Theoretical Studies on the Catalytic Mechanism and Cyclization Pattern of Fungal Polyketide Synthase Product Template Domain

Abstract

Aromatic polyketides are pharmaceutically significant natural products. Product template (PT) domains from fungal non‐reducing polyketide synthases (NR‐PKSs) control the regioselective aldol cyclization of highly reactive polyketide intermediates, whose instabilities have hindered experimental studies. The molecular basis of PT‐catalyzed cyclization and aromatization remain ambiguous. Herein, we conducted MD simulations and QM/MM calculations to explore the catalytic mechanism and the cyclization pattern of PksA PT specific for C4‐C9. The cyclization mediated by PksA PT is initiated by the C4 deprotonation followed by the aldol condensation in C4‐C9 register. PTs which evolve from ancient PKS dehydratases (DHs) perform dehydration reactions to aromatize the cyclized intermediates. In PksA PT, the catalytic His1345 and Asp1543 are responsible for aldol cyclization and aromatization steps, respectively. A keto‐enol tautomerism at C3 assists in the elimination reaction catalyzed by Asp1543 with a water molecule network, different from most DHs of PKSs. The C2‐C7 aldol cyclization in PksA PT can occur, but the subsequent dehydration step is kinetically unfeasible and thermodynamically unfavorable, suggesting that the aromatization steps determine the C4‐C9 cyclization mode of PksA PT. Overall, these computational investigations provide detailed mechanistic insights into the regiospecific intramolecular cyclization and aromatization controlled by fungal PksA PT.