Chalcone synthase (CHS) and stilbene synthase (STS) catalyse condensation reactions of p-coumaroyl-CoA and three C2 units from malonyl-CoA up to a common tetraketide intermediate but then catalyse different cyclization reactions to produce naringenin chalcone and resveratrol respectively. On the basis of sequence alignment with other condensing enzymes including 3-ketoacyl-(acyl carrier protein) synthases of polyketide and fatty-acid synthases, site-directed mutagenesis was performed on the active-site G372FGPG loops in CHS and STS. The CHS-P375G mutant showed a 6-fold decrease in overall condensing activity with selectively increased production of p-coumaroyltriacetic acid lactone (CTAL, the derailment product of the tetraketide intermediate). Meanwhile, resveratrol production by STS-P375G strongly decreased to give various products in the order CTAL > resveratrol≈ bisnoryangonin > naringenin. As a result, naringenin production (cross-reaction) by STS-P375G was close to 30% of resveratrol production. Both G374L mutants of CHS and STS showed no condensing activity with residual malonyl-CoA decarboxylase activity. These results suggested that the G372FGPG loop in CHS and STS contribute to a determination of the outcome during cyclization reactions by serving as a part of the active-site scaffold on which the stereochemistry of cyclization is performed. These observations provide the first biochemical indication that cyclization reactions are modulated by active-site geometry. The implications for the evolutionary relationship of these enzymes are also discussed.

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