A polyheteroatomic Diels–Alderase

Abstract

Now, Yi-Lei Zhao, Mehdi Mobli and Xudong Qu report a polyheteroatomic Diels–Alderase that catalyses the intramolecular cycloaddition of a C=O diene with a C=O dienophile. The researchers had previously worked on the elucidation of the biosynthesis of the polyketide antibiotic (−)-anthrabenzoxocinone ((−)-ABX). However, the construction of the oxygen-bridged tricyclic acetal of (−)-ABX from phenyldimethylanthrone (1) remained unclear. In the present work, investigations continued with knocking out genes of the biosynthetic gene cluster and analysing the reaction products. The results indicated that 1 was stereo-specifically reduced by the enzyme Abx₍₋₎E to compound 2 followed by its subsequent stereo-specific transformation into (−)-ABX by Abx₍₋₎F (pictured). This was confirmed by in vitro studies using the purified enzymes revealing a k_cat/K_M value of 3.43 ± 0.23 min⁻¹ μM⁻¹ for Abx₍₋₎F with 2. Mechanistic experiments all pointed to Abx₍₋₎F being a bifunctional enzyme that catalyses the formation of (−)-ABX by dehydration of compound 2 to a reactive o-quinone methide intermediate (Z, Ra)-3 and a subsequent dual-oxa HDA (pictured). Structural insights were gained by nuclear magnetic resonance spectroscopy and by solving the crystal structure of Abx₍₋₎F. This enabled the identification of the active side residues, which were then mutated to probe the reaction mechanism. With this approach Asp17 was found to act as a general base that mediates the dehydration to generate the o-quinone methide intermediate (Z, Ra)-3 and residues in the enzyme’s active site were identified that control the stereochemistry in the subsequent dual-oxa HDA.

This work expands the scope of biocatalytic Diels–Alder reactions and shows the power of enzymes to control reactive intermediates and stereoselectivity. Polyheteroatomic Diels–Alderases hold promise for the sustainable synthesis of complex bioactive molecules, with implications for drug discovery.