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Molecular cloning of CYP76A3, a novel cytochrome P450 from Petunia hybrida catalyzing the ω-hydroxylation of myristic acid

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Biologia Plantarum

Abstract

In higher plants, fatty acid hydroperoxides are intermediates in the synthesis of a diverse group of bioactive compounds. We used the reverse-transcriptase polymerase chain reaction to isolate a gene responsible for the oxidization of fatty acids from Petunia hybrida. A P450 cDNA that has not previously been isolated (CYP76A3) contained an open reading frame predicted to encode a polypeptide consisting of 507 amino acid residues. The cyp76A3 cDNA was expressed in Saccharomyces cerevisiae AH22 cells under the control of an alcohol dehydrogenase promoter and terminator. The recombinant yeast microsome containing the CYP76A3 hemoprotein was found to specifically catalyze ω-hydroxylation of myristic acid. A high level of the transcripts of the cyp76A3 gene was found in the leaves and roots of P. hybrida, but not in the stems and flowers.

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Abbreviations

dNTP:

deoxynucleotide triphosphate

HPLC:

high-performance liquid chromatography

MOPS:

3-(N-morpholino) propanesulfonic acid

PCR:

polymerase chain reaction

RT-PCR:

reverse-transcriptase polymerase chain reaction

SDS:

sodium dodecyl sulfate

SSC:

saline-sodium citrate

SSPE:

a mixture of 0.18 M NaCl, 10 mM NaPO4, and 1 mM EDTA at pH 7.5

TLC:

thin-layer chromatography

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Authors and Affiliations

  1. Research Center for Environmental Genomics, Kobe University, Rokkodaicho 1-1, Nada-ku, Kobe, 657-8501, Japan

    H. Imaishi & U. Ishitobi

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  1. H. Imaishi
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  2. U. Ishitobi
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Correspondence to H. Imaishi.

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Imaishi, H., Ishitobi, U. Molecular cloning of CYP76A3, a novel cytochrome P450 from Petunia hybrida catalyzing the ω-hydroxylation of myristic acid. Biol Plant 52, 242–250 (2008). https://doi.org/10.1007/s10535-008-0053-0

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  • DOI: https://doi.org/10.1007/s10535-008-0053-0

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