Alliin lyase (Alliinase) from garlic (Allium sativum). Biochemical characterization and cDNA cloning.
The garlic plant (Allium sativum) alliinase (EC 4.4.1.4), which catalyzes the synthesis of allicin, was purified to homogeneity from bulbs using various steps, including hydrophobic chromatography. Molecular and biochemical studies showed that the enzyme is a dimer of two subunits of MW 51.5 kDa each. Its Km using synthetic S-allylcysteine sulfoxide (+ isomer) as substrate was 1.1 mM, its pH optimum 6.5, and its isoelectric point 6.35. The enzyme is a glycoprotein containing 6% carbohydrate. N-terminal sequences of the intact polypeptide chain as well as of a number of peptides obtained after cyanogen bromide cleavage were obtained. Cloning of the cDNAs encoding alliinase was performed by a two-step strategy. In the first, a cDNA fragment (pAli-1-450 bp) was obtained by PCR using a mixed oligonucleotide primer synthesized according to a 6-amino acid segment near the N-terminal of the intact polypeptide. The second step involved screening of garlic lambda gt11 and lambda ZAPII cDNA libraries with pAli-1, which yielded two clones; one was nearly full length and the second was full length. These clones exhibited some degree of DNA sequence divergence, especially in their 3' noncoding regions, suggesting that they were encoded by separate genes. The nearly full length cDNA was fused in frame to a DNA encoding a signal peptide from alpha wheat gliadin, and expressed in Xenopus oocytes. This yielded a 50 kDa protein that interacted with the antibodies against natural bulb alliinase. Northern and Western blot analyses showed that the bulb alliinase was highly expressed in bulbs, whereas a lower expression level was found in leaves, and no expression was detected in roots. Strikingly, the roots exhibited an abundant alliinase activity, suggesting that this tissue expressed a distinct alliinase isozyme with very low homology to the bulb enzyme.
Rabinkov
A
A
Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot, Israel.
Zhu
X Z
XZ
Grafi
G
G
Galili
G
G
Mirelman
D
D
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D013437:Carbon-Sulfur Lyases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003488:Cyanogen Bromide / Q000378:metabolism; D003545:Cysteine / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000378:metabolism; D018076:DNA, Complementary / Q000737:chemistry*; D004220:Disulfides; D005260:Female; D005737:Garlic / Q000201:enzymology*; D005786:Gene Expression Regulation / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009693:Nucleic Acid Hybridization; D010946:Plants, Medicinal; D016133:Polymerase Chain Reaction; D011108:Polymers; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D011817:Rabbits; D013441:Sulfinic Acids / Q000378:metabolism; D014982:Xenopus laevis
D018076:DNA, Complementary; D004220:Disulfides; D011108:Polymers; D012333:RNA, Messenger; D013441:Sulfinic Acids; C006452:allicin; C006453:alliin; D013437:Carbon-Sulfur Lyases; C019243:alliin lyase; D003545:Cysteine; D003488:Cyanogen Bromide