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 isopentenyldiphosphate 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 anthrayuinone 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 11), with K, values for isopentenyl-POP of 5.1 pM and 1.0 pM, respectively. Both isoforms require MnZ+ or Mg2+ as cofactor, isoform I1 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 Mg" or in the presence of 0.5 mM MnZ+. A pH optimum of 7-7.8 was found for both forms and both were competitively inhibited by geranyl diphosphate (K, 96 pM for isoform I) and the transition state analogue 2-(dimethy1amino)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. robustu cells. Isoform I1 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.

Recent advances in steroidal saponins biosynthesis and in vitro production

“…2a). IPP isomerase was characterized in Taxus media , Tripterygium wilfordii (Tong et al 2016), Gossypium barbadense (Wang et al (Phillips et al 2008), Camptotheca acuminate (Pan et al 2008), Cinchona robusta (Ramos-Valdivia et al 1997) etc.…”

Mathematical Modeling-Guided Evaluation of Biochemical, Developmental, Environmental, and Genotypic Determinants of Essential Oil Composition and Yield in Peppermint Leaves

“…1; Rios-Estepa et al, 2008); (2) competitive inhibition of PR by (+)-menthofuran (Rios-Estepa et al, 2008); and (3) competitive inhibition of isopentenyl-diphosphate isomerase (enzyme 8 in Fig. 1) by geranyl diphosphate (Ramos-Valdivia et al, 1997). In a recent publication (Rios-Estepa et al, 2008), we reported that the (+)-menthofuran concentration in secretory cells was roughly 400 mM when plants were grown under greenhouse conditions, which increased dramatically under low-light stress conditions.…”

The Arabidopsis thaliana Type I Isopentenyl Diphosphate Isomerases Are Targeted to Multiple Subcellular Compartments and Have Overlapping Functions in Isoprenoid Biosynthesis

“…Hence, IDI gene duplication is relatively recent on the scale of higher plant evolution, and there may be differences in IPP isomerase properties or localization among different plant lineages (Ramos-Valdivia et al, 1997a. For example, in Cinchona robusta cell cultures that produce anthraquinones requiring a DMAPP unit, two IDI proteins are known with different kinetic characteristics and patterns of occurrence (Ramos-Valdivia et al, 1997b). These results are in contrast with this study, in which it was shown that the two Arabidopsis proteins have no significant differences in kinetic properties but have different patterns of subcellular localization.…”