title: Purification and characterization of two isoforms of isopentenyl-diphosphate isomerase from elicitor-treated Cinchona robusta cells.
abstract: In Cinchona robusta (Rubiaceae) cell suspension cultures, the activity of the enzyme isopentenyl-diphosphate isomerase (isopentenyl-POP isomerase) is transiently induced after addition of a homogenate of the phytopathogenic fungus Phytophthora cinnamomi. The enzyme catalyses the interconversion of isopentenyl-POP and dimethylallyl diphosphate (dimethylallyl-POP) and may be involved in the biosynthesis of anthraquinone phytoalexins that accumulate rapidly after elicitation of Cinchona cells. From elicitor-treated C. robusta cells, two isoforms of isopentenyl-POP isomerase have been purified to apparent homogeneity in four chromatographic steps. The purified forms are monomeric enzymes of 34 kDa (isoform I) and 29 kDa (isoform II), with Km values for isopentenyl-POP of 5.1 microM and 1.0 microM, respectively. Both isoforms require Mn2+ or Mg2+ as cofactor, isoform II showing a preference for Mn2+ with maximum activity at 1.5-2 mM. Isoform I was most active in the presence of 0.5-1.5 mM Mg2+ or in the presence of 0.5 mM Mn2+. A pH optimum of 7-7.8 was found for both forms and both were competitively inhibited by geranyl diphosphate (Ki 96 microM for isoform I) and the transition state analogue 2-(dimethylamino)ethyl diphosphate. Rechromatography of purified isoforms did not indicate any interconversion of both forms. Western blot analysis, using antibodies raised against isopentenyl-POP isomerase purified from Capsicum annuum, showed the presence of both isoforms in the crude protein extracts from C. robusta cells. Isoform II was specifically induced by elicitation, non-treated cells contained low activity of this isoform. The possible role of isopentenyl-POP isomerase in the biosynthesis of anthraquinones is discussed.
authors.affiliation: Division of Pharmacognosy, Leiden/Amsterdam Center for Drug Research, Gorlaeus Laboratories, Leiden, The Netherlands.
authors.forename: A C
authors.initials: AC
authors.lastname: Ramos-Valdivia
authors.forename: R
authors.initials: R
authors.lastname: van der Heijden
authors.forename: R
authors.initials: R
authors.lastname: Verpoorte
authors.forename: B
authors.initials: B
authors.lastname: Camara
mesh_terms: D000880:Anthraquinones / Q000378:metabolism; D019748:Carbon-Carbon Double Bond Isomerases / Q000302:isolation & purification* / Q000378:metabolism; D002478:Cells, Cultured; D002929:Cinchona / Q000201:enzymology* / Q000378:metabolism / Q000382:microbiology; D004795:Enzyme Stability; D045782:Hemiterpenes; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D010838:Phytophthora / Q000472:pathogenicity; D010946:Plants, Medicinal
chemical_list: D000880:Anthraquinones; D045782:Hemiterpenes; D007527:Isoenzymes; D019748:Carbon-Carbon Double Bond Isomerases; C021453:isopentenyldiphosphate delta-isomerase