Biochemical Characterization of Allantoinase from <Emphasis Type="Italic">Escherichia coli</Emphasis> BL21 |
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Authors: | Ya-Yeh Ho Hui-Chuan Hsieh Cheng-Yang Huang |
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Institution: | (1) Department of Biomedical Sciences, Chung Shan Medical University, No. 110, Sec. 1, Chien-Kuo N. Rd., Taichung City, Taiwan;(2) Department of Medical Research, Chung Shan Medical University Hospital, No. 110, Sec. 1, Chien-Kuo N. Rd., Taichung City, Taiwan; |
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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: Mn2+- ≫ Co2+- > Zn2+- > Ni2+- > Cd2+- ~Mg2+-activated enzyme; however, activity of ALLase (Mn2+-activated form) was also significantly inhibited with 5 mM Co2+, Zn2+, and Cd2+ ions. Activity of Mn2+-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 Mn2+-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 Mn2+ 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. |
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