9D2A

Crystal structure of (+)-sabinene synthase from Thuja plicata: condition 3

Summary for 9D2A

• Entry DOI: 10.2210/pdb9d2a/pdb
• Descriptor: Sabinene synthase, 1,2-ETHANEDIOL, COBALT (II) ION, ... (4 entities in total)
• Functional Keywords: biosynthesis, terpene, cyclization, lyase
• Biological source: Thuja plicata
• Total number of polymer chains: 4
• Total formula weight: 257541.88

Authors

Gaynes, M.N.,Christianson, D.W. (deposition date: 2024-08-08, release date: 2024-11-27, Last modification date: 2024-12-11)

Primary citation

Gaynes, M.N.,Osika, K.R.,Christianson, D.W.
Structure and Function of Sabinene Synthase, a Monoterpene Cyclase That Generates a Highly Strained [3.1.0] Bicyclic Product.
Biochemistry, 63:3147-3159, 2024

PubMed 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, (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.

PubMed: 39527408
DOI: 10.1021/acs.biochem.4c00476

Experimental method

X-RAY DIFFRACTION (2.21 Å)