biostudies-literatureSato MNIGMS NIH HHS223-232https://www.ebi.ac.uk/biostudies/studies/S-EPMC9302879biostudies-literatureUnknown4(3)We have previously reported the identification of CghA, a proposed Diels-Alderase responsible for the formation of the bicyclic octalin core of the fungal secondary metabolite Sch210972. Here we show the crystal structure of the CghA-product complex at a resolution of 2.0 Å. Our result provides the second structural determination of eukaryotic Diels-Alderases and adds yet another fold to the family of proteins reported to catalyse [4 + 2] cycloaddition reactions. Site-directed mutagenesis-coupled kinetic characterization and computational analyses allowed us to identify key catalytic residues and propose a possible catalytic mechanism. Most interestingly, we were able to rationally engineer CghA such that the mutant was able to catalyse preferentially the formation of the energetically disfavoured <i>exo</i> adduct. This work expands our knowledge and understanding of the emerging and potentially widespread class of natural enzymes capable of catalysing stereoselective Diels-Alder reactions and paves the way towards developing enzymes potentially useful in various bio/synthetic applications.Nature catalysisCatalytic mechanism and <i>endo</i>-to-<i>exo</i> selectivity reversion of an octalin-forming natural Diels-Alderase.PMC9302879R01 GM124480Tsunematsu YMaeda NSato MYokoyama MHara KHouk KNWatanabe KNarita KKishimoto STang YJamieson CSHashimoto HfalseCatalytic mechanism and <i>endo</i>-to-<i>exo</i> selectivity reversion of an octalin-forming natural Diels-Alderase.We have previously reported the identification of CghA, a proposed Diels-Alderase responsible for the formation of the bicyclic octalin core of the fungal secondary metabolite Sch210972. Here we show the crystal structure of the CghA-product complex at a resolution of 2.0 Å. Our result provides the second structural determination of eukaryotic Diels-Alderases and adds yet another fold to the family of proteins reported to catalyse [4 + 2] cycloaddition reactions. Site-directed mutagenesis-coupled kinetic characterization and computational analyses allowed us to identify key catalytic residues and propose a possible catalytic mechanism. Most interestingly, we were able to rationally engineer CghA such that the mutant was able to catalyse preferentially the formation of the energetically disfavoured <i>exo</i> adduct. This work expands our knowledge and understanding of the emerging and potentially widespread class of natural enzymes capable of catalysing stereoselective Diels-Alder reactions and paves the way towards developing enzymes potentially useful in various bio/synthetic applications.2021-01-01T00:00:00Z2021 Mar2025-04-25T19:45:12.517Z2025-04-06T08:09:50.147ZS-EPMC93028793587353210.1038/s41929-021-00577-2