Alliinase [S‐alk(en)yl‐ L‐cysteine sulfoxide lyase] from Allium tuberosum (Chinese chive)

Abstract

Alliinase [S-alk(en)yl-L-cysteine sulfoxide lyase], a pyridoxal-phosphate-(Pxy-P)-dependent enzyme, is responsible for the degradative conversion of S-alk(en)yl-L-cysteine sulfoxide to volatile odorous sulfurcontaining metabolites in Allium plants. We have purified alliinase from shoots of Allium tuberosum (Chinese chive) to apparent homogeneity by SDS/polyacrylamide gel electrophoresis. A cDNA clone encoding alliinase was isolated from a cDNA library constructed from whole plants of A. tuberosum by hybridization screening with a synthetic 50-residue oligonucleotide encoding a conserved region of the alliinases from onion and garlic. The isolated cDNA encoded a protein of 476 amino acid residues with a molecular mass of 54 083 Da. The deduced amino acid sequence exhibited 66Ϫ69% 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 cDNA. An expression vector of recombinant alliinase cDNA was constructed in yeast. The catalytically active protein was in the soluble fraction of transformed yeast. Site-directed mutagenesis experiments indicated that Lys280 was essential for the catalytic activity and, thus, a possible Pxy-P-binding residue. The mRNA expression of the alliinase gene comprising a multigene family in the shoots of green plants was twofold higher than that in the roots of green plants ; however, the expression in the shoots of etiolated plants was only 13% that in green shoots, although the expression in the roots was not remarkably different between in green and etiolated plants. Immunohistochemical investigation indicated that the alliinase protein is predominantly accumulated in the bundle sheath cells of shoots of A. tuberosum.Keywords : Allium; alliinase ; cDNA cloning; pyridoxal 5′-phosphate; sulfur metabolism.The plants belonging to genus Allium generally produce characteristic odorous sulfur-containing compounds. These volatile sulfur compounds are formed from non-volatile S-alk(en)yl-L-cysteine sulfoxide (ACS), as a precursor, through enzymatic degradation, which is catalyzed by so-called alliinase [S-alk-(en)yl-L-cysteine sulfoxide lyase] [1, 2]. In onion (Allium cepa) cells, alliinase is assumed to be localized in vacuoles, thereby being separated from ACS present in the cytosol [3]. Upon rupture of the cells, the enzyme can react with the substrate to yield sulfenic acid as the first sulfur-containing volatile compound (Scheme 1). Numerous sulfur-containing metabolites, such as thiosulfinates, thiols, disulfides, etc. are secondarily produced from sulfenic acid through non-enzymatic reactions [4]. These

Studies of a Novel Cysteine Sulfoxide Lyase fromPetiveria alliacea: The First Heteromeric Alliinase

“…garlic cloves (Mazelis and Crews, 1968), onion bulbs (Tobkin and Mazelis, 1979), leek (Allium porrum; Lohmuller et al, 1994), Chinese chive (Allium tuberosum; Manabe et al, 1998), and wild garlic/ramson (Allium ursinum; Landshuter et al, 1994). Previous reports have shown that garlic (Nock and Mazelis, 1986), leek (Won and Mazelis, 1989), onion (Tobkin and Mazelis, 1979), and Welsh onion (Allium fistulosum; Fujita et al, 1990) alliinases are glycoproteins, and all alliinases have been shown to be pyridoxal 5#-P (PLP) dependent.…”

“…Previous reports have shown that garlic (Nock and Mazelis, 1986), leek (Won and Mazelis, 1989), onion (Tobkin and Mazelis, 1979), and Welsh onion (Allium fistulosum; Fujita et al, 1990) alliinases are glycoproteins, and all alliinases have been shown to be pyridoxal 5#-P (PLP) dependent. Isolated alliinases vary in molecular mass depending on the source of the enzyme, ranging from 67 kD in Chinese chive (Manabe et al, 1998) to 580 kD in leek (Won and Mazelis, 1989). The number of enzyme subunits has been shown to differ depending on the source of the alliinase, ranging from one for Chinese chive to 12 for leek.…”

“…The number of enzyme subunits has been shown to differ depending on the source of the alliinase, ranging from one for Chinese chive to 12 for leek. The affinity of the enzyme for substrate, expressed as K m and typically determined using S-ethyl-L-Cys sulfoxide as the substrate, also varies depending on the alliinase source, ranging from 2.7 mM for Chinese chive (Manabe et al, 1998) to 13 mM for leek (Won and Mazelis, 1989). The reported pH optima also vary depending on the species and the buffer conditions used, ranging from 6.5 (garlic in phosphate buffer; Nock and Mazelis, 1986) to 8.5 (Chinese chive in Tris-HCl buffer; Manabe et al, 1998).…”

A New Member of Plant CS-lyases

“…Radiolabeled BOCL-175 was then used to screen a broccoli cDNA library prepared as described in Ref. 22 using RNA extracted from 6-month-old field-grown influorescences. Approximately 280,000 individual unamplified plaques were screened as described in Ref.…”

“…Approximately 280,000 individual unamplified plaques were screened as described in Ref. 22 and one full-length clone (BOCL-3), and several partial clones were isolated and sequenced in both directions, as described previously (11), after subcloning into pBluescript II.…”