Regiospecific Cytochrome P450 Limonene Hydroxylases from Mint (Mentha) Species: cDNA Isolation, Characterization, and Functional Expression of (−)-4S-Limonene-3-hydroxylase and (−)-4S-Limonene-6-hydroxylase

The oxygenation pattern of the cyclic monoterpenoids of commercial mint (Mentha) species is determined by regiospecific cytochrome P450-catalyzed hydroxylation of the common olefinic precursor (−)-4S-limonene. In peppermint (Mentha × piperita), C3-allylic hydroxylation leads to (−)-trans-isopiperitenol, whereas in spearmint, C6-allylic hydroxylation leads to (−)-trans-carveol. The microsomal limonene-6-hydroxylase was purified from the oil glands of spearmint, and amino acid sequences from the homogeneous enzyme were used to design PCR primers with which a 500-bp amplicon was prepared. This nondegenerate probe was employed to screen a spearmint oil gland cDNA library from which the corresponding full-length cDNA was isolated and subsequently confirmed as the C6-hydroxylase by functional expression using the baculovirus–Spodoptera system. The probe was also utilized to isolate two closely related full-length cDNA species from a peppermint oil gland cDNA library which were confirmed as the limonene-3-hydroxylase by functional expression as before. Deduced sequence analysis of these regiospecific cytochrome P450 monooxygenases indicates that both enzymes bear a typical amino-terminal membrane anchor, consistent with the microsomal location of the native forms, exhibit calculated molecular weights of 56,149 (spearmint) and about 56,560 (peppermint), and are very similar in primary sequence (70% identity and 85% similarity). The availability of these regiochemically distinct, yet very closely related, recombinant hydroxylases and their corresponding genes provides a unique model system for understanding structure–function relationships in cytochrome P450 substrate binding and catalysis, and a means for transgenic manipulation of monoterpene biosynthetic pathways in plants.