Identification of an enzyme-catalyzed [4+2]-cycloaddition required for macrocyclization of spirotetronate-containing polyketides

Spirotetronate is a unique skeleton found in only polyketide natural products that were isolated from actinobacteria. Polyketides containing this skeleton show various biological activities. Biosynthesis of spirotetoronate-containing polyketides has been proposed, which involves intramolecular [4+2]-cycloaddition between a terminal conjugated diene and an exocyclic olefin of tetronate-containing acyclic intermediate coupled with formation of macrocyclic structure consisting of only C−C bonds. This mechanism for macrocyclization is far different from that for typical lactone-forming systems coupled with polymethylene chain releasing from polyketide synthase by the thioester domain. However, whether the proposed [4+2]-cycloaddition involves a specific enzyme has remained elusive and such an enzyme, if it actually exists, has never been documented. To elucidate the mechanism of spirotetronate formation, we focused on the biosynthesis of versipelostatin (VST), an unusual 17-membered macrocyclic spirotetronate-containing polyketide. In this presentation, we report the isolation of the biosynthetic gene cluster of VST and demonstrate that a small enzyme, designated VstJ, which consists of only 142 amino acid residues, catalyzes the stereoselective [4+2]-cycloaddition for the formation of the spirotetronate structure in VST biosynthesis. Because VstJ homologues can be found in the biosynthetic gene clusters of other spirotetranate moiety-containing macrocyclic polyketide compounds, VstJ may prove to be a general enzyme for spirotetronate biosynthesis.