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.
Y. Nakashima, S. Y. Y. Hnin, Subin Kim et al.· Chemical and pharmaceutical...· 0 citations
Quorum sensing in Gram-positive bacteria commonly relies on posttranslationally modified peptide pheromones. In Bacillus subtilis, the prenyltransferase ComQ catalyzes tryptophan prenylation of the quorum-sensing peptide ComX, but the structural basis of this unique peptide modification has remained unclear. Here we identified a previously uncharacterized ComQ homolog, StheQ, and its cognate peptide substrate, StheX, from Sphaerobacter thermophilus and investigated their structural and functional relationship. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis demonstrated that StheQ catalyzes prenylation of the tryptophan residue located second from the C-terminus of StheX. Crystal structures of apo StheQ and its complexes with a farnesyl pyrophosphate analog revealed that StheQ adopts the all-α-helical fold of the trans-isoprenyl diphosphate synthase (IPPS) superfamily while possessing an active-site architecture adapted for peptide-based indole prenylation. The structures identified a single Mg2+-binding site associated with the first aspartic acid-rich motif and showed no evidence for metal coordination at the pseudo-second aspartic acid-rich motif. Site-directed mutagenesis, complex formation assays, and docking analyses identified a peptide-binding pocket adjacent to the active site and suggested that N215 contributes to productive positioning of the acceptor tryptophan. These findings establish the structural basis for peptide prenylation by a ComQ-family enzyme, providing insight into the evolution of peptide-based indole prenylation within the IPPS superfamily, and support the view that ComQ-family enzymes constitute a distinct functional branch specialized for peptide modification.
