Structural Characterization of Early Michaelis Complexes in the Reaction Catalyzed by (+)-Limonene Synthase from Citrus sinensis Using Fluorinated Substrate Analogues
| Autor: | Hongkun Lin, Karan Malik, Ramasamy P. Kumar, B.R. Morehouse, Isaac J. Krauss, Jason O. Matos, Daniel D. Oprian |
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| Rok vydání: | 2017 |
| Předmět: |
Models
Molecular 0301 basic medicine Allylic rearrangement Stereochemistry Recombinant Fusion Proteins Gene Expression Stereoisomerism Crystallography X-Ray Ligands 010402 general chemistry 01 natural sciences Biochemistry Article Protein Structure Secondary 03 medical and health sciences Polyisoprenyl Phosphates Protein Domains Catalytic Domain Cyclohexenes Escherichia coli Enzyme kinetics Cloning Molecular Enzyme Inhibitors Intramolecular Lyases Enzyme Assays Plant Proteins chemistry.chemical_classification ATP synthase biology Terpenes Ligand Substrate (chemistry) Lyase Organophosphates 0104 chemical sciences Kinetics 030104 developmental biology Enzyme chemistry biology.protein Diterpenes Apoproteins Limonene Citrus sinensis |
| Zdroj: | Biochemistry. 56:1716-1725 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/acs.biochem.7b00144 |
| Popis: | 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 KI values of 2.4 ± 0.5 and 39.5 ± 5.2 μM, respectively. The KI values are similar to the KM 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 (kcat values of 0.00054 ± 0.00005 and 0.00024 ± 0.00002 s−1, 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. |
| Databáze: | OpenAIRE |
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