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PDBsum entry 1e4m
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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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High resolution X-Ray crystallography shows that ascorbate is a cofactor for myrosinase and substitutes for the function of the catalytic base.
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Authors
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W.P.Burmeister,
S.Cottaz,
P.Rollin,
A.Vasella,
B.Henrissat.
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Ref.
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J Biol Chem, 2000,
275,
39385-39393.
[DOI no: ]
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PubMed id
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Abstract
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Myrosinase, an S-glycosidase, hydrolyzes plant anionic 1-thio-beta-d-glucosides
(glucosinolates) considered part of the plant defense system. Although
O-glycosidases are ubiquitous, myrosinase is the only known S-glycosidase. Its
active site is very similar to that of retaining O-glycosidases, but one of the
catalytic residues in O-glycosidases, a carboxylate residue functioning as the
general base, is replaced by a glutamine residue. Myrosinase is strongly
activated by ascorbic acid. Several binary and ternary complexes of myrosinase
with different transition state analogues and ascorbic acid have been analyzed
at high resolution by x-ray crystallography along with a
2-deoxy-2-fluoro-glucosyl enzyme intermediate. One of the inhibitors,
d-gluconhydroximo-1,5-lactam, binds simultaneously with a sulfate ion to form a
mimic of the enzyme-substrate complex. Ascorbate binds to a site distinct from
the glucose binding site but overlapping with the aglycon binding site,
suggesting that activation occurs at the second step of catalysis, i.e.
hydrolysis of the glycosyl enzyme. A water molecule is placed perfectly for
activation by ascorbate and for nucleophilic attack on the covalently trapped
2-fluoro-glucosyl-moiety. Activation of the hydrolysis of the glucosyl enzyme
intermediate is further evidenced by the observation that ascorbate enhances the
rate of reactivation of the 2-fluoro-glycosyl enzyme, leading to the conclusion
that ascorbic acid substitutes for the catalytic base in myrosinase.
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Figure 4.
Fig. 4. The binding of ascorbate. The structures have
been obtained on crystals soaked with ascorbic acid and
different inhibitors. Electron density maps as described for
Fig. 2. Water molecules are shown as red spheres. The refined
structures are shown, including ascorbate, water molecules,
sulfate ions, glycerol, inhibitors, and active site residues.
Hydrogen bonds involved in ascorbate recognition are shown as
dotted lines. a, soak with ascorbate, the glycerol molecule
comes from the cryoprotectant. b, ascorbate and
gluco-hydroximolactam. The ascorbate competes with the sulfate
ion that has both partial occupancies of 0.6 for the ascorbate
and 0.4 for the sulfate. c, ascorbate bound to the 2-F-glucosyl
enzyme. The water molecule that is activated by the ascorbate
for an attack on the C-1 carbon of the glucose is indicated by a
pink arrow.
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Figure 8.
Fig. 8. Schematic reaction mechanism of myrosinase in the
absence (a) and presence (b and c) of ascorbic acid. E, enzyme;
S, substrate; GE, glucosyl enzyme; A, ascorbate; k[1], k[2],
etc., dissociation constants not involving ascorbate; k[3'],
rate constant in presence of ascorbate; k[A1], k[A  1],
etc., dissociation constants of ascorbate. The less important
back reactions are not shown.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2000,
275,
39385-39393)
copyright 2000.
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Secondary reference #1
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Title
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The crystal structures of sinapis alba myrosinase and a covalent glycosyl-Enzyme intermediate provide insights into the substrate recognition and active-Site machinery of an s-Glycosidase.
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Authors
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W.P.Burmeister,
S.Cottaz,
H.Driguez,
R.Iori,
S.Palmieri,
B.Henrissat.
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Ref.
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Structure, 1997,
5,
663-675.
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PubMed id
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