The specific malonylesterase from chickpea (Cicer arietinum L.), hydrolyzing biochanin A 7-O- glucoside-6″-O-malonate (BGM), has been purified to apparent homogeneity and characterized recently (Hinderer et al., Arch. Biochem. Biophys. 248, 570-578 [1986]). Its substrate specificity as well as the high molecular mass of the native enzyme were further investigated. The 5-deoxy- isoflavone conjugate corresponding to BGM, the formononetin 7-O-glucoside-6,,-O-malonate (FGM), was shown to be a substrate of the malonylesterase essentially as BGM. By contrast, methyl-BGM, a diester of malonic acid was a poor substrate. The purified enzyme completely lacked thioesterase activity with malonyl-CoA as substrate. The inability of the malonylesterase to hydrolyze synthetic acetyl or propionyl esters was further demonstrated with a highly sensitive fluorometric assay using esters of 4-methylumbelliferone. The enzyme-catalyzed hydrolysis of BGM was stimulated in the presence of dissociated carboxylic acids like citrate which was most effective at 30 mM and pH 7.5-8.0.
The purified malonylesterase as well as the enzyme activity in crude extracts were totally excluded in gelchromatography with Ultrogel AcA 22. The enrichment of the enzyme activity in microsomal fractions gave strong evidence that the malonylesterase is membrane-bound in vivo. Stimulation of the enzyme activity in vitro by detergents indicates the presence of lipid material in the enzyme and the activity profiles obtained after sedimentation analyses suggest that purification of a distinct membrane-protein complex had been achieved.
Role of PAL and PPO Enzyme Activity in Host Pathogen interaction of Chickpea (Cicer Arietinum L) Root Tissue Infected with Fusarium Wilt
