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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.


SDS-PAGE analysis of the purified recombinant PKSs. M, molecular mass standards, (1) the purified ORS (3 μg), (2) total soluble proteins from E. coli expressing ORS, (3) the purified CHS (3 μg), (4) total soluble proteins from E. coli expressing CHS.
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Figure 6: SDS-PAGE analysis of the purified recombinant PKSs. M, molecular mass standards, (1) the purified ORS (3 μg), (2) total soluble proteins from E. coli expressing ORS, (3) the purified CHS (3 μg), (4) total soluble proteins from E. coli expressing CHS.

Mentions: The recombinant ORS and CHS were bacterially expressed and purified, to evaluate their catalytic functions. The SDS-PAGE analysis demonstrated that the recombinant enzymes were purified as homogeneous proteins with molecular masses of ∼48 and ∼45 kDa (Figure 6), which were suitable sizes for hexahistidine-tagged ORS and CHS, respectively. In addition, the gel filtration chromatographies estimated the native molecular masses of ORS and CHS to be ∼101 and ∼94 kDa, respectively, suggesting that the recombinant enzymes are homodimers, as in the cases of known type III PKSs (Austin and Noel, 2003; Abe and Morita, 2010).


A Novel Class of Plant Type III Polyketide Synthase Involved in Orsellinic Acid Biosynthesis from Rhododendron dauricum
SDS-PAGE analysis of the purified recombinant PKSs. M, molecular mass standards, (1) the purified ORS (3 μg), (2) total soluble proteins from E. coli expressing ORS, (3) the purified CHS (3 μg), (4) total soluble proteins from E. coli expressing CHS.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037138&req=5

Figure 6: SDS-PAGE analysis of the purified recombinant PKSs. M, molecular mass standards, (1) the purified ORS (3 μg), (2) total soluble proteins from E. coli expressing ORS, (3) the purified CHS (3 μg), (4) total soluble proteins from E. coli expressing CHS.
Mentions: The recombinant ORS and CHS were bacterially expressed and purified, to evaluate their catalytic functions. The SDS-PAGE analysis demonstrated that the recombinant enzymes were purified as homogeneous proteins with molecular masses of ∼48 and ∼45 kDa (Figure 6), which were suitable sizes for hexahistidine-tagged ORS and CHS, respectively. In addition, the gel filtration chromatographies estimated the native molecular masses of ORS and CHS to be ∼101 and ∼94 kDa, respectively, suggesting that the recombinant enzymes are homodimers, as in the cases of known type III PKSs (Austin and Noel, 2003; Abe and Morita, 2010).

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.