Purification and characterization of 1-aminocyclopropane-1-carboxylate N-malonyltransferase from etiolated mung bean hypocotyls

Abstract

1-Aminocyclopropane-1-carboxylate (ACC) N-malonyltransferase converts ACC, an immediate precursor of ethylene, to the presumably inactive product malonyl-ACC (MACC). This enzyme plays a role in ethylene production by reducing the level of free ACC in plant tissue. In this study, ACC N-malonyltransferase was purified 3660-fold from etiolated mutig bean (Vigna radiate) hypocotyls, with a 6% overall recovery. The final specific activity was about 83,000 nmol of MACC formed mg-1 protein h-1. The five-step purification protocol consisted of polyethylene glycol fractionation, Cibacron blue 3GA-agarose chromatography using salt gradient elution, Sephadex G-100 gel filtration, MonoQ anion-exchange chromatography, and Cibacron blue 3GA-agarose chromatography using malonyl-CoA plus ACC for elution. The molecular mass of the native enzyme determined by Sephadex G-100 chromatography was 50 ± 3 kD. Protein from the final purification step showed one major band at 55 kD after sodium dodecyl sulfate polyacrylamide gel electrophoresis, indicating that ACC N-malonyltransferase is a monomer. The mung bean ACC N-malonyltransferase has a pH optimum of 8.0, an apparent Km of 0.5 mM for ACC and 0.2 mM for malonyl-coenzyme A, and an Arrhenius activation energy of 70.29 kJ mol-1 degree-1.