Molecular cloning, functional expression, and tissue distribution of a potato sprout allene oxide synthase involved in a 9-lipoxygenase pathway.
Potato (Solanum tuberosum) plants are rich in 9-lipoxygenase, which converts linoleic acid and alpha-linolenic acid to 9S-hydroperoxy-10E,12Z-octadecadienoic acid (9-HPOD) and 9S-hydroperoxy-10E,12Z,15Z-octadecatrienoic acid (9-HPOT) respectively. The allene oxide synthase (AOS) involved in 9-HPOD/9-HPOT metabolism in potato, however, has not been characterized in detail. We cloned a cDNA encoding a novel AOS from potato sprouts by reverse transcriptase-PCR based on a partial sequence in the EST database. This AOS was successfully expressed in the yeast Pichia pastoris, and purified using Ni-NTA resin. The recombinant enzyme metabolized 9-HPOD, 9-HPOT, 13-HPOD, and 13-HPOT with reaction efficiencies of 2.5 x 10(7), 1.0 x 10(7), 2.5 x 10(6), and 7.6 x 10(6) M(-1) s(-1) respectively. The alpha-ketol formed from 9-HPOD was composed mainly of the 9R-enatimomer (90%). Besides sprouts, the mRNA of this AOS was detected in buds, flowers, and stems, but not in leaves, tubers, or roots of mature plants, suggesting that this enzyme has a tissue-specific function.
Kongrit
Darika
D
Division of Applied Resources Chemistry, Course of Bioresources Science, The United Graduate School of Agricultural Sciences, Tottori University, Tottori, Japan.
Jisaka
Mitsuo
M
Kobayasi
Kazuhiro
K
Nishigaichi
Yutaka
Y
Nishimura
Kohji
K
Nagaya
Tsutomu
T
Yokota
Kazushige
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D008401:Gas Chromatography-Mass Spectrometry; D019746:Intramolecular Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008084:Lipoxygenase / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D018749:RNA, Plant / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D011198:Solanum tuberosum / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
D018749:RNA, Plant; D011994:Recombinant Proteins; D008084:Lipoxygenase; D019746:Intramolecular Oxidoreductases; C021377:hydroperoxide isomerase