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Molecular characterization of polyketide synthases from Ginkgo biloba
Ginkgolic acid (GA), a constituent in Ginkgo biloba, is regarded as a promising drug candidate because this compound inhibits protein SUMOylation . GA is a salicylic acid derivative with a long carbon side chain, and is considered to be biosynthesized via polyketide pathway . To obtain a cDNA for a polyketide synthase (PKS) involved in GA biosynthesis, we performed degenerate PCR and rapid amplification of cDNA ends reactions. Consequently, two cDNA clones encoding PKSs, named GbPKS1 and GbPKS2, were cloned and sequenced. The amino acid sequence of GbPKS1 showed >90% identity with chalcone synthase (CHS), the ubiquitous plant PKS catalyzing the first committed step of flavonoid biosynthesis. Contrary, the amino acid identity between GbPKS2 and CHS was relatively low (˜40%), and Thr197, a conserved residue in the CHS active site, was substituted into Gly in GbPKS2. The catalytic functions of GbPKSs were characterized with bacterially expressed recombinant enzymes. As expected, the recombinant GbPKS1 catalyzed chalcone production from 4-coumaroyl-CoA and three molecules of malonyl-CoA. On the other hand, GbPKS2 did not accept 4-coumaroyl-CoA as a starter substrate, but selected palmitoleoyl-CoA, the expected starter substrate for GA biosynthetic reaction, to produce triketide pyrone via two-step condensation with malonyl-CoA. GbPKS2 did not synthesize GA, but this enzyme is of interest because long-chain acyl-CoA utilizing PKSs have been rarely found to date. In addition, RT-PCR analysis demonstrated GbPKS2 is specifically expressed in fruit pulp tissue where GA is accumulated.
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2. Austin, MB. et al. (2003) Nat. Prod. Rep. 20:79–110.