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PDBsum entry 2a75
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* Residue conservation analysis
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Enzyme class:
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E.C.3.2.1.18
- exo-alpha-sialidase.
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Reaction:
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Hydrolysis of alpha-(2->3)-, alpha-(2->6)-, alpha-(2->8)-glycosidic linkages of terminal sialic residues in oligosaccharides, glycoproteins, glycolipids, colominic acid and synthetic substrates.
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DOI no:
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J Biol Chem
281:4149-4155
(2006)
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PubMed id:
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Structural and Kinetic Analysis of Two Covalent Sialosyl-Enzyme Intermediates on Trypanosoma rangeli Sialidase.
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A.G.Watts,
P.Oppezzo,
S.G.Withers,
P.M.Alzari,
A.Buschiazzo.
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ABSTRACT
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Trypanosoma rangeli sialidase is a glycoside hydrolase (family GH33) that
catalyzes the cleavage of alpha-2-->3-linked sialic acid residues from
sialoglycoconjugates with overall retention of anomeric configuration. Retaining
glycosidases usually operate through a ping-pong mechanism, wherein a covalent
intermediate is formed between the carbohydrate and an active site carboxylic
acid of the enzyme. Sialidases, instead, appear to use a tyrosine as the
catalytic nucleophile, leaving the possibility of an essentially different
catalytic mechanism. Indeed, a direct nucleophilic role for a tyrosine was shown
for the homologous trans-sialidase from Trypanosoma cruzi, although itself not a
typical sialidase. Here we present the three-dimensional structures of the
covalent glycosyl-enzyme complexes formed by the T. rangeli sialidase with two
different mechanism-based inactivators at 1.9 and 1.7A resolution. To our
knowledge, these are the first reported structures of enzymatically competent
covalent intermediates for a strictly hydrolytic sialidase. Kinetic analyses
have been carried out on the formation and turnover of both intermediates,
showing that structural modifications to these inactivators can be used to
modify the lifetimes of covalent intermediates. These results provide further
evidence that all sialidases likely operate through a similar mechanism
involving the transient formation of a covalently sialylated enzyme.
Furthermore, we believe that the ability to "tune" the inactivation
and reactivation rates of mechanism-based inactivators toward specific enzymes
represents an important step toward developing this class of inactivators into
therapeutically useful compounds.
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Selected figure(s)
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Figure 1.
A, structures of the fluorinated sialic acid derivatives
2,3-difluoro-N-acetyl-neuraminic acid (1) and
2,3-difluoro-2-keto-3-deoxy-d-glycero-d-galacto-nonulosonic acid
(2) used as mechanism-based inactivators. B, a typical
glycosidase-catalyzed reaction showing glycosylation (k[1]) and
deglycosylation (k[2]) rate constants affected by the
mechanism-based inactivators.
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Figure 5.
3-Fluoro-KDN covalent complex with TrSA. Methionine 96 is
observed in three alternate conformations, partially filling the
cavity left by the absent N-acetyl group on the sialyl moiety.
In the unbound enzyme (PDB code 1N1T) or bound to N-acetyl
containing sialic acid derivatives, this Met is observed only in
one conformation (corresponding to conformer A in this
structure). The water network changes are also highlighted; W360
and W369 interact with important residues in the site. The
refined 2mF[o] – DF[c] map contoured at 1σ is shown.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2006,
281,
4149-4155)
copyright 2006.
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Figures were
selected
by an automated process.
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Literature references that cite this PDB file's key reference
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PubMed id
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Reference
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E.M.Vilei,
A.Johansson,
Y.Schlatter,
K.Redhead,
and
J.Frey
(2011).
Genetic and functional characterization of the NanA sialidase from Clostridium chauvoei.
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Vet Res,
42,
2.
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A.Albohy,
M.D.Li,
R.B.Zheng,
C.Zou,
and
C.W.Cairo
(2010).
Insight into substrate recognition and catalysis by the human neuraminidase 3 (NEU3) through molecular modeling and site-directed mutagenesis.
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Glycobiology,
20,
1127-1138.
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A.J.Oakley,
S.Barrett,
T.S.Peat,
J.Newman,
V.A.Streltsov,
L.Waddington,
T.Saito,
M.Tashiro,
and
J.L.McKimm-Breschkin
(2010).
Structural and functional basis of resistance to neuraminidase inhibitors of influenza B viruses.
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J Med Chem,
53,
6421-6431.
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PDB codes:
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A.Kumar,
A.Lomize,
K.K.Jin,
D.Carlton,
M.D.Miller,
L.Jaroszewski,
P.Abdubek,
T.Astakhova,
H.L.Axelrod,
H.J.Chiu,
T.Clayton,
D.Das,
M.C.Deller,
L.Duan,
J.Feuerhelm,
J.C.Grant,
A.Grzechnik,
G.W.Han,
H.E.Klock,
M.W.Knuth,
P.Kozbial,
S.S.Krishna,
D.Marciano,
D.McMullan,
A.T.Morse,
E.Nigoghossian,
L.Okach,
R.Reyes,
C.L.Rife,
N.Sefcovic,
H.J.Tien,
C.B.Trame,
H.van den Bedem,
D.Weekes,
Q.Xu,
K.O.Hodgson,
J.Wooley,
M.A.Elsliger,
A.M.Deacon,
A.Godzik,
S.A.Lesley,
and
I.A.Wilson
(2010).
Open and closed conformations of two SpoIIAA-like proteins (YP_749275.1 and YP_001095227.1) provide insights into membrane association and ligand binding.
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Acta Crystallogr Sect F Struct Biol Cryst Commun,
66,
1245-1253.
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PDB codes:
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E.C.Schulz,
P.Neumann,
R.Gerardy-Schahn,
G.M.Sheldrick,
and
R.Ficner
(2010).
Structure analysis of endosialidase NF at 0.98 A resolution.
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Acta Crystallogr D Biol Crystallogr,
66,
176-180.
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PDB code:
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J.Chan,
A.R.Lewis,
M.Gilbert,
M.F.Karwaski,
and
A.J.Bennet
(2010).
A direct NMR method for the measurement of competitive kinetic isotope effects.
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Nat Chem Biol,
6,
405-407.
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T.V.Vuong,
and
D.B.Wilson
(2010).
Glycoside hydrolases: catalytic base/nucleophile diversity.
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Biotechnol Bioeng,
107,
195-205.
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O.Demir,
and
A.E.Roitberg
(2009).
Modulation of catalytic function by differential plasticity of the active site: case study of Trypanosoma cruzi trans-sialidase and Trypanosoma rangeli sialidase.
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Biochemistry,
48,
3398-3406.
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A.Buschiazzo,
and
P.M.Alzari
(2008).
Structural insights into sialic acid enzymology.
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Curr Opin Chem Biol,
12,
565-572.
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D.B.Berkowitz,
K.R.Karukurichi,
R.de la Salud-Bea,
D.L.Nelson,
and
C.D.McCune
(2008).
Use of Fluorinated Functionality in Enzyme Inhibitor Development: Mechanistic and Analytical Advantages.
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J Fluor Chem,
129,
731-742.
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D.J.Vocadlo,
and
G.J.Davies
(2008).
Mechanistic insights into glycosidase chemistry.
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Curr Opin Chem Biol,
12,
539-555.
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G.Xu,
X.Li,
P.W.Andrew,
and
G.L.Taylor
(2008).
Structure of the catalytic domain of Streptococcus pneumoniae sialidase NanA.
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Acta Crystallogr Sect F Struct Biol Cryst Commun,
64,
772-775.
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PDB codes:
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S.L.Newstead,
J.A.Potter,
J.C.Wilson,
G.Xu,
C.H.Chien,
A.G.Watts,
S.G.Withers,
and
G.L.Taylor
(2008).
The structure of Clostridium perfringens NanI sialidase and its catalytic intermediates.
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J Biol Chem,
283,
9080-9088.
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PDB codes:
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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.
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Links
