title: Functional characterization of LePGT1, a membrane-bound prenyltransferase involved in the geranylation of p-hydroxybenzoic acid.
abstract: The AS-PT (aromatic substrate prenyltransferase) family plays a critical role in the biosynthesis of important quinone compounds such as ubiquinone and plastoquinone, although biochemical characterizations of AS-PTs have rarely been carried out because most members are membrane-bound enzymes with multiple transmembrane alpha-helices. PPTs [PHB (p-hydroxybenzoic acid) prenyltransferases] are a large subfamily of AS-PTs involved in ubiquinone and naphthoquinone biosynthesis. LePGT1 [Lithospermum erythrorhizon PHB geranyltransferase] is the regulatory enzyme for the biosynthesis of shikonin, a naphthoquinone pigment, and was utilized in the present study as a representative of membrane-type AS-PTs to clarify the function of this enzyme family at the molecular level. Site-directed mutagenesis of LePGT1 with a yeast expression system indicated three out of six conserved aspartate residues to be critical to the enzymatic activity. A detailed kinetic analysis of mutant enzymes revealed the amino acid residues responsible for substrate binding were also identified. Contrary to ubiquinone biosynthetic PPTs, such as UBIA in Escherichia coli which accepts many prenyl substrates of different chain lengths, LePGT1 can utilize only geranyl diphosphate as its prenyl substrate. Thus the substrate specificity was analysed using chimeric enzymes derived from LePGT1 and UBIA. In vitro and in vivo analyses of the chimeras suggested that the determinant region for this specificity was within 130 amino acids of the N-terminal. A 3D (three-dimensional) molecular model of the substrate-binding site consistent with these biochemical findings was generated.
authors.affiliation: Laboratory of Plant Gene Expression, Research Institute for Sustainable Humanosphere, Kyoto University, Uji 611-0011, Japan.
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Ohara
authors.forename: Ayumu
authors.initials: A
authors.lastname: Muroya
authors.forename: Nobuhiro
authors.initials: N
authors.lastname: Fukushima
authors.forename: Kazufumi
authors.initials: K
authors.lastname: Yazaki
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D017124:Conserved Sequence; D004122:Dimethylallyltranstransferase / Q000737:chemistry* / Q000378:metabolism*; D062385:Hydroxybenzoates / Q000737:chemistry / Q000378:metabolism*; D029684:Lithospermum / Q000201:enzymology* / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D062385:Hydroxybenzoates; D008565:Membrane Proteins; D010940:Plant Proteins; D004122:Dimethylallyltranstransferase