title: Structural Characterization of Early Michaelis Complexes in the Reaction Catalyzed by (+)-Limonene Synthase from Citrus sinensis Using Fluorinated Substrate Analogues.
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 KIvalues of 2.4 ± 0.5 and 39.5 ± 5.2 μM, respectively. The KIvalues are similar to the KMfor 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 (kcatvalues 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.
authors.affiliation: Department of Biochemistry, Brandeis University , Waltham, Massachusetts 02454, United States.
authors.forename: Ramasamy P
authors.initials: RP
authors.lastname: Kumar
authors.affiliation: Department of Biochemistry, Brandeis University , Waltham, Massachusetts 02454, United States.
authors.forename: Benjamin R
authors.identifier: 0000-0003-3352-5463
authors.initials: BR
authors.lastname: Morehouse
authors.affiliation: Department of Biochemistry, Brandeis University , Waltham, Massachusetts 02454, United States.
authors.forename: Jason O
authors.initials: JO
authors.lastname: Matos
authors.affiliation: Department of Biochemistry, Brandeis University , Waltham, Massachusetts 02454, United States.
authors.forename: Karan
authors.initials: K
authors.lastname: Malik
authors.affiliation: Department of Chemistry, Brandeis University , Waltham, Massachusetts 02454, United States.
authors.forename: Hongkun
authors.identifier: 0000-0001-7266-9350
authors.initials: H
authors.lastname: Lin
authors.affiliation: Department of Chemistry, Brandeis University , Waltham, Massachusetts 02454, United States.
authors.forename: Isaac J
authors.initials: IJ
authors.lastname: Krauss
authors.affiliation: Department of Biochemistry, Brandeis University , Waltham, Massachusetts 02454, United States.
authors.forename: Daniel D
authors.identifier: 0000-0002-6520-5459
authors.initials: DD
authors.lastname: Oprian
mesh_terms: D001059:Apoproteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D032084:Citrus sinensis / Q000737:chemistry* / Q000201:enzymology; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D053138:Cyclohexenes / Q000737:chemistry* / Q000378:metabolism; D004224:Diterpenes / Q000737:chemistry* / Q000378:metabolism; D057075:Enzyme Assays; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D019753:Intramolecular Lyases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D000077222:Limonene; D008958:Models, Molecular; D010755:Organophosphates / Q000737:chemistry* / Q000378:metabolism; D010940:Plant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011106:Polyisoprenyl Phosphates / Q000737:chemistry / Q000378:metabolism; D000072417:Protein Domains; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013237:Stereoisomerism; D013729:Terpenes / Q000737:chemistry* / Q000378:metabolism
chemical_list: C555699:2-fluorogeranylgeranyl diphosphate; D001059:Apoproteins; D053138:Cyclohexenes; D004224:Diterpenes; D004791:Enzyme Inhibitors; D008024:Ligands; D010755:Organophosphates; D010940:Plant Proteins; D011106:Polyisoprenyl Phosphates; D011993:Recombinant Fusion Proteins; D013729:Terpenes; D000077222:Limonene; D019753:Intramolecular Lyases; C040265:pinene cyclase I; C002963:geranylgeranyl pyrophosphate