Investigation of the mechanism of the SpnF-catalyzed [4+2]-cycloaddition reaction in the biosynthesis of spinosyn A

Abstract

<p id="d15076410e275">Computational and structural studies of putative Diels–Alderases and the reactions that they catalyze have provided testable models of the reaction coordinates and mechanisms of catalysis; however, there has yet to be a direct experimental interrogation of these hypotheses. Herein, the <span class="inline-formula"> <math id="i1" overflow="scroll"> <mi>α</mi> </math> </span>-secondary deuterium kinetic isotope effects at all sites of rehybridization on the nonenzymatic and SpnF-catalyzed [4+2]-cycloaddition during biosynthesis of spinosyn A are reported. Results implicate an intermediary state between formation of the first and second carbon–carbon bonds and provide evidence that conformational restriction of the substrate may play a role in catalysis. </p><p class="first" id="d15076410e285">The Diels–Alder reaction is one of the most common methods to chemically synthesize a six-membered carbocycle. While it has long been speculated that the cyclohexene moiety found in many secondary metabolites is also introduced via similar chemistry, the enzyme SpnF involved in the biosynthesis of the insecticide spinosyn A in <i>Saccharopolyspora spinosa</i> is the first enzyme for which catalysis of an intramolecular <span class="inline-formula"> <math id="i2" overflow="scroll"> <mrow> <mo stretchy="false">[</mo> <mrow> <mn>4</mn> <mo>+</mo> <mn>2</mn> </mrow> <mo stretchy="false">]</mo> </mrow> </math> </span>-cycloaddition has been experimentally verified as its only known function. Since its discovery, a number of additional standalone <span class="inline-formula"> <math id="i3" overflow="scroll"> <mrow> <mo stretchy="false">[</mo> <mrow> <mn>4</mn> <mo>+</mo> <mn>2</mn> </mrow> <mo stretchy="false">]</mo> </mrow> </math> </span>-cyclases have been reported as potential Diels–Alderases; however, whether their catalytic cycles involve a concerted or stepwise cyclization mechanism has not been addressed experimentally. Here, we report direct experimental interrogation of the reaction coordinate for the <span class="inline-formula"> <math id="i4" overflow="scroll"> <mrow> <mo stretchy="false">[</mo> <mrow> <mn>4</mn> <mo>+</mo> <mn>2</mn> </mrow> <mo stretchy="false">]</mo> </mrow> </math> </span>-carbocyclase SpnF via the measurement of <span class="inline-formula"> <math id="i5" overflow="scroll"> <mi>α</mi> </math> </span>-secondary deuterium kinetic isotope effects (KIEs) at all sites of <span class="inline-formula"> <math id="i6" overflow="scroll"> <mrow> <mrow> <mi>s</mi> <mpadded width="+1.7pt"> <msup> <mi>p</mi> <mn>2</mn> </msup> </mpadded> </mrow> <mo>→</mo> <mrow> <mi>s</mi> <msup> <mi>p</mi> <mn>3</mn> </msup> </mrow> </mrow> </math> </span> rehybridization for both the nonenzymatic and enzyme-catalyzed cyclization of the SpnF substrate. The measured KIEs for the nonenzymatic reaction are consistent with previous computational results implicating an intermediary state between formation of the first and second carbon–carbon bonds. The KIEs measured for the enzymatic reaction suggest a similar mechanism of cyclization within the enzyme active site; however, there is evidence that conformational restriction of the substrate may play a role in catalysis. </p>