Characterization of arginine decarboxylase from Dianthus caryophyllus

Arginine decarboxylase (ADC, EC 4.1.1.9) is a key enzyme in the biosynthesis of polyamines in higher plants, whereas ornithine decarboxylase represents the sole pathway of polyamine biosynthesis in animals. Previously, we characterized a genomic clone from Dianthus caryophyllus, in which the deduced polypeptide of ADC was 725 amino acids with a molecular mass of 78 kDa. In the present study, the ADC gene was subcloned into the pGEX4T1 expression vector in combination with glutathione S-transferase (GST). The fusion protein GST–ADC was water-soluble and thus was purified by sequential GSTrap–arginine affinity chromatography. A thrombin-mediated on-column cleavage reaction was employed to release free ADC from GST. Hiload superdex gel filtration FPLC was then used to obtain a highly purified ADC. The identity of the ADC was confirmed by immunoblot analysis, and its specific activity with respect to 14C-arginine decarboxylation reaction was determined to be 0.9 CO2 pkat mg–1 protein. Km and Vmax of the reaction between ADC and the substrate were 0.077 ± 0.001 mM and 6.0 ± 0.6 pkat mg–1 protein, respectively. ADC activity was reduced by 70% in the presence of 0.1 mM Cu2+ or CO2+, but was only marginally affected by Mg2+, or Ca2+ at the same concentration. Moreover, spermine at 1 mM significantly reduced its activity by 30%.