SpnF is a polyketide tailoring enzyme in the biosynthetic pathway of the insecticide spinosyn A produced by Saccaropolyspora spinosa. SpnF is unique, because it is the only know enzyme that specifically catalyzes a [4+2]-cycloaddition without introducing any other changes to its substrate. The same cycloaddition also takes place nonenzymatically, but at a much reduced rate. If the reaction catalyzed by SpnF is a concerted process with a single pericyclic transition state, then SpnF would be the first example of a naturally selected Diels-Alderase. In order to investigate this possibility, a-secondary deuterium kinetic isotope effects were measured at both points of rehybridization in the diene during both the nonenzymatic and SpnF-catalyzed [4+2]-cycloaddition reactions. This was accomplished using regiospecifically deuterated substrates and electrospray ionization, time-of-flight mass spectrometry to follow changes in deuterium enrichment of the substrate as the reaction progressed. The presentation will describe the methodology and theory underlying the measurement of these KIEs, address the key challenges regarding these measurements and offer a mechanistic discussion of their implications for understanding the SpnF catalyzed [4+2]-cycloaddition.

Award Address  (Arthur C. Cope Late Career Scholars Award sponsored by the Arthur C. Cope Fund). Mechanistic studies of SpnF-catalyzed [4+2] cycloaddition in spinosyn A biosynthesis

248th National Meeting (2014)

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