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BACKGROUND: Myrosinase is the enzyme responsible for the hydrolysis of a variety
of plant anionic 1-thio-beta-D-glucosides called glucosinolates. Myrosinase and
glucosinolates, which are stored in different tissues of the plant, are mixed
during mastication generating toxic by-products that are believed to play a role
in the plant defence system. Whilst O-glycosidases are extremely widespread in
nature, myrosinase is the only known S-glycosidase. This intriguing enzyme,
which shows sequence similarities with O-glycosidases, offers the opportunity to
analyze the similarities and differences between enzymes hydrolyzing S- and
O-glycosidic bonds. RESULTS: The structures of native myrosinase from white
mustard seed (Sinapis alba) and of a stable glycosyl-enzyme intermediate have
been solved at 1.6 A resolution. The protein folds into a (beta/alpha)8-barrel
structure, very similar to that of the cyanogenic beta-glucosidase from white
clover. The enzyme forms a dimer stabilized by a Zn2+ ion and is heavily
glycosylated. At one glycosylation site the complete structure of a
plant-specific heptasaccharide is observed. The myrosinase structure reveals a
hydrophobic pocket, ideally situated for the binding of the hydrophobic
sidechain of glucosinolates, and two arginine residues positioned for
interaction with the sulphate group of the substrate. With the exception of the
replacement of the general acid/base glutamate by a glutamine residue, the
catalytic machinery of myrosinase is identical to that of the cyanogenic
beta-glucosidase. The structure of the glycosyl-enzyme intermediate shows that
the sugar ring is bound via an alpha-glycosidic linkage to Glu409, the catalytic
nucleophile of myrosinase. CONCLUSIONS: The structure of myrosinase shows
features which illustrate the adaptation of the plant enzyme to the dehydrated
environment of the seed. The catalytic mechanism of myrosinase is explained by
the excellent leaving group properties of the substrate aglycons, which do not
require the assistance of an enzymatic acid catalyst. The replacement of the
general acid/base glutamate of O-glycosidases by a glutamine residue in
myrosinase suggests that for hydrolysis of the glycosyl-enzyme, the role of this
residue is to ensure a precise positioning of a water molecule rather than to
provide general base assistance.
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