A candidate cDNA clone for (-)-limonene-7-hydroxylase from Perilla frutescens.
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 1550bp (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.
Mau
Christopher J D
CJ
Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340, USA. cjmau@wsu.edu
Karp
Frank
F
Ito
Michiho
M
Honda
Gisho
G
Croteau
Rodney B
RB
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D008969:Molecular Sequence Data; D031344:Perilla frutescens / Q000166:cytology / Q000201:enzymology* / Q000235:genetics*; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D003577:Cytochrome P-450 Enzyme System