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 (Me ntha) species is determined by regiospecific cytochrome P450-catalyzed hydr oxylation of the common olefinic precursor (-)-4S-limonene, In peppermint ( Mentha x piperita), C3-allylic hydroxylation leads to (-)-trans-isopiperite nol, whereas in spearmint, CG-allylic hydroxylation leads to (-)-trans-carv eol, The microsomal limonene-6-hydroxylase was purified from the oil glands of spearmint, and amino acid sequences from the homogeneous enzyme were us ed to design PCR primers with which a 500-bp amplicon was prepared. This no ndegenerate probe was employed to screen a spearmint oil gland cDNA library from which the corresponding full-length cDNA was isolated and subsequentl y confirmed as the CG-hydroxylase by functional expression using the baculo virus-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 limonene3-hydroxylase by functional expression as before. Deduced sequence analysis of these regiospecific cytochrome P450 monooxygenases indicates that both enzymes bear a typical amino-terminal m embrane anchor, consistent with the microsomal location of the native forms , exhibit calculated molecular weights of 56,149 (spearmint) and about 56,5 60 (peppermint), and are very similar in primary sequence (70% identity and 85% similarity). The availability of these regiochemically distinct, yet v ery closely related, recombinant hydroxylases and their corresponding genes provides a unique model system for understanding structure-function relati onships in cytochrome P450 substrate binding and catalysis, and a means for transgenic manipulation of monoterpene biosynthetic pathways in plants. (C ) 1999 Academic Press.