Molecular cloning of allene oxide cyclase - The enzyme establishing the stereochemistry of octadecanoids and jasmonates

Allene oxide cyclase (EC 5.3.99.6) catalyzes the stereospecific cyclization of an unstable allene oxide to (9S,13S)-12 oxo-(10,15Z)-phytodienoic acid, the ultimate precursor of jasmonic acid. This dimeric enzyme has previousl y been purified, and two almost identical N-terminal peptides were found, s uggesting allene oxide cyclase to be a homodimeric protein. Furthermore, th e native protein was N-terminally processed. Using degenerate primers, a po lymerase chain reaction fragment could be generated from tomato, which was further used to isolate a full-length cDNA clone of 1 kilobase pair coding for a protein of 245 amino acids with a molecular mass of 26 kDa. Whereas e xpression of the whole coding region failed to detect allene oxide cyclase activity, a 5'-truncated protein showed high activity, suggesting that addi tional amino acids impair the enzymatic function. Steric analysis of the 12 -oxophytodienoic acid formed by the recombinant enzyme revealed exclusive ( >99%) formation of the 9S,13S enantiomer. Exclusive formation of this enant iomer was also found in wounded tomato leaves. Southern analysis and geneti c mapping revealed the existence of a single gene for allene oxide cyclase located on chromosome 2 of tomato. Inspection of the N terminus revealed th e presence of a chloroplastic transit peptide, and the location of allene o xide cyclase protein in that compartment could be shown by immunohistochemi cal methods. Concomitant with the jasmonate levels, the accumulation of ali ene oxide cyclase mRNA was transiently induced after wounding of tomato lea ves.