Structure and Function of Sabinene Synthase, a Monoterpene Cyclase That Generates a Highly Strained [3.1.0] Bicyclic Product
Year: 2024
Type: article
Abstract: 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, Thuja plicata (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 2+ or Co 2+, with more modest activity observed using Mg 2+ or Ni 2+ . 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.
Source: Biochemistry
Institution: University of Pennsylvania
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
Awards: G1021286281, G102163711, G107546177, G1285306620, G1313983767, G1664757510, G171842289, G1867732070, G2004402655, G2287611682, G241279073, G262962599, G2842389008, G2857925935, G3067052642, G3075337988, G3262563802, G357145406, G3708755560, G3728080647, G409777406, G4155925329, G4429385918, G498139845, G5535608740, G5607048534, G5765906131, G605814063, G6476969910, G6816383395, G6848031779, G6918803902, G6985549539, G7103625430, G7463374569, G78868261, G8701900045, G9000051782, G9000295541, Center for Biomolecular Structure (CBMS), Predoctoral Training at the Chemistry-Biology Interface, NE-CAT: A Resource for Advanced Macromolecular Crystallography, G969889393, G993149817
OpenAlex ID: W4404229990