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Fusicoccadiene synthase (PaFS) efficiently channels substrates between its prenyltransferase and cyclase domains, even without a covalent linker. Optimal channeling occurs when cyclase domains associate with the prenyltransferase octamer core.

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Area of Science:

  • Biochemistry
  • Enzymology
  • Structural Biology

Background:

  • Fusicoccadiene synthase (PaFS) from *Phomopsis amygdala* is a bifunctional enzyme.
  • It possesses prenyltransferase (PT) and cyclase domains linked by a flexible region.
  • The PT domain synthesizes geranylgeranyl diphosphate (GGPP), and the cyclase domain converts it to fusicoccadiene.

Purpose of the Study:

  • To investigate the role of the linker and domain proximity in substrate channeling.
  • To determine the structural basis for GGPP channeling in PaFS.
  • To assess the interaction preference between PaFS PT and cyclase domains.

Main Methods:

  • Construction and analysis of a 'linkerless' PaFS variant.
  • Cryoelectron microscopy to determine the structure of the linkerless construct.
  • GGPP competition experiments with other diterpene cyclases.

Main Results:

  • Covalent linkage is not essential for GGPP channeling, though it may enhance efficiency.
  • PaFS PT and cyclase domains show preferential interaction, irrespective of linkage.
  • The linkerless PaFS structure reveals flexible association of cyclase domains with the PT octamer.

Conclusions:

  • Optimal substrate channeling in PaFS is achieved through cyclase domain association with the PT octamer.
  • This association can occur transiently or be locked in place, with or without a covalent linker.
  • The findings provide insights into the mechanisms of substrate channeling in multi-domain enzymes.