A single amino acid substitution (F363I) converts the regiochemistry of the spearmint (−)-limonene hydroxylase from a C6- to a C3-hydroxylase

Abstract

The essential oils of peppermint and spearmint are distinguished by the position of oxygenation on the constituent p-menthane monoterpenes. Peppermint produces monoterpenes bearing an oxygen at C3, whereas spearmint produces monoterpenes bearing an oxygen at C6. Branching of the monoterpene biosynthetic pathways in these species is determined by two distinct cytochrome P450s that catalyze the regiospecific hydroxylation of (؊)-4S-limonene at C3 or C6 exclusively. cDNAs encoding the limonene-3-hydroxylase from peppermint and the limonene-6-hydroxylase from spearmint have been isolated, shown to be 70% identical at the amino acid level, and functionally expressed. A combination of domain swapping and reciprocal site-directed mutagenesis between these two enzymes demonstrated that the exchange of a single residue (F363I) in the spearmint limonene-6-hydroxylase led to complete conversion to the regiospecificity and catalytic efficiency of the peppermint limonene-3-hydroxylase.S pearmint (Mentha spicata) and peppermint (M. x piperita) are closely related mint species used for the commercial production of essential oils, the distinguishing characteristics of which are due to the position of oxygenation of the constituent p-menthane monoterpenes. Peppermint produces almost exclusively monoterpenes bearing an oxygen function at C3 [such as (Ϫ)-menthol responsible for the cooling sensation of peppermint], whereas spearmint produces almost exclusively monoterpenes bearing an oxygen function at C6 [such as (Ϫ)-carvone responsible for the typical spearmint note] (1) (Fig. 1). The regiospecificity of oxygenation is established very early in the monoterpene biosynthetic pathway in which the common olefinic precursor (Ϫ)-4S-limonene is hydroxylated exclusively at C3 to yield (Ϫ)-trans-isopiperitenol, which is subsequently transformed to the complex mixture of peppermint oil components (2). In spearmint, limonene is hydroxylated exclusively at C6 to yield (Ϫ)-trans-carveol and, following oxidation, (Ϫ)-carvone (2). These regiospecific hydroxylations are mediated by two distinct cytochrome P450 oxygenases that have been isolated from the epidermal oil glands of the respective mints and characterized (3).Recently, a reverse genetic approach allowed isolation of a cDNA encoding the spearmint limonene-6-hydroxylase (designated SM12, assigned as CYP71D18) and two closely related cDNAs encoding the peppermint limonene-3-hydroxylase (PM2͞CYP71D15 and PM17͞CYP71D13) from the corresponding, highly enriched mint oil gland cDNA libraries, and all three clones were functionally expressed using the baculovirusSpodoptera system (4). The two limonene-3-hydroxylases are 93% identical to each other in deduced amino acid sequence and exhibit 70% identity with the limonene-6-hydroxylase, and all three were assigned to the CYP71D subfamily of cytochrome P450 genes (5). Subsequent heterologous expression in Escherichia coli and Saccharomyces cerevisiae (6) has provided high yields of the functional enzymes to allow more detailed characterization of...

Comparative molecular modeling of Anopheles gambiae CYP6Z1, a mosquito P450 capable of metabolizing DDT

“…As reviewed for vertebrate P450s (43), the closely related pairs of CYP2A4 and CYP2A5, CYP2C4 and CYP2C5, and CYP3A4 and CYP3A5 with sequence identities of 98%, Ͼ95%, and Ͼ85%, respectively, do not metabolize the same substrates with similar rates or regioselectivities. Among plant P450s, spearmint CYP71D15 and peppermint CYP71D18 have different regiospecificities for limonene hydroxylation because of a single amino acid difference within the catalytic site (F363I in SRS5) (44,45). The closely related lepidopteran CYP6B1 and CYP6B3 also differ in their metabolism of plant allelochemicals (46).…”

Kinetic and Molecular Analysis of 5-Epiaristolochene 1,3-Dihydroxylase, a Cytochrome P450 Enzyme Catalyzing Successive Hydroxylations of Sesquiterpenes

“…SRS-5 and SRS-6 were of particular interest given their proximity to the active site in several P450 structures (51) and because specific amino acid positions within these regions have previously been correlated with regio-and stereospecific reaction mechanisms (Fig. 4A) (52)(53)(54)(55)(56)(57)(58)(59)(60)(61). A homology model of EAH derived from comparison with the mammalian CYP2C5 structure was constructed (Fig.…”

“…Swapping this region between two highly homologous plant P450 enzymes, limonene 6-hydroxylase and limonene 3-hydroxylase, followed by reciprocal site-directed mutagenesis, demonstrated that the corresponding position also contributes to the regiospecificity of monoterpene hydroxylation. Exchange of Phe 363 with Ile changes the regiospecificity of limonene 6-hydroxylase (CYP71D18) from hydroxylating limonene at C-6 to C-3, but the reciprocal mutation, I363F, completely inactivates limonene 3-hydroxylase activity (56). A similar modification of the corresponding site (position 371) in cinnamate hydroxylase (CYP73A1), another plant-specific P450 family, from Ile to Phe leads to a dramatic decrease in substrate binding and enzymatic activity, but without a major perturbation of protein folding (57).…”

“…Sequence alignments of P450 enzymes, especially those representing the SRS regions (50), active-site residues (69), and those alignments between highly homologous but functionally distinct enzymes (56), have proven extremely useful in identifying structural features and amino acids contributing to such specificities of these enzymes. Elucidation of such specificities in the successive hydroxylation reactions of P450 enzymes such as EAH should also benefit from this comparative type of approach.…”

Determinants governing the CYP74 catalysis: Conversion of allene oxide synthase into hydroperoxide lyase by site‐directed mutagenesis

“…The previously described P450 mutant forms [19][20][21][22][23][24], including the MtHPL mutant [18], possessed some alterations like the change of substrate specificity or regiospecificity, but not the qualitative transformations of catalysis (like the conversion of dehydrase (AOS) into isomerase (HPL) in the present work). ysis, either loses one hydrogen atom, affording the allene oxide (WT enzyme), or undergoes the rearrangement into the isomeric radical, which recombines with hydroxyl radical to form the hemiacetal, a primary HPL product (F295I and S297A mutant forms).…”