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cDNA cloning of rice lipoxygenase L‐2 and characterization using an active enzyme expressed from the cDNA in Escherichia coli

OHTA, Hiroyuki, SHIRANO, Yumiko, TANAKA, Kunisuke, MORITA, Yuhei, SHIBATA, Daisuke
European journal of biochemistry 1992 v.206 no.2 pp. 331-336
Escherichia coli, Magnoliopsida, amino acid sequences, amino acids, codons, complementary DNA, enzyme activity, heat stability, lipoxygenase, mature plants, molecular weight, nucleotides, pH, peptides, rice, seedlings
A full‐length cDNA of rice lipoxygenase L‐2 was cloned from 3‐day old seedlings. The identity of the clone was determined by amino acid sequencing of selected peptides of the purified enzyme and immunological characterization of an active enzyme that was produced from the cDNA in Escherichia coli by cultivation at 15°C. The nucleotide sequence showed a strong bias toward G and C in the selection of nucleotides, especially at the third position of the codons(93%G/C). The complete amino acid sequence of the enzyme was deduced from the nucleotide sequence. The molecular mass of the enzyme was calculated to be 96657 Da based on 865 amino acids. The amino acid sequence shares similarity with those of dicot lipoxygenases throughout the enzyme at a level of 50%. A hydropathy profile calculated from the amino acid sequence resembled those of dicot lipoxygenases, suggesting conservation of the secondary structure of these enzymes. The active enzyme, expressed in Escherichia coli, was charaterized for pH dependence of the enzyme activity, intramolecular specificity, heat stability and Km. The enzyme had the same properties as the L‐2 enzyme that was isolated from seedlings, but differed from the lipoxygenase L‐3 isolated from mature plants.