title: Functional and Structural Characterization of a (+)-Limonene Synthase from Citrus sinensis.
abstract: Terpenes make up the largest and most diverse class of natural compounds and have important commercial and medical applications. Limonene is a cyclic monoterpene (C10) present in nature as two enantiomers, (+) and (-), which are produced by different enzymes. The mechanism of production of the (-)-enantiomer has been studied in great detail, but to understand how enantiomeric selectivity is achieved in this class of enzymes, it is important to develop a thorough biochemical description of enzymes that generate (+)-limonene, as well. Here we report the first cloning and biochemical characterization of a (+)-limonene synthase from navel orange (Citrus sinensis). The enzyme obeys classical Michaelis-Menten kinetics and produces exclusively the (+)-enantiomer. We have determined the crystal structure of the apoprotein in an "open" conformation at 2.3 Å resolution. Comparison with the structure of (-)-limonene synthase (Mentha spicata), which is representative of a fully closed conformation (Protein Data Bank entry 2ONG ), reveals that the short H-α1 helix moves nearly 5 Å inward upon substrate binding, and a conserved Tyr flips to point its hydroxyl group into the active site.
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: Ramasamy P
authors.initials: RP
authors.lastname: Kumar
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: Sarah Naomi
authors.initials: SN
authors.lastname: Olsen
authors.affiliation: Department of Biochemistry, Brandeis University , Waltham, Massachusetts 02454, United States.
authors.forename: Sonya
authors.initials: S
authors.lastname: Entova
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 / 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; D011756:Diphosphates / Q000737:chemistry / Q000378:metabolism; D004224:Diterpenes / Q000737:chemistry / Q000378:metabolism; D057075:Enzyme Assays; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D019753:Intramolecular Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D000077222:Limonene; D031337:Mentha spicata / Q000737:chemistry / Q000201:enzymology; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / 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: D001059:Apoproteins; D053138:Cyclohexenes; D011756:Diphosphates; D004224:Diterpenes; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D013729:Terpenes; C511282:geranyl diphosphate; D000077222:Limonene; D019753:Intramolecular Lyases; C040265:pinene cyclase I