A candidate cDNA clone for (−)-limonene-7-hydroxylase from Perilla frutescens |
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Authors: | Christopher JD Mau Frank Karp Gisho Honda Rodney B Croteau |
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Institution: | a Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340, USA b Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29, Yoshida-Shimoadachi Sakyo, Kyoto 606-8501, Japan |
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Abstract: | Cytochrome P450 mono-oxygenases from peppermint, spearmint and perilla (all members of the family Lamiaceae) mediate the regiospecific hydroxylation of the parent olefin (−)-limonene to produce essential oil components oxygenated at C3, C6 and C7, respectively. Cloning, expression and mutagenesis of cDNAs encoding the peppermint limonene-3-hydroxylase and the spearmint limonene-6-hydroxylase have allowed the identification of a single amino acid residue which determines the regiospecificity of oxygenation by these two enzymes. A hybridization strategy provided a cytochrome P450 limonene hydroxylase cDNA from perilla with which to further evaluate the structural determinants of regiospecificity for oxygenation of the common substrate (−)-limonene. The perilla cDNA was a partial clone of 1550 bp (lacking the N-terminal membrane insertion domain), and shared 66% identity with the peppermint 3-hydroxylase and spearmint 6-hydroxylase at the amino acid level. The perilla cytochrome P450 was expressed in Escherichia coli as a chimeric protein fused with the N-terminal membrane insertion domain of the limonene-3-hydroxylase. The kinetically competent recombinant protein was characterized and shown to produce a mixture of C3-, C6- and C7-hydroxylated limonene derivatives with a distribution of 33%, 14% and 53%, respectively. |
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Keywords: | Perilla frutescens Lamiaceae Monoterpenoid (&minus )-Limonene-7-hydroxylase Perillyl alcohol Perillaldehyde Cytochrome P450 |
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