Molecular Insights into Bifunctional Ambruticin DH3 for Substrate Specificity and Catalytic Mechanism

Abstract Dehydratase (DH), a domain located at polyketide synthase (PKS) modules, commonly catalyzes the dehydration of β ‐hydroxy to an α , β ‐unsaturated acyl intermediate. As a unique bifunctional dehydratase, AmbDH3 (the DH domain of module 3 of the ambruticin PKS) is verified to be responsible for both dehydration and the following pyran‐forming cyclization. Besides, in vitro studies showed that its catalytic efficiency varies with different chiral substrates. However, the detailed molecular mechanism of AmbDH3 remains unclear. In this work, the structural rationale for the substrate specificity ( 2R/2S ‐ and 6R/6S ‐substrates) in AmbDH3 was elucidated and the complete reaction pathways including dehydration and cyclization were presented. Both MD simulations and binding free energy calculations indicated AmbDH3 had a stronger preference for 2R ‐substrates ( 2R6R ‐ 2 , 2R6S ‐ 3 ) than 2S ‐substrates ( 2S6R ‐ 1 ), and residue H51 and G61 around the catalytic pocket were emphasized by forming stable hydrogen bonds with 2R ‐substrates. In addition, AmbDH3’s mild tolerance at C6 was explained by comparison of substrate conformation and hydrogen bond network in 6S ‐ and 6R ‐substrate systems. The QM/MM results supported a consecutive one‐base dehydration and cyclization mechanism for 2R6S ‐ 3 substrate with the energy barrier of 25.2 kcal mol −1 and 24.5 kcal mol −1 , respectively. Our computational results uncover the substrate recognition and catalytic process of the first bifunctional dehydratase‐cyclase AmbDH3, which will shed light on the application of multifunctional DH domains in PKSs for diverse natural product analogs and benefit the chemoenzymatic synthesis of stereoselective pyran‐containing products.