ALLIINASE [S-ALK(EN)YL-L-CYSTEINE SULFOXIDE LYASE] FROM ALLIUM-TUBEROSUM (CHINESE CHIVE) - PURIFICATION, LOCALIZATION, CDNA CLONING AND HETEROLOGOUS FUNCTIONAL EXPRESSION

Alliinase [S-alk(en)yl-L-cysteine sulfoxide lyase], a pyridoxal-phosph ate-(Pxy-P)-dependent enzyme, is responsible for the degradative conve rsion of S-alk(en)yl-L-cysteine sulfoxide to volatile odorous sulfur-c ontaining metabolites in Allium plants. We have purified alliinase fro m shoots of Allium tuberosum (Chinese chive) to apparent homogeneity b y SDS/polyacrylamide gel electrophoresis. A cDNA clone encoding alliin ase was isolated from a cDNA library constructed from whole plants of A. tuberosum by hybridization screening with a synthetic 50-residue ol igonucleotide encoding a conserved region of the alliinases from onion and garlic. The isolated cDNA encoded a protein of 476 amino acid res idues with a molecular mass of 54083 Da. The deduced amino acid sequen ce exhibited 66-69% identities with those of reported alliinases from onion, garlic and shallot. The partial amino acid sequence, which was determined for a V8 protease-digested peptide fragment of the purified alliinase, was perfectly matched with the sequence deduced from the c DNA. An expression vector of recombinant alliinase cDNA was constructe d in yeast. The catalytically active protein was in the soluble fracti on of transformed yeast. Site-directed mutagenesis experiments indicat ed that Lys280 was essential for the catalytic activity and, thus, a p ossible Pxy-P-binding residue. The mRNA expression of the alliinase ge ne comprising a multigene family in the shoots of green plants was two fold higher than that in the roots of green plants; however, the expre ssion in the shoots of etiolated plants was only 13% that in green sho ots, although the expression in the roots was not remarkably different between in green and etiolated plants. Immunohistochemical investigat ion indicated that the alliinase protein is predominantly accumulated in the bundle sheath cells of shoots of A. tuberosum.