Characterization and functional analysis of type III polyketide synthases in Selaginella moellendorffii.

Main conclusion: The evolutionary conservation of type III polyketide synthases (PKS) in Selaginella has been elucidated, and the critical amino acid residues of the anther-specific chalcone synthase-like enzyme (SmASCL) have been identified. Selaginella species are the oldest known vascular plants and a valuable resource for the study of metabolic evolution in land plants. Polyketides, especially flavonoids and sporopollenin precursors, are essential prerequisites for plant land colonization. Although type III polyketide synthases (PKS) are widely studied in seed plants, the related enzymes in Selaginella remain poorly characterized. Here, eight type III PKSs were identified in the Selaginella moellendorffii genome and classified into three clusters. Two PKSs were selected for further research based on their phylogenetic relationships and protein sequence similarity. Functional studies revealed that they were chalcone synthase (SmCHS) and anther-specific CHS-like enzyme (SmASCL). These enzymes are involved in the biosynthesis of flavonoids and sporopollenin, respectively. Their sequence information and enzymatic activity are similar to the orthologs in other plants. Phylogenetic analysis revealed that the ASCL and CHS enzymes were separated into two clades from the Bryophyta. These results suggest that CHS and ASCL emerged in the first land plants and then remained conserved during plant evolution. To study the structural basis of the enzymatic function of SmASCL, a series of mutants were constructed. The number of condensation reactions catalyzed by the P210L/Y211D and I200V/G201T double mutants exceeds that of the wild-type enzyme. Our study provides insight into the characteristics and functions of type III PKSs in S. moellendorffii. It also offers clues for a deeper understanding of the relationship between active sites and the enzymatic function of ASCLs.