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.