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

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.


Multiple amino acid sequence alignment of M. sativa CHS2 and R. dauricum CHS and ORS. The catalytic triad residues are colored red, and the active site residues are indicated with asterisks. The varied active site residues in ORS are colored blue. The four inserted peptide sequences a–d in ORS are highlighted by green boxes.
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Figure 2: Multiple amino acid sequence alignment of M. sativa CHS2 and R. dauricum CHS and ORS. The catalytic triad residues are colored red, and the active site residues are indicated with asterisks. The varied active site residues in ORS are colored blue. The four inserted peptide sequences a–d in ORS are highlighted by green boxes.

Mentions: The cDNA encoding a novel type III PKS, ORS, was cloned and sequenced together with the CHS cDNA from young leaves of R. dauricum, by PCR using degenerate primers and RACE strategy. The CHS cDNA contained a 1,170-bp open reading frame encoding a 389-amino acid polypeptide with a molecular mass of 42,599 Da, and shared very high levels (∼90%) of identity to known CHS sequences. The identity between R. dauricum CHS and the structurally characterized M. sativa CHS2 (Ferrer et al., 1999) was also significant (∼87.6%; Figure 2). In addition, all of the amino acid residues in the CHS active site are conserved in R. dauricum CHS (Table 1). A phylogenetic tree analysis grouped R. dauricum CHS within the CHS clade (Figure 3). Based on this sequence information, the CHS cDNA is expected to encode an active CHS in R. dauricum.


A Novel Class of Plant Type III Polyketide Synthase Involved in Orsellinic Acid Biosynthesis from Rhododendron dauricum
Multiple amino acid sequence alignment of M. sativa CHS2 and R. dauricum CHS and ORS. The catalytic triad residues are colored red, and the active site residues are indicated with asterisks. The varied active site residues in ORS are colored blue. The four inserted peptide sequences a–d in ORS are highlighted by green boxes.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Multiple amino acid sequence alignment of M. sativa CHS2 and R. dauricum CHS and ORS. The catalytic triad residues are colored red, and the active site residues are indicated with asterisks. The varied active site residues in ORS are colored blue. The four inserted peptide sequences a–d in ORS are highlighted by green boxes.
Mentions: The cDNA encoding a novel type III PKS, ORS, was cloned and sequenced together with the CHS cDNA from young leaves of R. dauricum, by PCR using degenerate primers and RACE strategy. The CHS cDNA contained a 1,170-bp open reading frame encoding a 389-amino acid polypeptide with a molecular mass of 42,599 Da, and shared very high levels (∼90%) of identity to known CHS sequences. The identity between R. dauricum CHS and the structurally characterized M. sativa CHS2 (Ferrer et al., 1999) was also significant (∼87.6%; Figure 2). In addition, all of the amino acid residues in the CHS active site are conserved in R. dauricum CHS (Table 1). A phylogenetic tree analysis grouped R. dauricum CHS within the CHS clade (Figure 3). Based on this sequence information, the CHS cDNA is expected to encode an active CHS in R. dauricum.

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.