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A Customizable Approach for the Enzymatic Production and Purification of Diterpenoid Natural Products

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Mutational Analysis of (+)-Limonene Synthase.

William H Schiff¹, Daniel D Oprian¹

¹Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02454, United States.

|August 2, 2023

Summary

Limonene synthase uses multiple substrates, but linalyl diphosphate may be a key intermediate. Mutations reveal distinct enzyme activity patterns, suggesting a specific reaction mechanism for limonene production.

Area of Science:

Biochemistry
Enzymology
Organic Chemistry

Background:

Limonene is a monoterpene produced by limonene synthase.
The enzyme can utilize geranyl diphosphate (GPP), neryl diphosphate (NPP), and linalyl diphosphate (LPP) substrates.
The mechanistic relationship between these substrates is not fully understood.

Purpose of the Study:

To investigate the substrate utilization mechanism of (+)-limonene synthase ((+)-LS).
To elucidate the role of GPP, NPP, and LPP in the (+)-LS catalyzed reaction.
To understand the relationship between alternative substrates using site-directed mutagenesis.

Main Methods:

Site-directed mutagenesis of 23 amino acid positions in the (+)-LS active site.
Enzyme activity assays using GPP, NPP, and LPP as substrates.

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Analysis of mutation effects on enzyme kinetics and substrate preference.

Main Results:

Mutations generally reduced activity with GPP and NPP.
Mutations exhibited differential effects with LPP, falling into two groups.
One group lost activity with all substrates; another retained activity with LPP but not GPP/NPP.
No mutations were observed that abolished LPP activity while retaining GPP/NPP activity.

Conclusions:

Linalyl diphosphate (LPP) is likely an intermediate in the (+)-limonene synthase reaction when using GPP or NPP.
The substrate reactivity order (LPP > NPP > GPP) supports an LPP intermediate model.
Understanding this mechanism provides insights into terpene cyclization reactions.