Molecular basis for two stereoselective Diels-Alderases that produce decalin skeletons

Abstract

Enzymes catalyzing [4+2] cycloadditions are involved in the formation of complex structures found in natural products, and play key roles in the control of stereochemistry. Fsa2 and Phm7 catalyze intramolecular [4+2] cycloaddition to form enantiomeric decalin scaffolds during the biosynthesis of HIV-1 integrase inhibitors, equisetin, and an opposite stereochemical homolog, phomasetin. Here, we solved the X-ray crystal structures of substrate-free Fsa2 and Phm7, and an inhibitor-bound Phm7 to understand the molecular basis underlying stereoselective cycloaddition reactions. Based on the crystal structures, docking simulations followed by all-atom molecular dynamics simulations provided binding models demonstrating the folding of linear polyenoyl tetramic acid substrates in the binding pocket of these enzymes, which explain the stereoselectivity in the construction of decalin scaffolds. Site-directed mutagenesis studies verified the binding models and, in combination with density functional theory calculations, clarified how hydrophilic amino acid residues in the Phm7 pocket regulate and catalyze the stereoselective cycloaddition. This powerful combination of experimental and theoretical approaches highlights the distinct molecular mechanisms of enzyme-mediated [4+2] cycloaddition and its stereoselectivity.

Discovery of fungal onoceroid triterpenoids through domainless enzyme-targeted global genome mining

“…These domainless proteins were divided into groups based on their sequence similarity, and their hidden Markov model (HMM) profiles were generated for HMMER 11 analysis. The representative members of these protein groups are the terpene cyclase Pyr4, 12 the Diels-Alderase Fsa2, 13 the hetero Diels-Alderase AsR5, 14 the epoxide hydrolase CtvD, 15 and the isomerase Trt14. 16 Furthermore, several additional HMM profiles were created for the domains of polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs).…”