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PDBsum entry 2ff1
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References listed in PDB file
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Key reference
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Title
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Transition-State complex of the purine-Specific nucleoside hydrolase of t. Vivax: enzyme conformational changes and implications for catalysis.
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Authors
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W.Versées,
J.Barlow,
J.Steyaert.
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Ref.
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J Mol Biol, 2006,
359,
331-346.
[DOI no: ]
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PubMed id
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Abstract
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Nucleoside hydrolases cleave the N-glycosidic bond of ribonucleosides. Crystal
structures of the purine-specific nucleoside hydrolase from Trypanosoma vivax
have previously been solved in complex with inhibitors or a substrate. All these
structures show the dimeric T. vivax nucleoside hydrolase with an
"open" active site with a highly flexible loop (loop 2) in its
vicinity. Here, we present the crystal structures of the T. vivax nucleoside
hydrolase with both soaked (TvNH-ImmH(soak)) and co-crystallised (TvNH-ImmH(co))
transition-state inhibitor immucillin H (ImmH or
(1S)-1-(9-deazahypoxanthin-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol) to 2.1 A and
2.2 A resolution, respectively. In the co-crystallised structure, loop 2 is
ordered and folds over the active site, establishing previously unobserved
enzyme-inhibitor interactions. As such this structure presents the first
complete picture of a purine-specific NH active site, including leaving group
interactions. In the closed active site, a water channel of highly ordered water
molecules leads out from the N7 of the nucleoside toward bulk solvent, while
Trp260 approaches the nucleobase in a tight parallel stacking interaction.
Together with mutagenesis results, this structure rules out a mechanism of
leaving group activation by general acid catalysis, as proposed for
base-aspecific nucleoside hydrolases. Instead, the structure is consistent with
the previously proposed mechanism of leaving group protonation in the T. vivax
nucleoside hydrolase where aromatic stacking with Trp260 and an intramolecular
O5'-H8C hydrogen bond increase the pKa of the N7 sufficiently to allow
protonation by solvent. A mechanism that couples loop closure to the positioning
of active site residues is proposed based on a comparison of the soaked
structure with the co-crystallized structure. Interestingly, the dimer interface
area increases by 40% upon closure of loop 2, with loop 1 of one subunit
interacting with loop 2 of the other subunit, suggesting a relationship between
the dimeric form of the enzyme and its catalytic activity.
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Figure 3.
Figure 3. Comparison of the quaternary structure and
subunit interface of TvNH-ImmH(soak) (a) and TvNH-ImmH(co) (b).
Loop 1 is shown in blue, loop 2 in green. The Ca^2+ is shown as
a grey sphere, ImmH is represented as a CPK model. The Figure
was made with PyMOL.60
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Figure 4.
Figure 4. (a) Active site of the closed TvNH-ImmH(co)
structure. The residues Arg252 and Asp255 are provided by loop
2. The bound inhibitor, ImmH, is shown in yellow. The electron
density, contoured at 3s, of an F[o] -F[c] simulated annealed
omit map calculated without the inhibitor is also shown. The
Ca^2+ and water molecules are represented as grey and red
spheres, respectively. The "nucleophilic" water molecule, three
water molecules forming a water channel from the N7 of the
nucleobase toward bulk solvent, and a water molecule interacting
with the N1 of the nucleobase are shown. The numbering of the
B-subunit is used for the water molecules. The Figure was made
with PyMOL.60 (b) Schematic representation of interactions in
the active site of the TvNH-ImmH(co) structure. ImmH is shown in
green. The color codes for the Ca^2+ and water molecules are the
same as above.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2006,
359,
331-346)
copyright 2006.
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