An Aromatic Farnesyltransferase Functions in Biosynthesis of the Anti-HIV Meroterpenoid Daurichromenic Acid.

Rhododendron dauricum produces daurichromenic acid, an anti-HIV meroterpenoid, via oxidative cyclization of the farnesyl group of grifolic acid. The prenyltransferase (PT) that synthesizes grifolic acid is a farnesyltransferase in plant specialized metabolism. In this study, we demonstrated that the isoprenoid moiety of grifolic acid is derived from the 2-C-methyl-d-erythritol-4-phosphate pathway that takes place in plastids. We explored candidate sequences of plastid-localized PT homologs and identified a cDNA for this PT, RdPT1, which shares moderate sequence similarity with known aromatic PTs. RdPT1 is expressed exclusively in the glandular scales, where daurichromenic acid accumulates. In addition, the gene product was targeted to plastids in plant cells. The recombinant RdPT1 regiospecifically synthesized grifolic acid from orsellinic acid and farnesyl diphosphate, demonstrating that RdPT1 is the farnesyltransferase involved in daurichromenic acid biosynthesis. This enzyme strictly preferred orsellinic acid as a prenyl acceptor, whereas it had a relaxed specificity for prenyl donor structures, also accepting geranyl and geranylgeranyl diphosphates with modest efficiency to synthesize prenyl chain analogs of grifolic acid. Such a broad specificity is a unique catalytic feature of RdPT1 that is not shared among secondary metabolic aromatic PTs in plants. We discuss the unusual substrate preference of RdPT1 using a molecular modeling approach. The biochemical properties as well as the localization of RdPT1 suggest that this enzyme produces meroterpenoids in glandular scales cooperatively with previously identified daurichromenic acid synthase, probably for chemical defense on the surface of R. dauricum plants.

Daurichromenic acid and grifolic acid: Phytotoxic meroterpenoids that induce cell death in cell culture of their producer Rhododendron dauricum.

Daurichromenic acid (DCA) is a meroterpenoid with anti-HIV activities that is isolated from Rhododendron dauricum L. We recently reported that DCA is biosynthesized and accumulated in the apoplast of glandular scales attached on the surface of young leaves of R. dauricum. In the present study, we confirmed that a cell suspension culture of R. dauricum could not produce DCA and its precursor grifolic acid even after elicitation with methyl jasmonate and ß-cyclodextrin. In addition, exogenous supplementation of DCA and grifolic acid effectively induced cell death in the same culture, with apoptosis-associated phenomena such as cytoplasmic shrinkage, chromatin condensation, and genomic DNA degradation. These findings suggested that DCA and grifolic acid are phytotoxic metabolites that have to be sequestered in the apoplast to avoid self-poisoning.

Identification and Characterization of Daurichromenic Acid Synthase Active in Anti-HIV Biosynthesis.

Daurichromenic acid (DCA) synthase catalyzes the oxidative cyclization of grifolic acid to produce DCA, an anti-HIV meroterpenoid isolated from Rhododendron dauricum We identified a novel cDNA encoding DCA synthase by transcriptome-based screening from young leaves of R. dauricum The gene coded for a 533-amino acid polypeptide with moderate homologies to flavin adenine dinucleotide oxidases from other plants. The primary structure contained an amino-terminal signal peptide and conserved amino acid residues to form bicovalent linkage to the flavin adenine dinucleotide isoalloxazine ring at histidine-112 and cysteine-175. In addition, the recombinant DCA synthase, purified from the culture supernatant of transgenic Pichia pastoris, exhibited structural and functional properties as a flavoprotein. The reaction mechanism of DCA synthase characterized herein partly shares a similarity with those of cannabinoid synthases from Cannabis sativa, whereas DCA synthase catalyzes a novel cyclization reaction of the farnesyl moiety of a meroterpenoid natural product of plant origin. Moreover, in this study, we present evidence that DCA is biosynthesized and accumulated specifically in the glandular scales, on the surface of R. dauricum plants, based on various analytical studies at the chemical, biochemical, and molecular levels. The extracellular localization of DCA also was confirmed by a confocal microscopic analysis of its autofluorescence. These data highlight the unique feature of DCA: the final step of biosynthesis is completed in apoplastic space, and it is highly accumulated outside the scale cells.

Combinatorial Biosynthesis of (+)-Daurichromenic Acid and Its Halogenated Analogue.

Daurichromenic acid is a meroterpenoid with various pharmacological activities that is biosynthesized from grifolic acid in Rhododendron dauricum. Heterologous expression of grifolic acid synthases from Stachybotrys bisbyi and a daurichromenic acid synthase from R. dauricum in Aspergillus oryzae mediated three-step combinatorial biosynthesis of (+)-daurichromenic acid through enantioselective 6-endo-trig cyclization. Additional introduction of a halogenase from Fusarium sp. into the strain resulted in the biosynthesis of (+)-5-chlorodaurichromenic acid, which exceeds the antibacterial activity of the original compounds.

A Novel Class of Plant Type III Polyketide Synthase Involved in Orsellinic Acid Biosynthesis from Rhododendron dauricum.

Rhododendron dauricum L. produces daurichromenic acid, the anti-HIV meroterpenoid consisting of sesquiterpene and orsellinic acid (OSA) moieties. To characterize the enzyme responsible for OSA biosynthesis, a cDNA encoding a novel polyketide synthase (PKS), orcinol synthase (ORS), was cloned from young leaves of R. dauricum. The primary structure of ORS shared relatively low identities to those of PKSs from other plants, and the active site of ORS had a unique amino acid composition. The bacterially expressed, recombinant ORS accepted acetyl-CoA as the preferable starter substrate, and produced orcinol as the major reaction product, along with four minor products including OSA. The ORS identified in this study is the first plant PKS that generates acetate-derived aromatic tetraketides, such as orcinol and OSA. Interestingly, OSA production was clearly enhanced in the presence of Cannabis sativa olivetolic acid cyclase, suggesting that the ORS is involved in OSA biosynthesis together with an unidentified cyclase in R. dauricum.

Characterization of 12-Oxophytodienoic Acid Reductases from Rose-scented Geranium (Pelargonium graveolens).

Pelargonium graveolens L'Hér, also referred to as rose geranium, is a popular herbal plant with typical rosy fragrance largely based on the blend of monoterpenoid constituents. Among them, citronellol, which is biosynthesized from geraniol via double bond reduction, is the most abundant scent compound. In this study, three 12-oxophytodienoic acid reductases (PgOPRl-3) hive been cloned from P. graveolens, as -possible candidates for the double-bond reductase involved in citronellol biosynthesis. The bacterially expressed recombinant PgOPRs did not reduce geraniol to citronellol, but stereoselectively converted citral into (S)-citronellal in the presence of NADPH. Thus, the a,-unsaturated carbonyl moiety in the substrate is essential for the catalytic activity of PgOPRs; as reported for OPRs from other plants and structurally related yeast old yellow enzymes. PgOPRs promiscuously accepted linear and cyclic α,ß- uisaturated carbonyl substrates, including methacrolein, a typical reactive carbonyl compound. The possible biotechnological applications for PgOPRs in plant metabolic'engineering, based on their catalytic properties, are discussed herein.

Daurichromenic acid-producing oxidocyclase in the young leaves of Rhododendron dauricum.

Rhododendron dauricum L., a flowering tree popular in Hokkaido, produces daurichromenic acid (DCA), a terpenophenol with a potent anti-HIV activity. The DCA-producing enzyme, named DCA synthase, could be detected in the soluble protein fraction prepared from the young leaves of R. dauricum. DCA synthase catalyzed oxidocyclization of the farnesyl group of grifolic acid to form (+)-DCA as the major reaction product. The DCA synthase reaction proceeds without the need for any cofactors and coenzymes except for molecular oxygen. Interestingly, these catalytic properties of DCA synthase are quite similar to those reported for cannabinoid synthases in the marijuana plant Cannabis sativa L.