PURIFICATION AND CHARACTERIZATION OF 2 ISOFORMS OF ISOPENTENYL-DIPHOSPHATE ISOMERASE FROM ELICITOR-TREATED CINCHONA-ROBUSTA CELLS

In Cinchona robusta (Rubiaceae) cell suspension cultures, the activity of the enzyme isopentenyl-diphosphate isomerase (isopentenyl-POP isom erase) is transiently induced after addition of a homogenate of the ph ytopathogenic fungus Phytophthora cinnamomi. The enzyme catalyses the interconversion of isopentenyl-POP and dimethylallyl diphosphate (dime thylallyl-POP) and may be involved in the biosynthesis of anthraquinon e phytoalexins that accumulate rapidly after elicitation of cinchona c ells. From elicitor-treated C. robusta cells, two isoforms of isopente nyl-POP isomerase have been purified to apparent homogeneity in four c hromatographic steps. The purified forms are monomeric enzymes of 34 k Da (isoform I) and 29 kDa (isoform II), wit K-m values for isopentenyl -POP of 5.1 mu M and 1.0 mu M, respectively. both isoforms require Mn2 + or Mg2+ as cofactor, isoform II showing a preference for Mn2+ with m aximum 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 (K-i 96 mu M 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 isopentyl-POP isomerase purified from Capsicum annuum, sowed the presence of both is oforms 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 iso merase in the biosynthesis of anthraquinones is discussed.