title: Homogeneous purification and characterization of LePGT1--a membrane-bound aromatic substrate prenyltransferase involved in secondary metabolism of Lithospermum erythrorhizon.
abstract: Membrane-bound type prenyltransferases for aromatic substrates play crucial roles in the biosynthesis of various natural compounds. Lithospermum erythrorhizon p-hydroxybenzoate: geranyltransferase (LePGT1), which contains multiple transmembrane α-helices, is involved in the biosynthesis of a red naphthoquinone pigment, shikonin. Taking LePGT1 as a model membrane-bound aromatic substrate prenyltransferase, we utilized a baculovirus-Sf9 expression system to generate a high yield LePGT1 polypeptide, reaching ~ 1000-fold higher expression level compared with a yeast expression system. Efficient solubilization procedures and biochemical purification methods were developed to extract LePGT1 from the membrane fraction of Sf9 cells. As a result, 80 μg of LePGT1 was purified from 150 mL culture to almost homogeneity as judged by SDS/PAGE. Using purified LePGT1, enzymatic characterization, e.g. substrate specificity, divalent cation requirement and kinetic analysis, was done. In addition, inhibition experiments revealed that aromatic compounds having two phenolic hydroxyl groups effectively inhibited LePGT1 enzyme activity, suggesting a novel recognition mechanism for aromatic substrates. As the first example of solubilization and purification of this membrane-bound protein family, the methods established in this study will provide valuable information for the precise biochemical characterization of aromatic prenyltransferases as well as for crystallographic analysis of this novel enzyme family.
authors.affiliation: Laboratory of Plant Gene Expression, Research Institute for Sustainable Humanosphere, Kyoto University, Kyoto 611-0011, Japan.
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Ohara
authors.forename: Koji
authors.initials: K
authors.lastname: Mito
authors.forename: Kazufumi
authors.initials: K
authors.lastname: Yazaki
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000302:isolation & purification* / Q000378:metabolism*; D000818:Animals; D004122:Dimethylallyltranstransferase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D007700:Kinetics; D029684:Lithospermum / Q000201:enzymology* / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017354:Point Mutation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D061987:Sf9 Cells; D018411:Spodoptera; D013379:Substrate Specificity
chemical_list: D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D019883:Alkyl and Aryl Transferases; C452181:geranyl diphosphate 4-hydroxybenzoate geranyltransferase; D004122:Dimethylallyltranstransferase