Characterization of plant beta-ureidopropionase and functional overexpression in Escherichia coli.
Pyrimidine bases are rapidly catabolized in growing plant tissues. The final 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) for beta-ureidopropionate (the substrate derived from uracil) of 11 microM. Only one enantiomer of racemic beta-ureidoisobutyrate (derived from thymine) was processed with a K(m) of 6 microM. The enzyme was inactivated by dialysis against 1,10-phenanthroline and activity could be partially restored by addition of Zn(2+). Maize beta-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) = 0.5 microM). A gene for Arabidopsis beta-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 beta-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.
Walsh
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Dow AgroSciences, Discovery Research, 9330 Zionsville Road, Indianapolis, Indiana 46268, USA. tawalsh@dowagro.com
Green
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Larrinua
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Schmitzer
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D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics; D029681:Arabidopsis Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D017173:Caenorhabditis elegans / Q000201:enzymology; D003001:Cloning, Molecular; D003624:Darkness; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D007460:Iodoacetamide / Q000494:pharmacology; D008969:Molecular Sequence Data; D018520:Plant Shoots / Q000201:enzymology; D051381:Rats; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D003313:Zea mays / Q000201:enzymology*
D029681:Arabidopsis Proteins; D004791:Enzyme Inhibitors; D000581:Amidohydrolases; C021590:beta-ureidopropionase; D007460:Iodoacetamide