Coenzyme A Tethering Mediates Dehydratase Substrate Promiscuity and Reaction Specificity in Platensimycin, Platencin, and Platensilin Biosynthesis.

Biosynthetic studies of the platensimycin (PTM), platencin (PTN), and platensilin (PTL) family of natural products have revealed numerous insights into the chemistry and enzymology of diterpenoid biosynthesis. A deeper understanding of the PTM biosynthetic machinery would advance fundamental knowledge in natural product biosynthesis and facilitate future efforts to exploit these compounds as potential leads for biomedical applications. Herein, we report the functional characterization of the gene that encodes the dehydratase responsible for the formation of the C6-C7 enone moiety in PTM, PTN, and PTL. In vitro experiments, enabled by the semisynthesis of designer substrates and mimics, reveal that PtmU1 is selective for multiple coenzyme A (CoA)-tethered substrates but specific only to the (7)-hydroxyl group for dehydration. A combination of kinetics analyses, targeted mutagenesis, and in silico docking studies identifies an arginine-rich structural element of PtmU1 that acts as a dynamic CoA-binding anchor, revealing a unique strategy for CoA binding that presumably contributes to the substrate promiscuity and dehydration specificity of PtmU1. Finally, a bioinformatics investigation identifies PtmU1 homologues bearing this CoA-binding motif, suggesting that the observed biosynthetic chemistry might be broadly distributed. Taken together, these results shed new insights into PTM, PTN, and PTL biosynthesis and reveal a new perspective on the role of CoA in substrate binding and enzyme catalysis.