Physical characterization of alliinase, the flavor generating enzyme in onions

The characteristic flavor of onions occurs when the enzyme alliinase (EC 4.4.1.4) hydrolyses the S-alk(en)yl-L-cysteine sulfoxides to form sulfur containing volatiles, pyruvate and ammonia. In this paper we have new evidence on the molecular mass of native onion alliinase, its subunit size and its relationship to the predicted size from translation of the gene. Alliinase from bulbs was purified to homogeneity. Two isoforms of alliinase were detected on ion-exchange chromatography. Both isoforms were analyzed by gelfiltration FPLC. Alliinase activity was detected at molecular masses of 59, 166 kDa and 59, 170 and 393 kDa for isoforms 1 and 2, respectively. When analyzed by SDS-PAGE subunit sizes were determined to be 53.3 kDa for isoform I and 53.3 and 51.6 kDa for isoform 2, Amino terminal sequencing of both subunits confirmed they were alliinase. Alliinase was deglycosylated and the subunits migrated as a single band indicating a common protein backbone and varying glycosylation. Thus native alliinase is a monomer, trimer and multimer of the subunits, Molecular analysis of alliinase cDNA indicated that two genes and thus two protein subunits were expressed in onion bulb tissue. Translation is likely to have initiated from the third methionine of the leader sequence of the cDNA to give a protein backbone of 53.5 kDa in gradient SDS-PACE. The functional significance of heterogenous subunits, from different genes, and multimeric native alliinase remains unknown.