Purification and characterization of malonyl-coenzyme A: 21-hydroxypregnane 21-O-malonyltransferase (Dp21MaT) from leaves of Digitalis purpurea L.
With respect to the cardenolide pathway and the characterization of enzymes involved in the formation of cardenolides, a malonyltransferase, termed malonyl-coenzyme A: 21-hydroxypregnane 21-O-malonyltransferase (Dp21MaT) has been purified. The enzyme catalyses the transfer of the malonyl moiety from malonyl-coenzyme A to 21-hydroxypregnane substrates. Malonyltransferase activity was checked in several potential starting materials including fresh leaves and cell suspension cultures from different plants. Fresh Digitalis purpurea L. leaves turned out to be the best enzyme source. The purification protocol included ammonium sulphate precipitation, hydrophobic interaction chromatography on Phenylsepharose 6 FF, ion exchange chromatography on Source 30 Q, affinity chromatography on Cibacron Blue 3GA and gel filtration on Superdex 75. Gel filtration and native SDS-PAGE analysis showed that Dp21MaT exists as a monomer with a molecular mass of 27kDa. Its pI, as determined by isoelectric focusing, was 4.66. The enzyme showed maximal activity at pH 6.5 when incubated at 42 degrees C. The energy of activation was 29.28kJmol(-1), whereas that of inactivation was 48.57kJmol(-1). Dp21MaT was purified 252-fold with a yield of about 1%. Hanes plots of kinetic data indicated K(m) values of 99microM (V(max) 47.57microkatkg(-1)) and 28.44microM (V(max) 39.4microkatkg(-1) protein) for 3beta-benzoyloxy-5beta-pregnane-14beta,21-dihydroxy-20-one and malonyl-CoA, respectively.
Kuate
Serge Philibert
SP
Department of Pharmaceutical Biology, Institute of Biology, Friedrich-Alexander University of Erlangen-Nuremberg, Staudtstr. 5, D-91058 Erlangen, Germany.
Pádua
Rodrigo M
RM
Eisenbeiss
Wilhelm F
WF
Kreis
Wolfgang
W
D000217:Acyltransferases / Q000737:chemistry* / Q000187:drug effects / Q000302:isolation & purification*; D001205:Ascorbic Acid / Q000494:pharmacology; D004070:Digitalis / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008968:Molecular Conformation; D008970:Molecular Weight; D018515:Plant Leaves / Q000201:enzymology*; D012680:Sensitivity and Specificity; D013237:Stereoisomerism; D013379:Substrate Specificity; D013696:Temperature
D008670:Metals; D000217:Acyltransferases; C527691:malonyl-coenzyme A-21-hydroxypregnane 21-O-malonyltransferase; D001205:Ascorbic Acid