title: Substitution of Two Active-Site Residues Alters C9-Hydroxylation in a Class II Diterpene Synthase.
abstract: Diterpenes form a vast and diverse class of natural products of both ecological and economic importance. Class II diterpene synthase (diTPS) enzymes control the committed biosynthetic reactions underlying diterpene chemical diversity. Homology modelling with site-directed mutagenesis identified two active-site residues in the horehound (Marrubium vulgare) class II diTPS peregrinol diphosphate synthase (MvCPS1); residue substitutions abolished the unique MvCPS1-catalysed water-capture reaction at C9 and redirected enzyme activity toward formation of an alternative product, halima-5(10),13-dienyl diphosphate. These findings contributed new insight into the steric interactions that govern diTPS-catalysed regiospecific oxygenation reactions and highlight the feasibility of diTPS engineering to provide a broader spectrum of bioactive diterpene natural products.
authors.affiliation: Department of Plant Biology, University of California Davis, One Shields Avenue, Davis, CA, 95616, USA.
authors.forename: Sibongile
authors.initials: S
authors.lastname: Mafu
authors.affiliation: Department of Plant Biology, University of California Davis, One Shields Avenue, Davis, CA, 95616, USA.|Present address: The Scripps Institute, 10550 North Torrey Pines Road, La Jolla, CA, 92037, USA.
authors.forename: Emil
authors.initials: E
authors.lastname: Fischer
authors.affiliation: Department of Chemistry, University of California Davis, 1 Shields Avenue, Davis, CA, 95616, USA.
authors.forename: J Bennett
authors.initials: JB
authors.lastname: Addison
authors.affiliation: Department of Plant Biology, University of California Davis, One Shields Avenue, Davis, CA, 95616, USA.|Present address: Federal University of Ceara, Mister Hull Avenue, 60455-760, Fortaleza, Brazil.
authors.forename: Isabel
authors.initials: I
authors.lastname: Riberio Barbosana
authors.affiliation: Department of Plant Biology, University of California Davis, One Shields Avenue, Davis, CA, 95616, USA.
authors.forename: Philipp
authors.initials: P
authors.lastname: Zerbe
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D055162:Biocatalysis; D020134:Catalytic Domain; D004224:Diterpenes / Q000737:chemistry; D008401:Gas Chromatography-Mass Spectrometry; D006900:Hydroxylation; D009682:Magnetic Resonance Spectroscopy; D027522:Marrubium / Q000201:enzymology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D013237:Stereoisomerism
chemical_list: D004224:Diterpenes; D010940:Plant Proteins; D019883:Alkyl and Aryl Transferases; C108877:terpene synthase
keywords: diterpene synthase; enzyme catalysis; natural products; peregrinol diphosphate; terpenes