Characterization of plant beta-ureidopropionase and functional overexpression in Escherichia coli

Pyrimidine bases are rapidly catabolized in growing plant tissues. The fina l enzyme of the catabolic pathway, beta -ureidopropionase (beta -UP; EC 3.5 .1.6), was partially purified from the shoots of etiolated maize (Zea mays) seedlings. The enzyme had a K(m)or beta -ureidopropionate (the substrate d erived from uracil) of 11 muM. Only one enantiomer of racemic beta -ureidoi sobutyrate (derived from thymine) was processed with a K-m of 6 muM. The en zyme was inactivated by dialysis against 1,10-phenanthroline and activity c ould be partially restored by addition of Zn2+. Maize beta -UP was very sen sitive to inactivation by iodoacetamide. This could be prevented by additio n of substrate, indicating the presence of an active site Cys. The enzyme w as strongly inhibited by short chain aliphatic acids and aryl propionates, the most potent inhibitor of which was 2-(2, 6-dinitrophenoxy)-propionate ( I-50 = 0.5 muM). A gene for Arabidopsis beta -UP encodes a polypeptide of 4 05 amino acids and has about 55% homology with the enzymes from other eukar yotic organisms. Several highly conserved residues link the plant beta -UP with a larger class of prokaryotic and eukaryotic amidohydrolases. An Arabi dopsis cDNA truncated at the N terminus by 14 residues was cloned and overe xpressed in Escherichia coli. The recombinant enzyme (43.7 kD) was soluble, functional, and purified to homogeneity with yields of 15 to 20 mg per 30 g fresh weight of E. coli cells. The recombinant enzyme from Arabidopsis an d the native enzyme from maize had molecular masses of approximately 440 kD , indicating the enzyme is a decamer at pH 7.