Two Structures of Alliinase from Alliium sativum L.: Apo Form and Ternary Complex with Aminoacrylate Reaction Intermediate Covalently Bound to the PLP Cofactor Linda J.W. Shimon 1 ⁎, Aharon Rabinkov 2 , Irina Shin 2 , Talia Miron 2 David Mirelman 2 , Meir Wilchek, 2 and Felix Frolow 3 ⁎ 1 Department of Chemical Research Support, The Weizmann Institute of Science, Rehovot 76100, Israel 2 Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel 3 Department of Molecular Microbiology and Biotechnology and The Daniella Rich Institute for Structural Biology , Tel Aviv University, Ramat Aviv 69978, Israel Alliinase (alliin lyase EC 4.4.1.4), a PLP-dependent α, β-eliminating lyase, constitutes one of the major protein components of garlic (Alliium sativum L.) bulbs. The enzyme is a homodimeric glycoprotein and catalyzes the conversion of a specific non-protein sulfur-containing amino acid alliin ((+S)-allyl-L-cysteine sulfoxide) to allicin (diallyl thiosulfinate, the well known biologically active component of freshly crushed garlic), pyruvate and ammonia. The enzyme was crystallized in the presence of (+S)-allyl-L- cysteine, forming dendrite-like monoclinic crystals. In addition, intention- ally produced apo-enzyme was crystallized in tetragonal form. These structures of alliinase with associated glycans were resolved to 1.4 Å and 1.61 Å by molecular replacement. Branched hexasaccharide chains N-linked to Asn146 and trisaccharide chains N-linked to Asn328 are seen. The structure of hexasaccharide was found similar to “short chain complex vacuole type” oligosaccharide most commonly seen in plant glycoproteins. An unexpected state of the enzyme active site has been observed in the present structure. The electron density in the region of the cofactor made it possible to identify the cofactor moiety as aminoacrylate intermediate covalently bound to the PLP cofactor. It was found in the present structure to be stabilized by large number of interactions with surrounding protein residues. Moreover, the existence of the expected internal aldimine bond between the ε-amino group of Lys251 and the aldehyde of the PLP is ruled out on the basis of a distinct separation of electron density of Lys251. The structure of the active site cavity in the apo-form is nearly identical to that seen in the holo-form, with two sulfate ions, an acetate and several water molecules from crystallization conditions that replace and mimic the PLP cofactor. © 2006 Elsevier Ltd. All rights reserved. *Corresponding authors Keywords: alliinase; X-ray structure; pyridoxal 5'-phosphate aminoacrylate; plant enzyme glycosylation Introduction Garlic (Alliium sativum L.) is a plant grown throughout the world that is valued both as a food and as a folk-medicine. For generations, garlic has been known for its remarkable medicinal properties, which include antibiotic as well as hypolipidemic, antithrombotic, antihypertensive and anticancer activities. 1 The pungent and unmistakable smell of garlic is a result of the reaction of the enzyme alliinase (E.C. 4.4.1.4) and its substrate, the non- protein amino acid, alliin. 2 Under normal circum- stances, in the intact cells of the garlic bulbs, these two molecules are physically separated: the enzyme alliinase is compartmentalized in the vacuoles while the alliin is localized in the cell cytoplasm. 3 How- ever, as has been commonly observed, when the garlic bulbs are crushed or injured, the enzyme and its substrate are brought into contact so that the reaction shown in Scheme 1 can take place. E-mail addresses of the corresponding authors: Linda.shimon@weizmann.ac.il; mbfrolow@post.tau.ac.il doi:10.1016/j.jmb.2006.11.041 J. Mol. Biol. (2007) 366, 611–625 0022-2836/$ - see front matter © 2006 Elsevier Ltd. All rights reserved.