Molecular cloning of allene oxide cyclase. The enzyme establishing the stereochemistry of octadecanoids and jasmonates.
Allene oxide cyclase (EC ) 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 previously been purified, and two almost identical N-terminal peptides were found, suggesting allene oxide cyclase to be a homodimeric protein. Furthermore, the native protein was N-terminally processed. Using degenerate primers, a polymerase 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 expression of the whole coding region failed to detect allene oxide cyclase activity, a 5'-truncated protein showed high activity, suggesting that additional 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 enantiomer was also found in wounded tomato leaves. Southern analysis and genetic mapping revealed the existence of a single gene for allene oxide cyclase located on chromosome 2 of tomato. Inspection of the N terminus revealed the presence of a chloroplastic transit peptide, and the location of allene oxide cyclase protein in that compartment could be shown by immunohistochemical methods. Concomitant with the jasmonate levels, the accumulation of allene oxide cyclase mRNA was transiently induced after wounding of tomato leaves.
Ziegler
J
J
Leibniz Institute of Plant Biochemistry, Department of Natural Product Biotechnology, Weinberg 3, D-06120 Halle, Germany.
Stenzel
I
I
Hause
B
B
Maucher
H
H
Hamberg
M
M
Grimm
R
R
Ganal
M
M
Wasternack
C
C
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003517:Cyclopentanes / Q000737:chemistry / Q000378:metabolism*; D018076:DNA, Complementary; D019746:Intramolecular Oxidoreductases / Q000235:genetics*; D018551:Solanum lycopersicum / Q000201:enzymology; D008969:Molecular Sequence Data; D054883:Oxylipins; D013229:Stearic Acids / Q000737:chemistry / Q000378:metabolism*; D013237:Stereoisomerism; D013347:Subcellular Fractions / Q000201:enzymology
D003517:Cyclopentanes; D018076:DNA, Complementary; D054883:Oxylipins; D013229:Stearic Acids; C011006:jasmonic acid; D019746:Intramolecular Oxidoreductases; C021377:hydroperoxide isomerase