Mechanism

Myrosinase catalyzes the chemical reaction

a thioglucoside + H₂O ${\displaystyle \rightleftharpoons }$ a sugar + a thiol

Thus, the two substrates of this enzyme are thioglucoside and H₂O, whereas its two products are sugar and thiol.

In the presence of water, myrosinase cleaves off the glucose group from a glucosinolate. The remaining molecule then quickly converts to a thiocyanate, an isothiocyanate, or a nitrile; these are the active substances that serve as defense for the plant. The hydrolysis of glucosinolates by myrosinase can yield a variety of products, depending on various physiological conditions such as pH and the presence of certain cofactors. All known reactions have been observed to share the same initial steps. (See Figure 2.) First, the β-thioglucoside linkage is cleaved by myrosinase, releasing D-glucose. The resulting aglycone undergoes a spontaneous Lossen-like rearrangement, releasing a sulfate. The last step in the mechanism is subject to the greatest variety depending on the physiological conditions under which the reaction takes place. At neutral pH, the primary product is the isothiocyanate. Under acidic conditions (pH < 3), and in the presence of ferrous ions or epithiospecifer proteins, the formation of nitriles is favored instead.^[2][6]

Figure 2: Mechanism of glucosinolate hydrolysis by myrosinase.^[2]

Cofactors and inhibitors

Ascorbate is a known cofactor of myrosinase, serving as a base catalyst in glucosinolate hydrolysis.^[1][7] For example, myrosinase isolated from daikon (Raphanus sativus) demonstrated an increase in V max from 2.06 μmol/min per mg of protein to 280 μmol/min per mg of protein on the substrate, allyl glucosinolate (sinigrin) when in the presence of 500 μM ascorbate.^[4] Sulfate, a byproduct of glucosinolate hydrolysis, has been identified as a competitive inhibitor of myrosinase.^[4] In addition, 2-F-2-deoxybenzylglucosinolate, which was synthesized specifically to study the mechanism of myrosinase, inhibits the enzyme by trapping one of the glutamic acid residues in the active site, Glu 409.^[3][8]

Structure

Myrosinase exists as a dimer with subunits of 60-70 kDa each.^{[9] [10]} X-ray crystallography of myrosinase isolated from Sinapis alba revealed the two subunits are linked by a zinc atom.^[7] The prominence of salt bridges, disulfide bridges, hydrogen bonding, and glycosylation are thought to contribute to the enzyme’s stability, especially when the plant is under attack and experiences severe tissue damage.^[2] A feature of many β-glucosidases are catalytic glutamate residues at their active sites, but two of these have been replaced by a single glutamine residue in myrosinase.^[3][11] Ascorbate has been shown to substitute for the activity of the glutamate residues.^[1] (See Figure 3 for mechanism.)

Figure 3: Active site of myrosinase during the first step of glucosinolate hydrolysis. Here, ascorbate is used as a cofactor to substitute for the missing second catalytic glutamate in order to cleave the thio-linked glucose.^[3]

Compartmentalization in plants

The glucosinolate-myrosinase defensive system is packaged in the plant in a unique manner. Plants store myrosinase glucosinolates by compartmentalization, such that the latter is released and activated only when the plant is under attack. Myrosinase is stored largely as myrosin grains in the vacuoles of particular idioblasts called myrosin cells, but have also been reported in protein bodies or vacuoles, and as cytosolic enzymes that tend to bind to membranes.^[12][13] Glucosinolates are stored in adjacent but separate "S-cells." ^[14] When the plant experiences tissue damage, the myrosinase comes into contact with glucosinolates, quickly activating them into their potent, antibacterial form.^[2] The most potent of such products are isothiocyanates, followed by thiocyanates and nitriles.^[15]

Evolution

Plants known to have evolved a myrosinase-glucosinolate defense system include: white mustard (Sinapis alba), ^[9] garden cress (Lepidium sativum),^[16] wasabi (Wasabia japonica),^[17] and daikon (Raphanus sativus),^[18][19] as well as several members of the family Brassicaceae, including yellow mustard (Brassica juncea),^[20] rape seed (Brassica napus),^[21] and common dietary brassicas like broccoli, cauliflower, cabbage, bok choy, and kale. ^[2] The bitter aftertaste of many of these vegetables can often be attributed to the hydrolysis of glucosinolates upon tissue damage during food preparation or when consuming these vegetables raw.^[2] Papaya seeds use this method of defense, but not the fruit pulp itself.^[22]

Myrosinase has also been isolated from the cabbage aphid.^[23] This suggests coevolution of the cabbage aphid with its main food source. The aphid employs a similar defense strategy to plants. Like its main food source, the cabbage aphid compartmentalizes its native myrosinase and the glucosinolates it ingests. When the cabbage aphid is attacked and its tissues are damaged, its stored glucosinolates are activated, producing isothiocyanates and deterring predators from attacking other aphids.^[24]

Agriculture

Historically, crops like rapeseed that contained the glucosinolate-myrosinase system were deliberately bred to minimize glucosinolate content, since rapeseed in animal feed was proving toxic to livestock.^[25] The glucosinolate-myrosinase system has been investigated as a possible biofumigant to protect crops against pests. The potent glucosinolate hydrolysis products (GHPs) could be sprayed onto crops to deter herbivory. Another option would be to use techniques in genetic engineering to introduce the glucosinolate-myrosinase system in crops as a means of fortifying their resistance against pests.^[15]

Health effects

Isothiocyanates, the primary product of glucosinolate hydrolysis, have been known to prevent iodine uptake in the thyroid, causing goiters.^[26] Isothiocyanates in high concentrations may cause hepatotoxicity.^[4] There is insufficient scientific evidence that consuming cruciferous vegetables with increased intake of isothiocyanates affects the risk of human diseases.^[27]