Enzymatic Insights into the Unusual Formation of Benzolactone and Benzopyran in the Biosynthesis of Spiromarmycin

Spiromarmycin-type natural products are rare fungal tricyclic aromatic polyketides with remarkable biological activities. However, the enzymatic processes for assembling the lactone and pyran ring of spiromarmycin and its analogues are unclear. Here, we identify a rare benzolactone formation process differing from typical thioesterase domain-catalyzed transesterification. In this process, a short-chain dehydrogenase/reductase (SDR-Spm14)-catalyzed reduction on the phenylacetic acid core plays a critical role in promoting the lactonization process. Then, prenyltransferase (Spm5)-catalyzed prenylation on the benzolactone intermediate, along with monooxygenase (Spm6)-mediated epoxidation, produces an epoxide intermediate. Interestingly, a C6 hydroxy preset by a hydroxylase (Spm12) plays a role instead of an epoxide hydrolase, allowing a unique spontaneous anti-Baldwin cyclization and benzopyran formation for producing a toxic final product in the extracellular space and thus avoiding self-toxicity. Furthermore, structural analysis of Spm14 proteins in apo form and in complex with NADP + indicates dramatic conformational changes for substrate accommodation. Subsequent molecular docking and structure-based mutagenesis analyses reveal a Tyr-based catalytic mechanism and a Lys-assisted substrate binding mode for Spm14. Importantly, Spm14 shows promiscuous substrate specificity toward diverse phenyl ketones with high stereoselectivity, expanding the potential application of Spm14 in the drug precursor synthesis. This study advances the understanding of benzolactone and benzopyran biosynthesis and expands the biocatalytic toolbox for future drug discovery.