Structure and Function of Sabinene Synthase, a Monoterpene Cyclase That Generates a Highly Strained [3.1.0] Bicyclic Product.
Publication Type:
Journal ArticleSource:
Biochemistry, Volume 63, Issue 23, p.3147-3159 (2024)Keywords:
Alkyl and Aryl Transferases, Bicyclic Monoterpenes, Catalytic Domain, Crystallography, X-Ray, Intramolecular Lyases, Kinetics, Models, Molecular, Monoterpenes, Mutagenesis, Site-Directed, Plant Proteins, Protein ConformationAbstract:
<p>Sabinene is a plant natural product with a distinctive strained [3.1.0] bicyclic ring system that is used commercially as a spicy and pine-like fragrance with citrus undertones. This unusual monoterpene has also been studied as an antifungal and anti-inflammatory agent as well as a next-generation biofuel. In order to understand the molecular determinants of [3.1.0] bicyclic ring formation in sabinene biosynthesis, we now report three X-ray crystal structures of sabinene synthase from Western red cedar, (TpSS), with open and partially closed active site conformations at 2.21-2.72 Å resolution. We additionally report the complete biochemical characterization of sabinene synthase, including steady-state kinetics, active site mutagenesis, and product array profiling. The catalytic metal ion requirement is unexpectedly broad for a class I terpene cyclase: optimal catalytic activity was measured using Mn or Co, with more modest activity observed using Mg or Ni. Kinetic parameters were determined for both full-length TpSS and a deletion variant lacking the putative N-terminal plastidial targeting sequence, designated ΔTpSS. Monoterpene product profiles for both indicated similar product arrays independent of the catalytic metal ion used, with sabinene comprising nearly 90% of the total products generated. Site-directed mutagenesis was utilized to probe the function of active site residues, and several mutants yielded altered product arrays. Most notably, the G458A substitution converted ΔTpSS into a high-activity α-pinene synthase. α-Pinene contains a bicyclic [3.1.1] ring system; structural and mechanistic analyses suggest a molecular rationale for the reprogrammed transannulation reaction, leading to the alternative bicyclic product.</p>