The Diels-Alder reaction is a [4+2] cycloaddition reaction in which a cyclohexene ring is formed between a 1,3-diene and an electron-deficient alkene via a single pericyclic transition state. This reaction has been proposed as a key transformation in the biosynthesis of many cyclohexene-containing secondary metabolites.  However, only few purified enzymes have thus far been implicated in biotransformations that are consistent with a Diels-Alder reaction. Although the stereochemical outcomes of these reactions indicate that the product formation could be enzyme-guided in each case, these enzymes typically demonstrate more than one catalytic activity leaving their specific influence on the cycloaddition step uncertain. In our studies of the biosynthesis of spinosyn A, a tetracyclic polyketide-derived insecticide from Saccharopolyspora spinosa, we identified a cyclase, SpnF, that catalyzes a transannular [4+2] cycloaddition to form the cyclohexene ring in spinosyn A. Kinetic analysis demonstrates that SpnF specifically accelerates the ring formation reaction. This work establishes SpnF as the first example characterized in vitro of a stand-alone enzyme solely committed to the catalysis of a [4+2] cycloaddition reaction.  A second enzyme, SpnL, was also identified as responsible for the final cross-bridging step that completes the tetracyclic core of spinosyn A in a manner consistent with a Rauhut-Currier reaction. The mode of formation of the complex perhydro-as-indacene moiety in spinosyn A is now fully established.

Thursday, January 26, 2012