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

Abstract

Daurichromenic acid (DCA) synthase catalyzes the oxidative cyclization of grifolic acid to produce DCA, an anti-HIV meroterpenoid isolated from We identified a novel cDNA encoding DCA synthase by transcriptome-based screening from young leaves of 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 , 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, 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 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.

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

“…The unique feature of DCA biosynthesis is relative to its tissue distribution and localization; DCA synthase is distributed exclusively in the glandular scales, the multicellular epidermal tissues covering young leaves of R. dauricum. 4 In addition, as DCA synthase is a secreted protein, the biosynthetic reaction presumably proceeds extracellularly, leading to the accumulation of DCA in the apoplast of glandular scales as we reported previously. 4 With respect to this unique extracellular biosynthesis and accumulation of DCA, we proposed the possibility that DCA would serve as a chemical defense component outside of the scale cells, the outermost layer of plants.…”

“…4 In addition, as DCA synthase is a secreted protein, the biosynthetic reaction presumably proceeds extracellularly, leading to the accumulation of DCA in the apoplast of glandular scales as we reported previously. 4 With respect to this unique extracellular biosynthesis and accumulation of DCA, we proposed the possibility that DCA would serve as a chemical defense component outside of the scale cells, the outermost layer of plants. In addition, we suspected that DCA is excluded from cells because it might be toxic to plant cells, as in the case of structurally related meroterpenoids, such as cannabinoids.…”

“…2,3 Concerning the biosynthesis of DCA, we have recently reported that DCA is produced from grifolic acid by DCA synthase, a flavoprotein oxidase with a covalently attached flavin adenine dinucleotide (FAD) cofactor. 4 DCA synthase catalyzes the oxidative cyclization of farnesyl moiety of grifolic acid, in a FAD-and oxygen-dependent manner, to produce DCA and hydrogen peroxide, as shown in Fig. 1.…”

“…15 In addition, secreted class III peroxidases, 16 commonly distributed in the plant kingdom, would participate in detoxification of exogenously added H 2 O 2 in the R. dauricum cell culture. Nevertheless, because DCA is highly accumulated in plants, 4 a large amount of H 2 O 2 would also be produced as a by-product during DCA biosynthesis. Thus, it is reasonable that DCA synthase reactions take place extracellularly in specialized tissues to avoid cellular damage by H 2 O 2 as well as DCA.…”

“…19,20 Meroterpenoids are hybrid natural products that harbor isoprenoid moieties in their molecules, many of which have characteristic biological activities. 4 It has been reported that a number of meroterpenoids, such as prenylated flavonoids, 21 stilbenoids, 9 and furanocoumarins, 22 are secreted into apoplastic compartments after their biosynthesis to avoid self-poisoning, and probably to serve as self-defense. This process can be seen in cannabinoids, as we reported previously: they possess phytotoxic properties and are localized in the storage cavities of glandular trichomes.…”