbc.M206360200

Date:

05-Sep-02 Release date: 02-Oct-02

Protein chains

P22887  (NDKC_DICDI) -  Nucleoside diphosphate kinase, cytosolic from Dictyostelium discoideum

Seq: 155 a.a.

Struc: 150 a.a.*

* PDB and UniProt seqs differ at 3 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.2.7.4.6 - nucleoside-diphosphate kinase.
• Reaction:
  1. a ribonucleoside 5'-diphosphate + ATP = a ribonucleoside 5'-triphosphate + ADP
  2. a 2'-deoxyribonucleoside 5'-diphosphate + ATP = a 2'-deoxyribonucleoside 5'-triphosphate + ADP

ribonucleoside 5'-diphosphate + ATP (Bound ligand (Het Group name = ABT) matches with 63.16% similarity) = ribonucleoside 5'-triphosphate + ADP

2'-deoxyribonucleoside 5'-diphosphate + ATP (Bound ligand (Het Group name = ABT) matches with 63.16% similarity) = 2'-deoxyribonucleoside 5'-triphosphate + ADP

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

reference

DOI no: 10.1074/jbc.M206360200

J Biol Chem 277:39953-39959 (2002)

PubMed id: 12171931

Improving nucleoside diphosphate kinase for antiviral nucleotide analogs activation.

S.Gallois-Montbrun, B.Schneider, Y.Chen, V.Giacomoni-Fernandes, L.Mulard, S.Morera, J.Janin, D.Deville-Bonne, M.Veron.

ABSTRACT

Antiviral nucleoside analog therapies rely on their incorporation by viral DNA polymerases/reverse transcriptase leading to chain termination. The analogs (3'-deoxy-3'-azidothymidine (AZT), 2',3'-didehydro-2',3'-dideoxythymidine (d4T), and other dideoxynucleosides) are sequentially converted into triphosphate by cellular kinases of the nucleoside salvage pathway and are often poor substrates of these enzymes. Nucleoside diphosphate (NDP) kinase phosphorylates the diphosphate derivatives of the analogs with an efficiency some 10(4) lower than for its natural substrates. Kinetic and structural studies of Dictyostelium and human NDP kinases show that the sugar 3'-OH, absent from all antiviral analogs, is required for catalysis. To improve the catalytic efficiency of NDP kinase on the analogs, we engineered several mutants with a protein OH group replacing the sugar 3'-OH. The substitution of Asn-115 in Ser and Leu-55 in His results in an NDP kinase mutant with an enhanced ability to phosphorylate antiviral derivatives. Transfection of the mutant enzyme in Escherichia coli results in an increased sensitivity to AZT. An x-ray structure at 2.15-A resolution of the Dictyostelium enzyme bearing the serine substitution in complex with the R(p)-alpha-borano-triphosphate derivative of AZT shows that the enhanced activity reflects an improved geometry of binding and a favorable interaction of the 3'-azido group with the engineered serine.

Selected figure(s)

Figure 3.
Fig. 3. Phosphotransfer efficiency of NDPK-A and mutants for nucleoside analogs. The rates of phosphorylation of pure recombinant NDP kinases (1 µM) were measured at the pre-steady state in a fluorescence stopped-flow with d4TTP. The catalytic efficiency (expressed in M 1s 1) was determined from the variation of the rate as a function of analog as shown in Fig. 2: NDPK-A ( ), N115S ( ), L55H ( ), Dictyostelium ( ), and L55H-N115S ( ). B, catalytic efficiencies of NDPK-A ( ) and the mutants L55H ( ), N115S ( ), and L55H-N115S ( ) for several antiviral analog triphosphate derivatives. The values were extracted from stopped-flow experiments as shown in A for d4TTP.

Figure 4.
Fig. 4. Structure of EI-N119S in complex with RB-AZT-TP. A, the x-ray structure of the triple mutant of Dictyostelium NDP kinase (yellow bonds) in complex with the R[p] stereoisomer of the -borano analog of AZT triphosphate is compared with that of the wild type protein in complex with TDP (21) (blue bonds). The orientation is as in Fig. 1, and the mutated side chains at positions 64, 119, and 122 are in ball-and-stick representation. B, the same structure (yellow bonds) is compared with that of the complex with AZT diphosphate (Ref. 20, purple bonds). Movements of the sugar ring, of the azido group (N3), and of the Lys-16 side chain may explain the improved efficiency of the enzyme bearing the N119S mutation. Despite these changes and of the -borano (BH3) substitution, the - and -phosphates superimpose exactly.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2002, 277, 39953-39959) copyright 2002.

Figures were selected by an automated process.