Myrosinase

Myrosinase (EC 3.2.1.147, thioglucoside glucohydrolase, sinigrinase, and sinigrase) is a family of enzymes involved in plant defense against herbivores. The three-dimensional structure has been elucidated and is available in the PDB (see links in the infobox). Myrosinase (EC 3.2.1.147, thioglucoside glucohydrolase, sinigrinase, and sinigrase) is a family of enzymes involved in plant defense against herbivores. The three-dimensional structure has been elucidated and is available in the PDB (see links in the infobox). A member of the glycoside hydrolase family, myrosinase possesses several similarities with the more ubiquitous O-glycosidases. However, myrosinase is the only known enzyme found in nature that can cleave a thio-linked glucose. Its known biological function is to catalyze the hydrolysis of a class of compounds called glucosinolates. Myrosinase is regarded as a defense-related enzyme and is capable of hydrolyzing glucosinolates into various compounds, some of which are toxic. Myrosinase catalyzes the chemical reaction 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. Ascorbate is a known cofactor of myrosinase, serving as a base catalyst in glucosinolate hydrolysis.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.Sulfate, a byproduct of glucosinolate hydrolysis, has been identified as a competitive inhibitor of myrosinase.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. Myrosinase exists as a dimer with subunits of 60-70 kDa each. X-ray crystallography of myrosinase isolated from Sinapis alba revealed the two subunits are linked by a zinc atom. 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.A feature of many ß-glucosidases are catalytic glutamate residues at their active sites, but two of these have been replaced by a single glutamine residue in myrosinase. Ascorbate has been shown to substitute for the activity of the glutamate residues. (See Figure 3 for mechanism.) Myrosinase and its natural substrate, glucosinolate, are known to be part of the plant’s defense response. When the plant is attacked by pathogens, insects, or other herbivore, the plant uses myrosinase to convert glucosinolates, which are otherwise-benign, into toxic products like isothiocyanates, thiocyanates, and nitriles.