Functional Expression of Regiospecific Cytochrome P450 Limonene Hydroxylases from Mint (Mentha spp.) in Escherichia coli and Saccharomyces cerevisiae

The oxygenation pattern of the essential oil monoterpenes of commercial mint (Mentha) species is determined by regiospecific cytochrome P450-catalyzed hydroxylation of the common olefinic precursor (−)-4S-limonene. In spearmint (M. spicata), C6-allylic hydroxylation leads to (−)-trans-carveol and thence (−)-carvone, whereas in peppermint (M. × piperita), C3-allylic hydroxylation leads to (−)-trans-isopiperitenol and ultimately (−)-menthol. cDNAs encoding the C6-hydroxylase and C3-hydroxylase from spearmint and peppermint, respectively, were isolated by a combination of reverse genetic and homology-based cloning methods (S. Lupien, F. Karp, M. Wildung, and R. Croteau, Arch. Biochem. Biophys. 368, 181–192, 1999). Although both hydroxylase genes were confirmed by functional expression using the baculovirus–Spodoptera system, too little protein was available by this approach to permit detailed study of the structure–function relationships of these catalysts, especially the substrate binding determinants that underlie the regiochemistry and stereochemistry of the reactions. Therefore, heterologous overexpression systems based on Escherichia coli and Saccharomyces cerevisiae were developed to produce several N-terminally modified versions of the recombinant hydroxylases. Ancillary methods for the solubilization, purification, and reconstitution (with recombinant spearmint cytochrome P450 reductase) of the limonene hydroxylases were also devised, with which substrate binding behavior and precise regiochemistry and stereochemistry of product formation were determined.