The [4+2] cycloaddition remains one of the most intriguing transformations in synthetic and natural products chemistry. In nature, however, there are remarkably few enzymes known to have this activity. We herein report an unprecedented enzymatic [4+2] cyclization cascade that has a central role in the biosynthesis of pyrroindomycins, which are pentacyclic spirotetramate natural products. Beginning with a linear intermediate that contains two pairs of 1,3-diene and alkene groups, the dedicated cyclases PyrE3 and PyrI4 act in tandem to catalyze the formation of two cyclohexene rings in the dialkyldecalin system and the tetramate spiro-conjugate of the molecules. The two cyclizations are completely enzyme dependent and proceed in a regio- and stereoselective manner to establish the enantiomerically pure pentacyclic core. Analysis of a related spirotetronate pathway confirms that homologs are functionally exchangeable, establishing the generality of these findings and explaining how nature creates diverse active molecules with similar rigid scaffolds.
The Diels-Alder [4 + 2] cycloaddition reaction is one of the most powerful and elegant organic synthesis methods for forming 6-membered molecules and has been known for nearly a century. However, whether and how enzymes catalyze this type of reaction is still not completely clear. Here we focus on PyrI4, an enzyme found in the biosynthetic pathway of pyrroindomycins where it catalyzes the formation of a spiro-conjugate via an enzyme-dependent exo-selective [4 + 2] cycloaddition reaction. We report the crystal structures of PyrI4 alone and in complex with its product. Comparative analysis of these structures, combined with biochemical analysis, lead us to propose a unique trapping mechanism whereby the lid-like action of the N-terminal tail imposes conformational constraints on the β barrel catalytic core, which enhances the proximity and polarization effects of reactive groups (1,3-diene and alkene) to drive cyclization in a regio- and stereo-specific manner. This work represents an important step toward the wider application of enzyme-catalyzed [4 + 2] cyclization for synthetic purposes.
Avermectins (AVEs), which are widely used for the treatment of agricultural parasitic diseases, belong to a family of 6,6-spiroketal moiety-containing, macrolide natural products. AVE biosynthesis is known to employ a type I polyketide synthase (PKS) system to assemble the molecular skeleton for further functionalization. It remains unknown how and when spiroketal formation proceeds, particularly regarding the role of AveC, a unique protein in the pathway that shares no sequence homology to any enzyme of known function. Here, we report the unprecedented, dual function of AveC by correlating its activity with spiroketal formation and modification during the AVE biosynthetic process. The findings in this study were supported by characterizing extremely unstable intermediates, products and their spontaneous derivative products from the simplified chemical profile and by comparative analysis of in vitro biotransformations and in vivo complementations mediated by AveC and MeiC (the counterpart in biosynthesizing the naturally occurring, AVE-like meilingmycins). AveC catalyzes the stereospecific spiroketalization of a dihydroxy-ketone polyketide intermediate and the optional dehydration to determine the regiospecific saturation characteristics of spiroketal diversity. These reactions take place between the closures of the hexene ring and 16-membered macrolide and the formation of the hexahydrobenzofuran unit. MeiC can replace the spirocyclase activity of AveC, but it lacks the independent dehydratase activity. Elucidation of the generality and specificity of AveC-type proteins allows for the rationalization of previously published results that were not completely understood, suggesting that enzyme-mediated spiroketal formation was initially underestimated, but is, in fact, widespread in nature for the control of stereoselectivity.
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