myrosinase

Myrosinase is regarded as a defense-related enzyme and is capable of hydrolyzing glucosinolates into various compounds, some of which are toxic.[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC157743/ A wound- and methyl jasmonate-inducible transcript coding for a myrosinase-associated protein with similarities to an early nodulin]

Myrosinase catalyzes the chemical reaction

:a thioglucoside + H₂O $\backslash rightleftharpoons$ 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.{{cite journal | vauthors = Lambrix V, Reichelt M, Mitchell-Olds T, Kliebenstein DJ, Gershenzon J | title = The Arabidopsis epithiospecifier protein promotes the hydrolysis of glucosinolates to nitriles and influences Trichoplusia ni herbivory | journal = The Plant Cell | volume = 13 | issue = 12 | pages = 2793–2807 | date = December 2001 | pmid = 11752388 | pmc = 139489 | doi = 10.1105/tpc.010261 }}

File:Myrosinase general mechanism.png

Ascorbate is a known cofactor of myrosinase, serving as a base catalyst in glucosinolate hydrolysis.{{cite journal | vauthors = Burmeister WP, Cottaz S, Driguez H, Iori R, Palmieri S, Henrissat B | title = 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 | journal = Structure | volume = 5 | issue = 5 | pages = 663–675 | date = May 1997 | pmid = 9195886 | doi = 10.1016/s0969-2126(97)00221-9 | doi-access = free }}

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.{{cite journal | vauthors = Cottaz S, Rollin P, Driguez H |year=1997 |title=Synthesis of 2-deoxy-2-fluoroglucotropaeolin, a thioglucosidase inhibitor |journal=Carbohydrate Research |volume=298 |issue=1–2 |pages=127–130 |doi=10.1016/s0008-6215(96)00294-7}}

Myrosinase exists as a dimer with subunits of 60-70 kDa each.{{cite journal | vauthors = Björkman R, Janson JC | title = Studies on myrosinases. I. Purification and characterization of a myrosinase from white mustard seed (Sinapis alba, L.) | journal = Biochimica et Biophysica Acta (BBA) - Enzymology | volume = 276 | issue = 2 | pages = 508–518 | date = August 1972 | pmid = 5068825 | doi = 10.1016/0005-2744(72)91011-X }}

{{cite journal | vauthors = Pessina A, Thomas RM, Palmieri S, Luisi PL | title = An improved method for the purification of myrosinase and its physicochemical characterization | journal = Archives of Biochemistry and Biophysics | volume = 280 | issue = 2 | pages = 383–389 | date = August 1990 | pmid = 2369130 | doi = 10.1016/0003-9861(90)90346-Z }}

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 β-glucosidases are catalytic glutamate residues at their active sites, but two of these have been replaced by a single glutamine residue in myrosinase.{{cite journal | vauthors = Henrissat B, Davies GJ | title = Glycoside hydrolases and glycosyltransferases. Families, modules, and implications for genomics | journal = Plant Physiology | volume = 124 | issue = 4 | pages = 1515–1519 | date = December 2000 | pmid = 11115868 | pmc = 1539306 | doi = 10.1104/pp.124.4.1515 }} Ascorbate has been shown to substitute for the activity of the glutamate residues. (See Figure 3 for mechanism.)

File:Glucosinolate hydrolysis with ascorbate cofactor at active site of myrosinase.png hydrolysis. Here, ascorbate is used as a cofactor to substitute for the missing second catalytic glutamate in order to cleave the thio-linked glucose.]]

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 herbivores, the plant uses myrosinase to convert glucosinolates, which are otherwise-benign, into toxic products like isothiocyanates, thiocyanates, and nitriles.

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.{{cite journal | vauthors = Lüthy B, Matile P | year = 1984 | title = The mustard oil bomb: Rectified analysis of the subcellular organisation of the myrosinase system | journal = Biochemie und Physiologie der Pflanzen| volume = 179 | issue = 1–2| pages = 5–12 | doi=10.1016/s0015-3796(84)80059-1}}{{cite journal | vauthors = Andréasson E, Bolt Jørgensen L, Höglund AS, Rask L, Meijer J | title = Different myrosinase and idioblast distribution in Arabidopsis and Brassica napus | journal = Plant Physiology | volume = 127 | issue = 4 | pages = 1750–1763 | date = December 2001 | pmid = 11743118 | pmc = 133578 | doi = 10.1104/pp.010334 }} Glucosinolates are stored in adjacent but separate "S-cells."

{{cite journal | vauthors = Koroleva OA, Davies A, Deeken R, Thorpe MR, Tomos AD, Hedrich R | title = Identification of a new glucosinolate-rich cell type in Arabidopsis flower stalk | journal = Plant Physiology | volume = 124 | issue = 2 | pages = 599–608 | date = October 2000 | pmid = 11027710 | pmc = 59166 | doi = 10.1104/pp.124.2.599 }} When the plant experiences tissue damage, the myrosinase comes into contact with glucosinolates, quickly activating them into their potent, antibacterial form. The most potent of such products are isothiocyanates, followed by thiocyanates and nitriles.{{cite journal | vauthors = Gimsing AL, Kirkegaard JA |year=2009 |title=Glucosinolates and biofumigation: fate of glucosinolates and their hydrolysis products in soil |journal=Phytochemistry Reviews |volume=8 |pages=299–310 |doi=10.1007/s11101-008-9105-5 |s2cid =30626061 }}

Plants known to have evolved a myrosinase-glucosinolate defense system include: white mustard (Sinapis alba),

garden cress (Lepidium sativum),{{cite journal | vauthors = Durham PL, Poulton JE | title = Effect of Castanospermine and Related Polyhydroxyalkaloids on Purified Myrosinase from Lepidium sativum Seedlings | journal = Plant Physiology | volume = 90 | issue = 1 | pages = 48–52 | date = May 1989 | pmid = 16666767 | pmc = 1061675 | doi = 10.1104/pp.90.1.48 }}

wasabi (Wasabia japonica),{{cite journal | vauthors = Ohtsuru M, Kawatani H |year=1979 |journal=Agricultural and Biological Chemistry |volume=43 |issue =11 |pages=2249–2255| doi = 10.1271/bbb1961.43.2249 |title=Studies on the myrosinase from Wasabia japonica: Purification and some properties of wasabi myrosinase.|doi-access=free }} and daikon (Raphanus sativus),{{cite journal | vauthors = Iversen TH, Baggerud C |year=1980 |journal=Zeitschrift für Pflanzenphysiologie |volume=97 |issue =5 |pages=399–407 |title = Myrosinase activity in differentiated and undifferentiated plants of Brassiaceae Z. |doi=10.1016/s0044-328x(80)80014-6}}{{cite journal| vauthors = El-Sayed ST, Jwanny EW, Rashad MM, Mahmoud AE, Abdallah NM |title=Glycosidases in plant tissues of some brassicaceae screening of different cruciferous plants for glycosidases production|journal=Applied Biochemistry and Biotechnology|volume=55|issue=3|year=1995|pages=219–230|issn=0273-2289|doi=10.1007/BF02786861|s2cid=84375704}}

as well as several members of the family Brassicaceae, including

yellow mustard (Brassica juncea),{{cite journal | vauthors = Masaru O, Tadao H |year = 1972|journal = Agricultural and Biological Chemistry |volume = 36|issue =13|pages = 2495–2503|doi = 10.1271/bbb1961.36.2495|title=Molecular Properties of Multiple Forms of Plant Myrosinase|doi-access = free}}

rape seed (Brassica napus),{{cite journal | vauthors = Lönnerdal B, Janson JC |year=1973 |journal=Biochimica et Biophysica Acta (BBA) - Enzymology |volume=315 |issue =2 |pages=421–429|doi=10.1016/0005-2744(73)90272-6 |title=Studies on myrosinases. II. Purification and characterization of a myrosinase from rapeseed (Brassica napus L.)}} and common dietary brassicas like broccoli, cauliflower, cabbage, bok choy, and kale.

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. Papaya seeds use this method of defense, but not the fruit pulp itself.{{cite journal | vauthors = Nakamura Y, Yoshimoto M, Murata Y, Shimoishi Y, Asai Y, Park EY, Sato K, Nakamura Y | display-authors = 6 | title = Papaya seed represents a rich source of biologically active isothiocyanate | journal = Journal of Agricultural and Food Chemistry | volume = 55 | issue = 11 | pages = 4407–4413 | date = May 2007 | pmid = 17469845 | doi = 10.1021/jf070159w }}

Myrosinase has also been isolated from the cabbage aphid.{{cite journal | vauthors = Husebye H, Arzt S, Burmeister WP, Härtel FV, Brandt A, Rossiter JT, Bones AM | title = Crystal structure at 1.1 Angstroms resolution of an insect myrosinase from Brevicoryne brassicae shows its close relationship to beta-glucosidases | journal = Insect Biochemistry and Molecular Biology | volume = 35 | issue = 12 | pages = 1311–1320 | date = December 2005 | pmid = 16291087 | doi = 10.1016/j.ibmb.2005.07.004 }} 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.{{cite journal | vauthors = Bridges M, Jones AM, Bones AM, Hodgson C, Cole R, Bartlet E, Wallsgrove R, Karapapa VK, Watts N, Rossiter JT | display-authors = 6 | title = Spatial organization of the glucosinolate-myrosinase system in brassica specialist aphids is similar to that of the host plant | journal = Proceedings. Biological Sciences | volume = 269 | issue = 1487 | pages = 187–191 | date = January 2002 | pmid = 11798435 | pmc = 1690872 | doi = 10.1098/rspb.2001.1861 }}

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.{{cite journal | vauthors = Brabban AD, Edwards C | title = Isolation of glucosinolate degrading microorganisms and their potential for reducing the glucosinolate content of rapemeal | journal = FEMS Microbiology Letters | volume = 119 | issue = 1–2 | pages = 83–88 | date = June 1994 | pmid = 8039675 | doi = 10.1111/j.1574-6968.1994.tb06871.x | doi-access = free }}

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.

Isothiocyanates, the primary product of glucosinolate hydrolysis, have been known to prevent iodine uptake in the thyroid, causing goiters.{{cite journal | vauthors = Bones AM, Rossiter JT |year=1996 |title=The myrosinase-glucosinolate system, its organisation and biochemistry |journal=Physiologia Plantarum |volume=97 |pages=194–208 |doi=10.1111/j.1399-3054.1996.tb00497.x }} Isothiocyanates in high concentrations may cause hepatotoxicity. There is insufficient scientific evidence that consuming cruciferous vegetables with increased intake of isothiocyanates affects the risk of human diseases.{{cite web |title=Isothiocyanates |url=https://lpi.oregonstate.edu/mic/dietary-factors/phytochemicals/isothiocyanates |publisher=Micronutrient Information Center, Linus Pauling Institute, Oregon State University |access-date=26 June 2022 |date=1 April 2017}}

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{{Sugar hydrolases}}

Category:EC 3.2.1