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

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ABSTRACT

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.

No MeSH data available.


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Analysis of reaction products afforded by the recombinant ORS from acetyl-CoA and malonyl-CoA. (A) HPLC elution profiles of (a) the standard reaction using 20 μg of ORS, (b) the control reaction with heat-denatured ORS, and (c–e) the reactions using 20 μg (∼0.42 nmol) of ORS, along with 7, 20, or 50 μg (∼0.58, 1.7, and 4.2 nmol) of C. sativa OAC, respectively. The percentages of each product under standard assay conditions are shown in the parentheses. Note that each product has different molar extinction coefficients, and thus the peak intensities are not equal to the product percentages in the parentheses. (B) Orcinol and OSA product amounts from reactions without or with the indicated amounts of C. sativa OAC. Data are means ± SD of triplicate determinations. (C) OAC-dependent orcinol decrease and OSA increase, as compared with the product amounts under standard assay conditions. Data are means ± SD of triplicate determinations. n.s., not significant by Student’s t-test (P > 0.05).
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Figure 7: Analysis of reaction products afforded by the recombinant ORS from acetyl-CoA and malonyl-CoA. (A) HPLC elution profiles of (a) the standard reaction using 20 μg of ORS, (b) the control reaction with heat-denatured ORS, and (c–e) the reactions using 20 μg (∼0.42 nmol) of ORS, along with 7, 20, or 50 μg (∼0.58, 1.7, and 4.2 nmol) of C. sativa OAC, respectively. The percentages of each product under standard assay conditions are shown in the parentheses. Note that each product has different molar extinction coefficients, and thus the peak intensities are not equal to the product percentages in the parentheses. (B) Orcinol and OSA product amounts from reactions without or with the indicated amounts of C. sativa OAC. Data are means ± SD of triplicate determinations. (C) OAC-dependent orcinol decrease and OSA increase, as compared with the product amounts under standard assay conditions. Data are means ± SD of triplicate determinations. n.s., not significant by Student’s t-test (P > 0.05).

Mentions: In contrast to CHS, an HPLC analysis confirmed that the recombinant ORS produced five reaction products (products 1–5) from acetyl-CoA and malonyl-CoA (Figure 7A). The LC-ESI-MS analysis of the reaction mixture identified these products as tetraacetic acid lactone (1), triacetic acid lactone (2), orcinol (3), OSA (4), and phloroacetophenone (5), based on direct comparisons of their retention time, HR-MS, MS/MS, and UV-VIS data to those obtained for authentic samples, as listed in Supplementary Table 3. The ratio of the products under the standard conditions was 2.5% (1), 10.7% (2), 83.4% (3), 3.0% (4), and 0.4% (5), respectively, suggesting that ORS predominantly produces orcinol (3). In the ORS reaction, orcinol is not the decarboxylation product of enzymatically synthesized OSA, since the product ratio between oricinol and OSA was always constant regardless of the incubation period. It should be noted that ORS is the first plant type III PKS that produces aromatic polyketides from an acetate-derived tetraketide (methyl tetra-β-ketide) intermediate. Although ORS contains the T197G substitution that could affect the polyketide size (Abe et al., 2005), we could not detect large reaction products, such as pentaketides, in the ORS reaction mixture.


A Novel Class of Plant Type III Polyketide Synthase Involved in Orsellinic Acid Biosynthesis from Rhododendron dauricum
Analysis of reaction products afforded by the recombinant ORS from acetyl-CoA and malonyl-CoA. (A) HPLC elution profiles of (a) the standard reaction using 20 μg of ORS, (b) the control reaction with heat-denatured ORS, and (c–e) the reactions using 20 μg (∼0.42 nmol) of ORS, along with 7, 20, or 50 μg (∼0.58, 1.7, and 4.2 nmol) of C. sativa OAC, respectively. The percentages of each product under standard assay conditions are shown in the parentheses. Note that each product has different molar extinction coefficients, and thus the peak intensities are not equal to the product percentages in the parentheses. (B) Orcinol and OSA product amounts from reactions without or with the indicated amounts of C. sativa OAC. Data are means ± SD of triplicate determinations. (C) OAC-dependent orcinol decrease and OSA increase, as compared with the product amounts under standard assay conditions. Data are means ± SD of triplicate determinations. n.s., not significant by Student’s t-test (P > 0.05).
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Figure 7: Analysis of reaction products afforded by the recombinant ORS from acetyl-CoA and malonyl-CoA. (A) HPLC elution profiles of (a) the standard reaction using 20 μg of ORS, (b) the control reaction with heat-denatured ORS, and (c–e) the reactions using 20 μg (∼0.42 nmol) of ORS, along with 7, 20, or 50 μg (∼0.58, 1.7, and 4.2 nmol) of C. sativa OAC, respectively. The percentages of each product under standard assay conditions are shown in the parentheses. Note that each product has different molar extinction coefficients, and thus the peak intensities are not equal to the product percentages in the parentheses. (B) Orcinol and OSA product amounts from reactions without or with the indicated amounts of C. sativa OAC. Data are means ± SD of triplicate determinations. (C) OAC-dependent orcinol decrease and OSA increase, as compared with the product amounts under standard assay conditions. Data are means ± SD of triplicate determinations. n.s., not significant by Student’s t-test (P > 0.05).
Mentions: In contrast to CHS, an HPLC analysis confirmed that the recombinant ORS produced five reaction products (products 1–5) from acetyl-CoA and malonyl-CoA (Figure 7A). The LC-ESI-MS analysis of the reaction mixture identified these products as tetraacetic acid lactone (1), triacetic acid lactone (2), orcinol (3), OSA (4), and phloroacetophenone (5), based on direct comparisons of their retention time, HR-MS, MS/MS, and UV-VIS data to those obtained for authentic samples, as listed in Supplementary Table 3. The ratio of the products under the standard conditions was 2.5% (1), 10.7% (2), 83.4% (3), 3.0% (4), and 0.4% (5), respectively, suggesting that ORS predominantly produces orcinol (3). In the ORS reaction, orcinol is not the decarboxylation product of enzymatically synthesized OSA, since the product ratio between oricinol and OSA was always constant regardless of the incubation period. It should be noted that ORS is the first plant type III PKS that produces aromatic polyketides from an acetate-derived tetraketide (methyl tetra-β-ketide) intermediate. Although ORS contains the T197G substitution that could affect the polyketide size (Abe et al., 2005), we could not detect large reaction products, such as pentaketides, in the ORS reaction mixture.

View Article: PubMed Central - PubMed

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus