Molecular evaluation of a spearmint mutant altered in the expression of limonene hydroxylases that direct essential oil monoterpene biosynthesis.
Gamma irradiation of Scotch spearmint created a mutant line, 643-10-74, which has an altered essential oil reminiscent of peppermint because the monoterpene metabolites in the oil glands of the mutant are predominantly oxygenated at the C3 position of the p-menthane ring instead of the C6 position normally found in spearmint. The limonene hydroxylase genes responsible for directing the regiochemistry of oxygenation were cloned from Scotch spearmint and mutant 643 and expressed in Escherichia coli. The limonene bydroxylase from the wild-type parent hydroxylated the C6 position while the enzyme from the mutant oxygenated the C3 position. Comparison of the amino acid sequences with other limonene hydroxylases showed that the mutant enzyme was more closely related to the peppermint limonene-3-hydroxylases than to the spearmint limonene-6-hydroxylases. Because of the sequence differences between the Scotch spearmint and mutant 643 limonene hydroxylases, it is most likely that the mutation did not occur within the structural gene for limonene hydroxylase but rather at a regulatory site within the genome that controls the expression of one or the other regiospecific variants.
Institute of Biological Chemistry, Washington State University, 385 Clark Hall, Pullman, WA 99164-6340, USA.
Cinzia
C
Bertea
Michel
M
Schalk
Christopher J D
CJ
Mau
Frank
F
Karp
Mark R
MR
Wildung
Rodney
R
Croteau
D000595:Amino Acid Sequence; D002849:Chromatography, Gas; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D031337:Mentha spicata / Q000201:enzymology* / Q000235:genetics*; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D039821:Monoterpenes / Q000378:metabolism*; D009822:Oils, Volatile / Q000737:chemistry; D010940:Plant Proteins; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013237:Stereoisomerism
D018076:DNA, Complementary; D039821:Monoterpenes; D009822:Oils, Volatile; D010940:Plant Proteins; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C120915:limonene-3-hydroxylase; C120916:limonene-6-hydroxylase