Direct Evidence of an Enzyme-Generated LPP Intermediate in (+)-Limonene Synthase Using a Fluorinated GPP Substrate...

Area of Science:

• Biochemistry
• Enzymology
• Organic Chemistry

Background:

• Linalyl diphosphate (LPP) is a proposed intermediate in monoterpene cyclization by terpene synthases.
• The conformation of geranyl diphosphate (GPP) substrate is thought to necessitate LPP formation for cyclization.
• Directly observing LPP has been challenging due to complex reaction mechanisms.

Purpose of the Study:

• To synthesize a fluorinated analog of geranyl diphosphate (DFGPP) to trap and visualize the proposed linalyl diphosphate intermediate (DFLPP).
• To provide direct experimental evidence for the role of LPP in enzymatic monoterpene cyclization.

Main Methods:

• Synthesis of 8,9-difluorogeranyl diphosphate (DFGPP) as a substrate analog.
• Steady-state kinetic analysis using (+)-limonene synthase (LS).
• X-ray crystallography of apo-LS soaked with DFGPP under limited turnover conditions.

Main Results:

• DFGPP cyclization was completely blocked, yielding only difluoromyrcene.
• X-ray crystallography revealed trapped DFLPP in the enzyme active site, confirmed by electron density fitting.
• The observed DFLPP conformation supported its role as an intermediate.

Conclusions:

• Directly observed DFLPP provides strong evidence for LPP as an obligate intermediate in monoterpene cyclization.
• The study validates current mechanistic models of terpene synthase activity.
• The fluorinated analog approach is effective for trapping and characterizing enzyme intermediates.