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

Byung sun Jeon, Mark W. Ruszczycky, William Russell, Geng Min Lin, Namho Kim, Sei hyun Choi, Shao An Wang, Yung nan Liu, John W. Patrick, David H. Russell, Hung wen Liu

Research output: Contribution to journalArticle

17 Scopus citations

Abstract

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 [4 + 2]-cycloaddition has been experimentally verified as its only known function. Since its discovery, a number of additional standalone [4 + 2]-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 [4 + 2]-carbocyclase SpnF via the measurement of α-secondary deuterium kinetic isotope effects (KIEs) at all sites of sp2 → sp3 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.

Original languageEnglish (US)
Pages (from-to)10408-10413
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume114
Issue number39
DOIs
StatePublished - Sep 26 2017
Externally publishedYes

Keywords

  • Bayesian analysis
  • Diels–Alderase
  • Enzyme
  • Isotope effect
  • Spinosyn

ASJC Scopus subject areas

  • General

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    Jeon, B. S., Ruszczycky, M. W., Russell, W., Lin, G. M., Kim, N., Choi, S. H., Wang, S. A., Liu, Y. N., Patrick, J. W., Russell, D. H., & Liu, H. W. (2017). Investigation of the mechanism of the SpnF-catalyzed [4+2]-cycloaddition reaction in the biosynthesis of spinosyn A. Proceedings of the National Academy of Sciences of the United States of America, 114(39), 10408-10413. https://doi.org/10.1073/pnas.1710496114