Characterization of Plant β-Ureidopropionase and Functional Overexpression in Escherichia coli

Abstract

Pyrimidine bases are rapidly catabolized in growing plant tissues. The final enzyme of the catabolic pathway, ␤-ureidopropionase (␤-UP; EC 3.5.1.6), was partially purified from the shoots of etiolated maize (Zea mays) seedlings. The enzyme had a K m for ␤-ureidopropionate (the substrate derived from uracil) of 11 m. Only one enantiomer of racemic ␤-ureidoisobutyrate (derived from thymine) was processed with a K m of 6 m. The enzyme was inactivated by dialysis against 1,10-phenanthroline and activity could be partially restored by addition of Zn 2ϩ . Maize ␤-UP was very sensitive to inactivation by iodoacetamide. This could be prevented by addition of substrate, indicating the presence of an active site Cys. The enzyme was strongly inhibited by short chain aliphatic acids and aryl propionates, the most potent inhibitor of which was 2-(2, 6-dinitrophenoxy)-propionate (I 50 ϭ 0.5 m). A gene for Arabidopsis ␤-UP encodes a polypeptide of 405 amino acids and has about 55% homology with the enzymes from other eukaryotic organisms. Several highly conserved residues link the plant ␤-UP with a larger class of prokaryotic and eukaryotic amidohydrolases. An Arabidopsis cDNA truncated at the N terminus by 14 residues was cloned and overexpressed 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 and the native enzyme from maize had molecular masses of approximately 440 kD, indicating the enzyme is a decamer at pH 7.

Dual function of pyrimidine metabolism in potato (Solanum tuberosum) plants: pyrimidine salvage and supply of β‐alanine to pantothenic acid synthesis

“…It is synthesized by the condensation of b-alanine and pantoic acid (Genschel et al 1999). In higher plants, three possible pathways of b-alanine biosynthesis have been reported; these are the spermidine route (Terano andSuzuki 1978, Rastogi andDavies 1989), the propionate route (Hatch andStumpf 1962, Rathinasabapathi 2002) and the uracil route (Walsh et al 2001, Duhazé et al 2003. Our results strongly suggest that the uracil route, at least, contributes to pantothenic acid synthesis in potato leaves.…”

Yeast β-Alanine Synthase Shares a Structural Scaffold and Origin with Dizinc-dependent Exopeptidases

“…Furthermore, kinetic studies demonstrated that rat ␤AS is an allosteric enzyme with positive cooperativity toward the substrate N-carbamyl-␤-alanine, which triggers a change in the oligomeric state from homohexamer to homododecamer, whereas the presence of the reaction product ␤-alanine induces the dissociation into inactive trimers (16). The two characterized plant ␤ASs exist as homodecamers at pH 7, with subunit molecular masses comparable with those of the mammalian enzymes (43-45 kDa) (15). In contrast, Sk␤AS is a homodimer, and kinetic studies do not support an allosteric regulation of the enzyme.…”

“…The presence of a dimetal site in this enzyme became apparent after calculation of the initial electron density map, which showed two connected peaks of strong density. The high structural similarity of Sk␤AS to dizinc-dependent exopeptidases, the results of the metal analyses performed for other ␤ASs (14,15), and the type of ligands coordinating the metal ions suggested zinc as the most probable type of metal. After inclusion of two zinc ions/subunit in the model, the distances to the coordinating residues refined to those usually observed in zinc-binding sites.…”

“…Metal analyses performed for the rat and maize enzymes suggested that both contain Zn 2ϩ ions (14,15). Furthermore, kinetic studies demonstrated that rat ␤AS is an allosteric enzyme with positive cooperativity toward the substrate N-carbamyl-␤-alanine, which triggers a change in the oligomeric state from homohexamer to homododecamer, whereas the presence of the reaction product ␤-alanine induces the dissociation into inactive trimers (16).…”

Crystallization and preliminary X-ray data analysis of β-alanine synthase fromDrosophila melanogaster

“…Eukaryotic AS has been purified and characterized from a number of sources (Sanno et al, 1970;Wasternack et al, 1979;Tamaki et al, 1987;Gojković et al, 2001;Walsh et al, 2001;Waldmann et al, 2005). Their subunit molecular weights range from 42 to 50 kDa.…”