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Dynamically Complex [6+4] and [4+2] Cycloadditions in the Biosynthesis of Spinosyn A.

ABSTRACT: SpnF, an enzyme involved in the biosynthesis of spinosyn A, catalyzes a transannular Diels-Alder reaction. Quantum mechanical computations and dynamic simulations now show that this cycloaddition is not well described as either a concerted or stepwise process, and dynamical effects influence the identity and timing of bond formation. The transition state for the reaction is ambimodal and leads directly to both the observed Diels-Alder and an unobserved [6+4] cycloadduct. The potential energy surface bifurcates and the cycloadditions occur by dynamically stepwise modes featuring an "entropic intermediate". A rapid Cope rearrangement converts the [6+4] adduct into the observed [4+2] adduct. Control of nonstatistical dynamical effects may serve as another way by which enzymes control reactions.

SUBMITTER: Patel A

PROVIDER: S-EPMC5367884 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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Dynamically Complex [6+4] and [4+2] Cycloadditions in the Biosynthesis of Spinosyn A.

Patel Ashay A Chen Zhuo Z Yang Zhongyue Z Gutiérrez Osvaldo O Liu Hung-wen HW Houk K N KN Singleton Daniel A DA

Journal of the American Chemical Society 20160309 11

SpnF, an enzyme involved in the biosynthesis of spinosyn A, catalyzes a transannular Diels-Alder reaction. Quantum mechanical computations and dynamic simulations now show that this cycloaddition is not well described as either a concerted or stepwise process, and dynamical effects influence the identity and timing of bond formation. The transition state for the reaction is ambimodal and leads directly to both the observed Diels-Alder and an unobserved [6+4] cycloadduct. The potential energy sur ...[more]

PMID: 26909570

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Evidence for an Enzyme-Catalyzed Rauhut-Currier Reaction during the Biosynthesis of Spinosyn A.

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The Global Regulator PhoU Positively Controls Growth and Butenyl-Spinosyn Biosynthesis in Saccharopolyspora pogona

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Enzyme-catalysed [4+2] cycloaddition is a key step in the biosynthesis of spinosyn A.

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Enzyme-catalysed [6+4] cycloadditions in the biosynthesis of natural products.

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| S-EPMC6944468 | biostudies-literature

Pseudoaglycone of spinosyn a.

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Project description:Butenyl-spinosyn produced by Saccharopolyspora pogona exhibits strong insecticidal activity and a broad pesticidal spectrum. Currently, important functional genes involved in butenyl-spinosyn biosynthesis remain unknown, which leads to difficulty in efficient understanding of its regulatory mechanism and improving its production by metabolic engineering. Here, we present data supporting a role of the SenX3-RegX3 system in regulating the butenyl-spinosyn biosynthesis. EMSAs and qRT-PCR demonstrated that RegX3 positively controls butenyl-spinosyn production in an indirect way. Integrated proteomic and metabolomic analysis, regX3 deletion not only strengthens the basal metabolic ability of S. pogona in the mid-growth phase but also promotes the flow of the acetyl-CoA produced via key metabolic pathways into the TCA cycle rather than the butenyl-spinosyn biosynthetic pathway, which ultimately leads to continued growth but reduced butenyl-spinosyn production. The strategy demonstrated here may be valuable for revealing the regulatory role of the SenX3-RegX3 system in the biosynthesis of other natural products.

| S-EPMC7414002 | biostudies-literature

Strain-promoted alkyne-azide cycloadditions (SPAAC) reveal new features of glycoconjugate biosynthesis.

Project description:We have shown that 4-dibenzocyclooctynol (DIBO), which can easily be obtained by a streamlined synthesis approach, reacts exceptionally fast in the absence of a Cu(I) catalyst with azido-containing compounds to give stable triazoles. Chemical modifications of DIBO, such as oxidation of the alcohol to a ketone, increased the rate of strain promoted azide-alkyne cycloadditions (SPAAC). Installment of a ketone or oxime in the cyclooctyne ring resulted in fluorescent active compounds whereas this property was absent in the corresponding cycloaddition adducts; this provides the first example of a metal-free alkyne-azide fluoro-switch click reaction. The alcohol or ketone functions of the cyclooctynes offer a chemical handle to install a variety of different tags, and thereby facilitate biological studies. It was found that DIBO modified with biotin combined with metabolic labeling with an azido-containing monosaccharide can determine relative quantities of sialic acid of living cells that have defects in glycosylation (Lec CHO cells). A combined use of metabolic labeling/SPAAC and lectin staining of cells that have defects in the conserved oligomeric Golgi (COG) complex revealed that such defects have a greater impact on O-glycan sialylation than galactosylation, whereas sialylation and galactosylation of N-glycans was similarly impacted. These results highlight the fact that the fidelity of Golgi trafficking is a critical parameter for the types of oligosaccharides being biosynthesized by a cell. Furthermore, by modulating the quantity of biosynthesized sugar nucleotide, cells might have a means to selectively alter specific glycan structures of glycoproteins.

| S-EPMC3151320 | biostudies-literature

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

Project description: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 Saccharopolyspora spinosa is the first enzyme for which catalysis of an intramolecular [Formula: see text]-cycloaddition has been experimentally verified as its only known function. Since its discovery, a number of additional standalone [Formula: see text]-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 [Formula: see text]-carbocyclase SpnF via the measurement of [Formula: see text]-secondary deuterium kinetic isotope effects (KIEs) at all sites of [Formula: see text] 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.

| S-EPMC5625926 | biostudies-literature

Chemoenzymatic synthesis of spinosyn A.

Project description:Following the biosynthesis of polyketide backbones by polyketide synthases (PKSs), post-PKS modifications result in a significantly elevated level of structural complexity that renders the chemical synthesis of these natural products challenging. We report herein a total synthesis of the widely used polyketide insecticide spinosyn?A by exploiting the prowess of both chemical and enzymatic methods. As more polyketide biosynthetic pathways are characterized, this chemoenzymatic approach is expected to become readily adaptable to streamlining the synthesis of other complex polyketides with more elaborate post-PKS modifications.

| S-EPMC4266379 | biostudies-literature

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