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PDBsum entry 1mn7
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Contents |
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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Improving nucleoside diphosphate kinase for antiviral nucleotide analogs activation.
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Authors
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S.Gallois-Montbrun,
B.Schneider,
Y.Chen,
V.Giacomoni-Fernandes,
L.Mulard,
S.Morera,
J.Janin,
D.Deville-Bonne,
M.Veron.
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Ref.
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J Biol Chem, 2002,
277,
39953-39959.
[DOI no: ]
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PubMed id
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Abstract
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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.
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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.
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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.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2002,
277,
39953-39959)
copyright 2002.
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