Structural Basis of Starter-Substrate Selectivity Governed by a Single Residue in Orcinol Synthase

Orcinol synthase (RdORS) from Rhododendron dauricum is a plant type III polyketide synthase involved in the biosynthesis of orsellinic acid-derived metabolites. In contrast to the related Cannabis sativa tetraketide synthase (CsTKS), which preferentially accepts medium-chain acyl-CoAs, RdORS selectively utilizes short-chain starter substrates. Here, we investigated the structural basis underlying this substrate selectivity by combining X-ray crystallography, mutational analysis, and biochemical characterization. The crystal structure of RdORS revealed that its catalytic cavity is substantially smaller than that of CsTKS because of a bulky tryptophan (Trp) 357 residue positioned at the cavity bottom. In vitro enzymatic assays demonstrated that wild-type RdORS efficiently generated tetraketide-derived products from acetyl- and butyryl-CoAs with three malonyl-CoAs, whereas productive tetraketide formation progressively diminished as starter-substrate chain length increased. Structural analysis of the RdORS Trp357S mutant revealed marked cavity expansion without perturbation of the overall catalytic framework. Correspondingly, the Trp357S substitution enabled RdORS to utilize medium-chain acyl-CoAs up to decanoyl-CoA, thereby partially recapitulating the substrate preference of CsTKS. Thus, our results provided direct structural evidence that Trp357 is a key structural determinant underlying the distinct starter-substrate preferences of RdORS.