Mechanistic studies of SpnF- and SpnL-catalyzed cyclization reactions in Spinosyn A biosynthesis
Creators: name: Jeon, Byung-Sun
name: 0000-0003-4189-6774
Publisher: name: The University of Texas at Austin
Year: 2017
Subjects: Spinosyn
SpnF
SpnL

description: Spinosyn A produced by actinomycete bacteria Saccaropolyspora spinosa is a particularly interesting natural product due to its structural features and biological activity. The aglycone core of spinosyn A contains an unique perhydro-as-indacene ring system that is the result of a reaction catalyzed by the biosynthetic enzymes SpnF and SpnL. The works described in this dissertation focus on the mechanisms of cyclization reactions catalyzed by SpnF and SpnL. SpnF is a unique tailoring enzyme, because its only known function is to catalyze a [4+2] cycloaddition to form a cyclohexene moiety without introducing any other changes to its substrate. The ring formation also takes place nonenzymatically but at a much reduced rate. If ring formation takes place in a concerted process with a pericyclic, aromatic transition state, then SpnF would be the first example of a naturally selected Diels-Alderase. In order to investigate its mechanism, α-secondary deuterium kinetic isotope effects (KIEs) were measured at all sites of proposed rehybridization for both the nonenzymatic and enzymatic reactions with chemoenzymatically synthesized substrate analogs. The measured KIEs for the nonenzymatic reaction are consistent with computational studies implying the existence of an intermediary state between the first bond formation and the second. A similar pattern for KIEs associated with the enzymatic reaction indicates that nonenzymatic cycloaddition and enzymatic cycloaddition might occur via similar mechanisms. However, a sizable inverse KIE at C4 suggests that conformational constraint of the substrate in the active site may play a role in SpnF catalysis. In addition, how SpnF accelerates the cyclization was studied to assess the effects of entropic preorganization and enthalpic transition state stabilization. Three mechanistic probes, linear, C13-C14 Unc and C2-C3 Unc were synthesized and tested. Except C2-C3 Unc, transformation did not take place under nonenzymatic and enzymatic conditions. MS analysis showed that C2-C3 Unc was transformed to dehydrated products at a much reduced rate compared to SpnM natural substrate. Thus, both functional groups at C1 and C15 appear to be important for SpnF catalysis, though the C15 carbonyl functionality appears to be more important than the C1 ester. Meanwhile, a by-product of the SpnM-catalyzed dehydration was structurally elucidated via extensive NMR analysis. The by-product, a regioisomer of the SpnF substrate has a trans-[s-trans]-cis configuration, whereas the SpnF substrate has a trans-[s-trans]-trans configuration. SpnL, another tailoring enzyme, catalyzes a cyclization reaction that completes the aglycone core structure. To distinguish between two possible mechanisms, namely, a Rauhut-Currier type mechanism and a Michael addition mechanism, deuterated substrate analogs and fluoro-containing substrate analogs were synthesized. An isotope tracer experiment and KIE measurement suggested that the Rauhut-Currier type mechanism is the more favored route compared to the Michael addition mechanism. Also, detection of a covalent adduct with the C13F analog by MS further supports the hypothesis that SpnL appears to operate via a Rauhut-Currier type reaction for the cyclization. Mass analysis of trypsin-digested fragments revealed that Cys60 and Cys205 might work as a nucleophile in SpnL catalysis.