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

Background: Myrosinase is the enzyme responsible for the hydrolysis of a variety of plant anionic 1-thio-beta-o-glucosides called glucosinol ates. Myrosinase and glucosinolates, which are stored in different tis sues of the plant, are mixed during mastication generating toxic by-pr oducts that are believed to play a role in the plant defence system. W hilst O-glycosidases are extremely widespread in nature, myrosinase is the only known S-glycosidase. This intriguing enzyme, which shows seq uence similarities with O-glycosidases, offers the opportunity to anal yze the similarities and differences between enzymes hydrolyzing S- an d O-glycosidic bonds. Results: The structures of native myrosinase fro m white mustard seed (Sinapis alba) and of a stable glycosyl-enzyme in termediate have been solved at 1.6 Angstrom resolution. The protein fo lds into a (beta/alpha)(8)-barrel structure, very similar to that of t he cyanogenic beta-glucosidase from white clover. The enzyme forms a d imer stabilized by a Zn2+ ion and is heavily glycosylated. At one glyc osylation site the complete structure of a plant-specific heptasacchar ide is observed. The myrosinase structure reveals a hydrophobic pocket , ideally situated for the binding of the hydrophobic sidechain of glu cosinolates, and two arginine residues positioned for interaction with the sulphate group of the substrate. With the exception of the replac ement of the general acid/base glutamate by a glutamine residue, the c atalytic machinery of myrosinase is identical to that of the cyanogeni c beta-glucosidase. The structure of the glycosyl-enzyme intermediate shows that the sugar ring is bound via an alpha-glycosidic linkage to Glu409, the catalytic nucleophite of myrosinase. Conclusions: The stru cture of myrosinase shows features which illustrate the adaptation of the plant enzyme to the dehydrated environment of the seed. The cataly tic mechanism of myrosinase is explained by the excellent leaving grou p properties of the substrate aglycons, which do not require the assis tance of an enzymatic acid catalyst, The replacement of the general ac id/base glutamate of O-glycosidases by a glutamine residue in myrosina se suggests that for hydrolysis of the glycosyl-enzyme, the role of th is residue is to ensure a precise positioning of a water molecule rath er than to provide general base assistance.