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The Active Principle of Garlic at Atomic Resolution

2002; Elsevier BV; Volume: 277; Issue: 48 Linguagem: Inglês

10.1074/jbc.m208669200

1083-351X

E. Bartholomeus Kuettner, Rolf Hilgenfeld, M.S. Weiss,

Phytochemical compounds biological activities

Despite the fact that many cultures around the world value and utilize garlic as a fundamental component of their cuisine as well as of their medicine cabinets, relatively little is known about the plant's protein configuration that is responsible for the specific properties of garlic. Here, we report the three-dimensional structure of the garlic enzyme alliinase at 1.5 Å resolution. Alliinase constitutes the major protein component in garlic bulbs, and it is able to cleave carbon-sulfur bonds. The active enzyme is a pyridoxal-5′-phosphate-dependent homodimeric glycoprotein and belongs to the class I family of pyridoxal-5′-phosphate-dependent enzymes. In addition, it contains a novel epidermal growth factor-like domain that makes it unique among all pyridoxal-5′-phosphate-dependent enzymes. Despite the fact that many cultures around the world value and utilize garlic as a fundamental component of their cuisine as well as of their medicine cabinets, relatively little is known about the plant's protein configuration that is responsible for the specific properties of garlic. Here, we report the three-dimensional structure of the garlic enzyme alliinase at 1.5 Å resolution. Alliinase constitutes the major protein component in garlic bulbs, and it is able to cleave carbon-sulfur bonds. The active enzyme is a pyridoxal-5′-phosphate-dependent homodimeric glycoprotein and belongs to the class I family of pyridoxal-5′-phosphate-dependent enzymes. In addition, it contains a novel epidermal growth factor-like domain that makes it unique among all pyridoxal-5′-phosphate-dependent enzymes. Garlic (Allium sativum) has been known and utilized as a spice and herbal remedy for more than 4000 years. Most likely originating from Central Asia, the plant made its way viathe Old Sumerians, the Egyptian high cultures, the Greeks, and the Romans into modern European and American cuisine, pharmacy, and culture. Today, a myriad of pharmacological properties are attributed to garlic or its ingredients, ranging from blood lipid level and blood pressure lowering and inhibition of blood clotting to antiviral, antifungal, and antimicrobial activities and even cancerostatic effects (1Siegel G. Walter A. Engel S. Walper A. Michel F. Wien. Med. Wochenschr. 1999; 149: 217-224PubMed Google Scholar, 2Block E. Angew. Chem. Int. Ed. Engl. 1992; 31: 1135-1178Crossref Scopus (974) Google Scholar, 3Tsai Y. Cole L.L. Davis L.E. Lockwood S.J. Simmons V. Wild G.C. Planta Med. 1985; 51: 460-461Crossref PubMed Scopus (99) Google Scholar, 4Agarwal K.C. Med. Res. Rev. 1996; 16: 111-124Crossref PubMed Scopus (535) Google Scholar). Although these pharmacological effects are little understood, it is clear that most of them rely on sulfur-containing garlic components. Many of the active compounds have been identified in garlic and other Allium species and found to belong to one of three groups: dithiines, allyl sulfides, and ajoenes (Fig. 1 A). Their organic chemistry has been studied (2Block E. Angew. Chem. Int. Ed. Engl. 1992; 31: 1135-1178Crossref Scopus (974) Google Scholar), and in some cases their therapeutic effects have been elucidated (4Agarwal K.C. Med. Res. Rev. 1996; 16: 111-124Crossref PubMed Scopus (535) Google Scholar). All of these compounds originate from thiosulfinates such as allicin (Fig. 1 B), which in turn are produced by the action of the enzyme alliinase (EC4.4.1.4) on cysteine derivatives.Garlic alliinase was first described in 1947 (5Stoll A. Seebeck E. Experientia. 1947; 3: 114-115Crossref PubMed Scopus (45) Google Scholar). It is a homodimeric enzyme of 2 × 448 amino acid residues and a total molecular weight of 103,000. The enzyme contains one pyridoxal-5′-phosphate (PLP) 1The abbreviations used for: PLP, pyridoxal-5′-phosphate; aAT, aromatic amino acid-aminotransferase; AA, aminoacrylate; ACC, 1-aminocyclopropane-1-carboxylate; EGF, epidermal growth factor. 1The abbreviations used for: PLP, pyridoxal-5′-phosphate; aAT, aromatic amino acid-aminotransferase; AA, aminoacrylate; ACC, 1-aminocyclopropane-1-carboxylate; EGF, epidermal growth factor.cofactor per subunit and belongs to the class I of PLP-dependent enzymes (6Schneider G. Käck H. Lindqvist Y. Structure. 2000; 8: R1-R6Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar). With its C-S lyase activity, alliinase is able to cleave the Cβ-Sγ bond of sulfoxide derivatives of cysteine to produce allicin (Fig.1 B). Because of the vacuolar location of the enzyme and the presence of its substrate in the cytosol, the alliinase/alliin system has been discussed as a primitive defense mechanism of the plant (7Ellmore G.S. Feldberg R.S. Am. J. Bot. 1994; 81: 89-94Crossref Scopus (70) Google Scholar). Based on multiple sequence alignments of various alliinase sequences, the presence of a unique epidermal growth factor (EGF)-like domain was proposed (8Kuettner E.B. Hilgenfeld R. Weiss M.S. Arch. Biochem. Biophys. 2002; 402: 192-200Crossref PubMed Scopus (35) Google Scholar). The protein also contains four putative N-glycosylation sites at Asn19, Asn146, Asn191, and Asn328 (9van Damme E.J.M. Smeets K. Torrekens S. van Leuven F. Peumans W.J. Eur. J. Biochem. 1992; 209: 751-757Crossref PubMed Scopus (70) Google Scholar). Recently, diffraction quality crystals of the natural form of the enzyme have been obtained (8Kuettner E.B. Hilgenfeld R. Weiss M.S. Arch. Biochem. Biophys. 2002; 402: 192-200Crossref PubMed Scopus (35) Google Scholar, 10Shimon L.J.W. Rabinkov A. Miron T. Mirelman D. Wilchek M. Frolow F. Acta Crystallogr. Sec. D. 2002; 58: 1335-1337Crossref PubMed Scopus (7) Google Scholar).Here, we report the three-dimensional structure of garlic alliinase at 1.5 Å resolution. Alliinase is only the second garlic protein for which a structure determination has been carried out. We confirm the presence of the proposed EGF-like domain, rationalize the reaction mechanism, and provide some explanation for the substrate selectivity of alliinase. Garlic (Allium sativum) has been known and utilized as a spice and herbal remedy for more than 4000 years. Most likely originating from Central Asia, the plant made its way viathe Old Sumerians, the Egyptian high cultures, the Greeks, and the Romans into modern European and American cuisine, pharmacy, and culture. Today, a myriad of pharmacological properties are attributed to garlic or its ingredients, ranging from blood lipid level and blood pressure lowering and inhibition of blood clotting to antiviral, antifungal, and antimicrobial activities and even cancerostatic effects (1Siegel G. Walter A. Engel S. Walper A. Michel F. Wien. Med. Wochenschr. 1999; 149: 217-224PubMed Google Scholar, 2Block E. Angew. Chem. Int. Ed. Engl. 1992; 31: 1135-1178Crossref Scopus (974) Google Scholar, 3Tsai Y. Cole L.L. Davis L.E. Lockwood S.J. Simmons V. Wild G.C. Planta Med. 1985; 51: 460-461Crossref PubMed Scopus (99) Google Scholar, 4Agarwal K.C. Med. Res. Rev. 1996; 16: 111-124Crossref PubMed Scopus (535) Google Scholar). Although these pharmacological effects are little understood, it is clear that most of them rely on sulfur-containing garlic components. Many of the active compounds have been identified in garlic and other Allium species and found to belong to one of three groups: dithiines, allyl sulfides, and ajoenes (Fig. 1 A). Their organic chemistry has been studied (2Block E. Angew. Chem. Int. Ed. Engl. 1992; 31: 1135-1178Crossref Scopus (974) Google Scholar), and in some cases their therapeutic effects have been elucidated (4Agarwal K.C. Med. Res. Rev. 1996; 16: 111-124Crossref PubMed Scopus (535) Google Scholar). All of these compounds originate from thiosulfinates such as allicin (Fig. 1 B), which in turn are produced by the action of the enzyme alliinase (EC4.4.1.4) on cysteine derivatives. Garlic alliinase was first described in 1947 (5Stoll A. Seebeck E. Experientia. 1947; 3: 114-115Crossref PubMed Scopus (45) Google Scholar). It is a homodimeric enzyme of 2 × 448 amino acid residues and a total molecular weight of 103,000. The enzyme contains one pyridoxal-5′-phosphate (PLP) 1The abbreviations used for: PLP, pyridoxal-5′-phosphate; aAT, aromatic amino acid-aminotransferase; AA, aminoacrylate; ACC, 1-aminocyclopropane-1-carboxylate; EGF, epidermal growth factor. 1The abbreviations used for: PLP, pyridoxal-5′-phosphate; aAT, aromatic amino acid-aminotransferase; AA, aminoacrylate; ACC, 1-aminocyclopropane-1-carboxylate; EGF, epidermal growth factor.cofactor per subunit and belongs to the class I of PLP-dependent enzymes (6Schneider G. Käck H. Lindqvist Y. Structure. 2000; 8: R1-R6Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar). With its C-S lyase activity, alliinase is able to cleave the Cβ-Sγ bond of sulfoxide derivatives of cysteine to produce allicin (Fig.1 B). Because of the vacuolar location of the enzyme and the presence of its substrate in the cytosol, the alliinase/alliin system has been discussed as a primitive defense mechanism of the plant (7Ellmore G.S. Feldberg R.S. Am. J. Bot. 1994; 81: 89-94Crossref Scopus (70) Google Scholar). Based on multiple sequence alignments of various alliinase sequences, the presence of a unique epidermal growth factor (EGF)-like domain was proposed (8Kuettner E.B. Hilgenfeld R. Weiss M.S. Arch. Biochem. Biophys. 2002; 402: 192-200Crossref PubMed Scopus (35) Google Scholar). The protein also contains four putative N-glycosylation sites at Asn19, Asn146, Asn191, and Asn328 (9van Damme E.J.M. Smeets K. Torrekens S. van Leuven F. Peumans W.J. Eur. J. Biochem. 1992; 209: 751-757Crossref PubMed Scopus (70) Google Scholar). Recently, diffraction quality crystals of the natural form of the enzyme have been obtained (8Kuettner E.B. Hilgenfeld R. Weiss M.S. Arch. Biochem. Biophys. 2002; 402: 192-200Crossref PubMed Scopus (35) Google Scholar, 10Shimon L.J.W. Rabinkov A. Miron T. Mirelman D. Wilchek M. Frolow F. Acta Crystallogr. Sec. D. 2002; 58: 1335-1337Crossref PubMed Scopus (7) Google Scholar). Here, we report the three-dimensional structure of garlic alliinase at 1.5 Å resolution. Alliinase is only the second garlic protein for which a structure determination has been carried out. We confirm the presence of the proposed EGF-like domain, rationalize the reaction mechanism, and provide some explanation for the substrate selectivity of alliinase. We gratefully acknowledge the provision of data collection facilities at the x-ray diffraction beamline at ELETTRA (Trieste, Italy) and the EMBL beamline BW7B at Deutsches Elektronen Synchrotron (DESY; Hamburg, Germany). R. H. thanks the Fonds der Chemischen Industrie.