High Resolution X-ray Crystallography Shows That Ascorbate Is a Cofactor for Myrosinase and Substitutes for the Function of the Catalytic Base

Abstract

Myrosinase, an S-glycosidase, hydrolyzes plant anionic 1-thio-␤-D-glucosides (glucosinolates) considered part of the plant defense system. Although O-glycosidases are ubiquitous, myrosinase is the only known Sglycosidase. Its active site is very similar to that of retaining O-glycosidases, but one of the catalytic residues in O-glycosidases, a carboxylate residue functioning as the general base, is replaced by a glutamine residue. Myrosinase is strongly activated by ascorbic acid. Several binary and ternary complexes of myrosinase with different transition state analogues and ascorbic acid have been analyzed at high resolution by x-ray crystallography along with a 2-deoxy-2-fluoro-glucosyl enzyme intermediate. One of the inhibitors, D-gluconhydroximo-1,5-lactam, binds simultaneously with a sulfate ion to form a mimic of the enzyme-substrate complex. Ascorbate binds to a site distinct from the glucose binding site but overlapping with the aglycon binding site, suggesting that activation occurs at the second step of catalysis, i.e. hydrolysis of the glycosyl enzyme. A water molecule is placed perfectly for activation by ascorbate and for nucleophilic attack on the covalently trapped 2-fluoroglucosyl-moiety. Activation of the hydrolysis of the glucosyl enzyme intermediate is further evidenced by the observation that ascorbate enhances the rate of reactivation of the 2-fluoro-glycosyl enzyme, leading to the conclusion that ascorbic acid substitutes for the catalytic base in myrosinase.Glucosinolates are anionic ␤-D-S-glucosides found prominently in plants of the genus Brassica (cabbage, mustard, rapeseed, and other Cruciferae). They constitute a large family of S-glucosides that differ by their aglycon (Ref. 1 and Fig. 1a). The same plants produce myrosinase (thioglucoside glucohydrolase, EC 3.2.3.1), an S-glucosidase hydrolyzing glucosinolates. Myrosinase and glucosinolates are stored in different compartments of the plant, especially in the seeds. Mixing of enzyme and substrate (for example through mastication) induces glucosinolate hydrolysis. The biological function of myrosinase and glucosinolates is only partly elucidated; it has been suggested that they represent a defense system of the plant. Glucosinolates may as well serve to store inactive precursors of hormones such as 3-indolylacetic acid. 3-Indolyl acetonitrile and related indoles (2) are released from indolyl glucosinolates by myrosinase (3). Cleavage of indol-3-ylmethyl glucosinolate (glucobrassicin) by myrosinase in the presence of ascorbic acid produces ascorbigen, a condensation product of ascorbic acid with 3-hydroxymethylindole (4). Thus, the myrosinase system could be involved in the storage and in the inactivation of ascorbic acid. A detailed review on myrosinase is given by Bones and Rossiter (3).Myrosinase hydrolyzes the S-glycosides with retention of the anomeric configuration (5). Retaining glycosidases operate by a double displacement at the anomeric center promoted by two carboxylic residues acting as acid/base and as nucleophile, respec...

Phyllotreta striolataflea beetles use host plant defense compounds to create their own glucosinolate-myrosinase system

“…S6). The S. alba enzyme is thought to mediate hydrolysis by the good leaving group character of the glucosinolate aglycone and the use of the cofactor ascorbate as a base (33). The glucose recognition site is identical among the three enzymes; however, only two of the seven residues forming the aglycone binding site in plant myrosinases are identical in the P. striolata myrosinase (Fig.…”

Molecular basis of substrate specificity in family 1 glycoside hydrolases

“…Nevertheless, myrosinases from GH family1 lack the acid/base residue. Besides that, ascorbate is a co-factor for myrosinases and replaces the catalytic base (4).…”

Klotho Is a Novel β-Glucuronidase Capable of Hydrolyzing Steroid β-Glucuronides

“…For myrosinase, it has been shown that the acid/base residue is not necessary in the first step of catalysis because of the excellent leaving group ability of the aglycone of the natural substrates (glucosinolates). For the second step, Burmeister and colleagues (24) have shown by high resolution x-ray crystallography that ascorbate is a cofactor for myrosinase and substitutes for the function of the catalytic base.…”