Myrosinases (thioglucoside glucohydrolase,
EC 3.2.3.1) are typical enzymes of the Brassicaceae family that also contains
their substrates, the glucosinolates, in variable concentrations and sites depending
on plant organs and tissues. In undamaged tissues, enzyme and substrate(s) are
confined in different sides of the cell. Therefore, the myrosinase-glucosinolate
system, although in various arrangements and concentrations, is always present
in all Brassicaceae organs where, when activated following tissue damage, it
plays a defensive role against generalized pathogens (Bones and Rossiter, 1996;
Chew, 1988; Louda and Mole, 1991; Rosa et al., 1997). Myrosinase(s) and glucosinolates
when are mixed during a mechanical wounding or pathogen attack of plant tissues
generate cytotoxic byproducts that play an important role in the Brassicaceae
defense system. Myrosinases catalyze the cleavage of the S-Glucose bond of glucosinolates,
which are a variety of anionic 1-thio-ß-D-glucosides, via acid/base-catalyzed
reaction with the release of the aglycon and the formation of the glycosyl enzyme
intermediate. Myrosinases are glycopolypeptides containing various thiol groups,
disulfide and salt bridges and, depending on the source, have multiple forms
with different molecular weights (135-480 kD), number of subunits (2-12) and
a high percentage of carbohydrate (up to 22.5%), mostly hexoses (Björkman,
1976; Bones and Rossiter, 1996). The main myrosinase isoenzyme isolated from
ripe seeds of Sinapis alba, which is the typical source of this enzyme, consists
of two identical subunits with a molecular weight of 71.7 kD (Björkman
and Janson, 1972; Pessina et al., 1990), containing 499 residues, stabilized
by a Zn2+ion bound on a 2-fold axis, with tetrahedral coordination. This myrosinase
isoenzyme has three disulfide bridges per sub-unit and 21 carbohydrate residues
distributed in 10 glycosylation sites on the surface (Burmeister et al., 1997).
Glucosinolates have a common structure with four main elements, which are the
thioglucosidic bond, the sulfate anion, the glucosidic residue, and a side aglycon
chain of aliphatic, aromatic or heteroaromatic type (Figure 1(1)). At present
about 120 glucosinolates have been isolated and characterized (Fahey et al.
2001). In their native form, glucosinolates have a low biological activity,
while their derived products: isothiocyanates (3), thiones (5), nitriles (6)
and epithionitriles (7), obtained by myrosinase-catalyzed hydrolysis constitute
an important group of bioactive molecules of vegetable origin. The enzymatic
catalysis of glucosinolates so far has mainly been studied for its antinutritional
effects in animal feed, although in recent years these compounds have been considered
valuable for their interesting biological and chemical properties. Some authors
consider these molecules useful, not only for their activity against bacteria,
fungi, nematodes, tumor cell growth, and in cancer prevention (Fenwick et al.,
1983; Lazzeri et al., 1993; Huang et al., 1994; Leoni et al. 1997)(10,11,12,13),
but also because some of them could be used as important intermediates in chemical
synthesis (Gueyrard et al., 2000)(14). The mechanism of glucosinolates enzymatic
hydrolysis has been studied using the main myrosinase isoenzyme isolated from
white mustard (Sinapis alba) seeds and various types of glucosinolates, desulfo-glucosinolates
and some synthetic competitive inhibitors (Iori et al., 1996; Cottaz et al.
1996)(15,16).
Myrosinase has been isolated also from several other cruciferous plant sources, mainly from ripe seeds, viz. rapeseed (Brassica napus) (Lönnerdal and Janson 1972)(17), yellow mustard (Brassica juncea) (Ohtsuru and Hata, 1972)(18), wasabi (Wasabi japonica) (Ohtsuru and Kawatani, 1979)(19), and seedlings viz. water cress (Durham and Poulton, 1990)(20), daikon (Raphanus sativus) (Shikita et al., 1999)(21) and Crambe abyssinica (MYRc) (Tookey (1973; Bernardi et al. 2003) (22).
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