title: Biochemical characterization of allantoinase from Escherichia coli BL21.
abstract: Bacterial allantoinase (ALLase; EC 3.5.2.5), which catalyzes the conversion of allantoin into allantoate, possesses a binuclear metal center in which two metal ions are bridged by a posttranslationally carboxylated lysine. Here, we characterized ALLase from Escherichia coli BL21. Purified recombinant ALLase exhibited no activity but could be activated when preincubating with some metal ions before analyzing its activity, and was in the order: Mn(2+)- ≫ Co(2+)- > Zn(2+)- > Ni(2+)- > Cd(2+)- ~Mg(2+)-activated enzyme; however, activity of ALLase (Mn(2+)-activated form) was also significantly inhibited with 5 mM Co(2+), Zn(2+), and Cd(2+) ions. Activity of Mn(2+)-activated ALLase was increased by adding the reducing agent dithiothreitol (DTT), but was decreased by treating with the sulfhydryl modifying reagent N-ethylmaleimide (NEM). Inhibition of Mn(2+)-activated ALLase by chelator 8-hydroxy-5-quinolinesulfonic acid (8-HQSA), but not EDTA, was pH-dependent. Analysis of purified ALLase by gel filtration chromatography revealed a mixture of monomers, dimers, and tetramers. Substituting the putative metal binding residues His59, His61, Lys146, His186, His242, and Asp315 with Ala completely abolished the activity of ALLase, even preincubating with Mn(2+) ions. On the basis of these results, as well as the pH-activity profile, the reaction mechanism of ALLase is discussed and compared with those of other cyclic amidohydrolases.
authors: Department of Biomedical Sciences, Chung Shan Medical University, Taichung City, Taiwan.
authors: Ya-Yeh
authors: YY
authors: Ho
authors: Hui-Chuan
authors: HC
authors: Hsieh
authors: Cheng-Yang
authors: CY
authors: Huang
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D004229:Dithiothreitol; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D019216:Metals, Heavy / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D029968:Escherichia coli Proteins; D019216:Metals, Heavy; D011994:Recombinant Proteins; D000581:Amidohydrolases; C019659:allantoinase; D004229:Dithiothreitol