Publications

• Monoterpene biosynthesis: specificity of the hydroxylations of (-)-limonene by enzyme preparations from peppermint (Mentha piperita), spearmint (Mentha spicata), and perilla (Perilla frutescens) leaves.

  Karp F., Mihaliak C.A., Harris J.L., Croteau R.

  Microsomal preparations from the epidermal oil glands of Mentha piperita, Mentha spicata, and Perilla frutescens leaves catalyze the NADPH- and O2-dependent allylic hydroxylation of the monoterpene olefin (-)-limonene at C-3, C-6, and C-7, respectively, to produce the corresponding alcohols, (-)-t ... >> More

  Microsomal preparations from the epidermal oil glands of Mentha piperita, Mentha spicata, and Perilla frutescens leaves catalyze the NADPH- and O2-dependent allylic hydroxylation of the monoterpene olefin (-)-limonene at C-3, C-6, and C-7, respectively, to produce the corresponding alcohols, (-)-trans-isopiperitenol, (-)-trans-carveol, and (-)-perillyl alcohol. These transformations are the key steps in the biosynthesis of oxygenated monoterpenes in the respective species, and the responsible enzyme systems meet most of the established criteria for cytochrome P450-dependent mixed function oxygenases. The reactions catalyzed are completely regiospecific and, while exhibiting only a modest degree of enantioselectivity, are highly specific for limonene as substrate. Of numerous monoterpene olefins tested, including several positional isomers of limonene, only the 8,9-dihydro analog served as an alternate substrate for ring (C-3 and C-6) hydroxylation, but not side chain (C-7) hydroxylation. In addition to the regiospecificity of the allylic hydroxylation, these enzymes are also readily distinguishable based on differential inhibition by substituted imidazoles. << Less

  Arch Biochem Biophys 276:219-226(1990) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Molecular evaluation of a spearmint mutant altered in the expression of limonene hydroxylases that direct essential oil monoterpene biosynthesis.

  Bertea C., Schalk M., Mau C.J.D., Karp F., Wildung M.R., Croteau R.

  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 ... >> More

  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. << Less

  Phytochemistry 64:1203-1211(2003) [PubMed] [EuropePMC]

• Hydroxylation of limonene enantiomers and analogs by recombinant (-)-limonene 3- and 6-hydroxylases from mint (Mentha) species: evidence for catalysis within sterically constrained active sites.

  Wuest M., Little D.B., Schalk M., Croteau R.

  Limonene enantiomers and substrate analogs, including specifically fluorinated derivatives, were utilized to probe active site interactions with recombinant (-)-(4S)-limonene-3-hydroxylase (CYP71D13) and (-)-(4S)-limonene-6-hydroxylase (CYP71D18) from mint (Mentha) species. (-)-(4S)-Limonene is hy ... >> More

  Limonene enantiomers and substrate analogs, including specifically fluorinated derivatives, were utilized to probe active site interactions with recombinant (-)-(4S)-limonene-3-hydroxylase (CYP71D13) and (-)-(4S)-limonene-6-hydroxylase (CYP71D18) from mint (Mentha) species. (-)-(4S)-Limonene is hydroxylated by both enzymes at the designated C3- and C6-allylic positions, with strict regio- and stereospecificity and without detectable allylic rearrangement, to give the corresponding products (-)-trans-isopiperitenol and (-)-trans-carveol. CYP71D13-catalyzed hydroxylation of (+)-(4R)-limonene also yields the corresponding trans-3-hydroxylated product ((+)-transisopiperitenol); however, the C6-hydroxylase converts (+)-(4R)-limonene to a completely different product profile dominated by the enantiopure cis-6-hydroxylated product (+)-cis-carveol along with several minor products, including both enantiomers of the trans-6-hydroxylated product ((+/-)-trans-carveol), indicating allylic rearrangement during catalysis. These results demonstrate that the regiospecificity and facial stereochemistry of oxygen insertion is dictated by the absolute configuration of the substrate. Fluorinated limonene analogs are also tightly bound by both enzymes and hydroxylated at the topologically congruent positions in spite of the polarizing effect of the fluorine atom on substrate reactivity. This strict retention of oxygenation geometry suggests a rigid substrate orientation imposed by multiple hydrophobic active site contacts. Structurally simplified substrate analogs are hydroxylated at slower rates and with substantial loss of regiospecificity, consistent with a loss of active site complementarity. Evaluation of the product profiles generated allowed assessment of the role of hydrophobic contacts in orienting the substrate relative to the activated oxygen species. << Less

  Arch. Biochem. Biophys. 387:125-136(2001) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.