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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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Summary of the reactions catalyzed by ORS, from acetyl-CoA as a starter substrate.aIn the presence of C. sativa OAC, methyl tetra-β-ketide CoA, released from the ORS active site, undergoes aldol condensation to form a C2–C7 linkage. The following aromatization and thioester hydrolysis could take place in solution to yield OSA. In the absence of OAC, the tetraketide CoA would be spontaneously cyclized into orcinol.
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Figure 10: Summary of the reactions catalyzed by ORS, from acetyl-CoA as a starter substrate.aIn the presence of C. sativa OAC, methyl tetra-β-ketide CoA, released from the ORS active site, undergoes aldol condensation to form a C2–C7 linkage. The following aromatization and thioester hydrolysis could take place in solution to yield OSA. In the absence of OAC, the tetraketide CoA would be spontaneously cyclized into orcinol.

Mentions: The simplest scenario explaining the OAC-dependent product change is illustrated, based on the reported biochemical properties of OAC (Gagne et al., 2012; Yang et al., 2016) (Figure 10). Like C. sativa TKS (Taura et al., 2009), ORS produces and releases considerable amounts of a tetraketide (methyl tetra-β-ketide CoA), perhaps as the “real” major product. In the absence of OAC, the tetraketide is non-enzymatically cyclized to orcinol by decarboxylative aldol condensation, via a reaction scheme in the order of (1) thioester hydrolysis, (2) aldol condensation accompanied by decarboxylation, and (3) aromatization, as proposed for various alkylresorcinols and stilbene biosynthetic reactions (Austin et al., 2004; Funa et al., 2006; Taura et al., 2009; Cook et al., 2010). In contrast, OAC accepts the non-physiological substrate, methyl tetra-β-ketide CoA, to form the C2–C7 linkage before the thioester cleavage (Figure 10), as in the case of olivetolic acid biosynthesis. Since OAC lacks aromatase and thioesterase domains (Yang et al., 2016), the following aromatization and thioester hydrolysis would take place in solution, to form OSA (Figure 10). The OAC active site contains a hydrophobic pentyl-binding pocket that is important for the recognition and binding of the physiological substrate, pentyl tetra-β-ketide CoA (Yang et al., 2016). Thus, methyl tetra-β-ketide CoA, supplied by ORS, might not be a preferable substrate for OAC, and the OAC-dependent aldol condensation competes with the spontaneous orcinol formation even when an excess amount of OAC is present, as shown in Figure 7. Nevertheless, this study provided the first evidence that OAC can partly accept methyl tetra-β-ketide CoA as a substrate, suggesting the possibility that the rational modification of the OAC active site, especially the pentyl-binding pocket, based on the crystal structure (Yang et al., 2016) could create mutant OACs with novel substrate specificities. In contrast to orcinol, the production of tetraacetic acid lactone and phloroacetophenone was not affected by adding OAC (Figure 7A). These tetraketide-derived products are likely to be synthesized in the active site of ORS, rather than outside the enzyme. It is also notable that, unlike C. sativa TKS, ORS could synthesize resorcylic acid OSA in the absence of a cyclase, with a catalytic efficiency similar to those of the O. sativa ARASs. These results suggested the multifunctional nature of the ORS active site, which can catalyze both the C1–C6 Claisen and C2–C7 aldol reactions, in addition to the C1–C5oxy lactonization of the same methyl tetra-β-ketide intermediate (Figure 10).


A Novel Class of Plant Type III Polyketide Synthase Involved in Orsellinic Acid Biosynthesis from Rhododendron dauricum
Summary of the reactions catalyzed by ORS, from acetyl-CoA as a starter substrate.aIn the presence of C. sativa OAC, methyl tetra-β-ketide CoA, released from the ORS active site, undergoes aldol condensation to form a C2–C7 linkage. The following aromatization and thioester hydrolysis could take place in solution to yield OSA. In the absence of OAC, the tetraketide CoA would be spontaneously cyclized into orcinol.
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Related In: Results  -  Collection

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Figure 10: Summary of the reactions catalyzed by ORS, from acetyl-CoA as a starter substrate.aIn the presence of C. sativa OAC, methyl tetra-β-ketide CoA, released from the ORS active site, undergoes aldol condensation to form a C2–C7 linkage. The following aromatization and thioester hydrolysis could take place in solution to yield OSA. In the absence of OAC, the tetraketide CoA would be spontaneously cyclized into orcinol.
Mentions: The simplest scenario explaining the OAC-dependent product change is illustrated, based on the reported biochemical properties of OAC (Gagne et al., 2012; Yang et al., 2016) (Figure 10). Like C. sativa TKS (Taura et al., 2009), ORS produces and releases considerable amounts of a tetraketide (methyl tetra-β-ketide CoA), perhaps as the “real” major product. In the absence of OAC, the tetraketide is non-enzymatically cyclized to orcinol by decarboxylative aldol condensation, via a reaction scheme in the order of (1) thioester hydrolysis, (2) aldol condensation accompanied by decarboxylation, and (3) aromatization, as proposed for various alkylresorcinols and stilbene biosynthetic reactions (Austin et al., 2004; Funa et al., 2006; Taura et al., 2009; Cook et al., 2010). In contrast, OAC accepts the non-physiological substrate, methyl tetra-β-ketide CoA, to form the C2–C7 linkage before the thioester cleavage (Figure 10), as in the case of olivetolic acid biosynthesis. Since OAC lacks aromatase and thioesterase domains (Yang et al., 2016), the following aromatization and thioester hydrolysis would take place in solution, to form OSA (Figure 10). The OAC active site contains a hydrophobic pentyl-binding pocket that is important for the recognition and binding of the physiological substrate, pentyl tetra-β-ketide CoA (Yang et al., 2016). Thus, methyl tetra-β-ketide CoA, supplied by ORS, might not be a preferable substrate for OAC, and the OAC-dependent aldol condensation competes with the spontaneous orcinol formation even when an excess amount of OAC is present, as shown in Figure 7. Nevertheless, this study provided the first evidence that OAC can partly accept methyl tetra-β-ketide CoA as a substrate, suggesting the possibility that the rational modification of the OAC active site, especially the pentyl-binding pocket, based on the crystal structure (Yang et al., 2016) could create mutant OACs with novel substrate specificities. In contrast to orcinol, the production of tetraacetic acid lactone and phloroacetophenone was not affected by adding OAC (Figure 7A). These tetraketide-derived products are likely to be synthesized in the active site of ORS, rather than outside the enzyme. It is also notable that, unlike C. sativa TKS, ORS could synthesize resorcylic acid OSA in the absence of a cyclase, with a catalytic efficiency similar to those of the O. sativa ARASs. These results suggested the multifunctional nature of the ORS active site, which can catalyze both the C1–C6 Claisen and C2–C7 aldol reactions, in addition to the C1–C5oxy lactonization of the same methyl tetra-β-ketide intermediate (Figure 10).

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