5UV2

Crystal Structure of (+)-Limonene Synthase Complexed with 2-Fluoroneryl Diphosphate

Summary for 5UV2

• Entry DOI: 10.2210/pdb5uv2/pdb
• Related: 5UV0 5UV1
• Descriptor: (+)-limonene synthase, MANGANESE (II) ION, (2E)-2-fluoro-3,7-dimethylocta-2,6-dien-1-yl trihydrogen diphosphate, ... (4 entities in total)
• Functional Keywords: terpene synthase, enantiomer, terpene synthase fold, monoterpene, fluorinated analog, lyase
• Biological source: Citrus sinensis (Sweet orange)
• Total number of polymer chains: 1
• Total formula weight: 70938.23

Authors

Prem Kumar, R.,Malik, K.,Oprian, D.D. (deposition date: 2017-02-17, release date: 2017-03-22, Last modification date: 2023-10-04)

Primary citation

Kumar, R.P.,Morehouse, B.R.,Matos, J.O.,Malik, K.,Lin, H.,Krauss, I.J.,Oprian, D.D.
Structural Characterization of Early Michaelis Complexes in the Reaction Catalyzed by (+)-Limonene Synthase from Citrus sinensis Using Fluorinated Substrate Analogues.
Biochemistry, 56:1716-1725, 2017

PubMed Abstract: The stereochemical course of monoterpene synthase reactions is thought to be determined early in the reaction sequence by selective binding of distinct conformations of the geranyl diphosphate (GPP) substrate. We explore here formation of early Michaelis complexes of the (+)-limonene synthase [(+)-LS] from Citrus sinensis using monofluorinated substrate analogues 2-fluoro-GPP (FGPP) and 2-fluoroneryl diphosphate (FNPP). Both are competitive inhibitors for (+)-LS with K values of 2.4 ± 0.5 and 39.5 ± 5.2 μM, respectively. The K values are similar to the K for the respective nonfluorinated substrates, indicating that fluorine does not significantly perturb binding of the ligand to the enzyme. FGPP and FNPP are also substrates, but with dramatically reduced rates (k values of 0.00054 ± 0.00005 and 0.00024 ± 0.00002 s, respectively). These data are consistent with a stepwise mechanism for (+)-LS involving ionization of the allylic GPP substrate to generate a resonance-stabilized carbenium ion in the rate-limiting step. Crystals of apo-(+)-LS were soaked with FGPP and FNPP to obtain X-ray structures at 2.4 and 2.2 Å resolution, respectively. The fluorinated analogues are found anchored in the active site through extensive interactions involving the diphosphate, three metal ions, and three active-site Asp residues. Electron density for the carbon chains extends deep into a hydrophobic pocket, while the enzyme remains mostly in the open conformation observed for the apoprotein. While FNPP was found in multiple conformations, FGPP, importantly, was in a single, relatively well-defined, left-handed screw conformation, consistent with predictions for the mechanism of stereoselectivity in the monoterpene synthases.

PubMed: 28272876
DOI: 10.1021/acs.biochem.7b00144

Experimental method

X-RAY DIFFRACTION (2.2 Å)