Premium
Homogeneous purification and characterization of Le PGT 1 – a membrane‐bound aromatic substrate prenyltransferase involved in secondary metabolism of Lithospermum erythrorhizon
Author(s) -
Ohara Kazuaki,
Mito Koji,
Yazaki Kazufumi
Publication year - 2013
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.12239
Subject(s) - prenyltransferase , chemistry , biochemistry , divalent , enzyme , sf9 , biosynthesis , substrate (aquarium) , biology , recombinant dna , organic chemistry , gene , ecology , spodoptera
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.