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.

Plumbagin, a Natural Product with Potent Anticancer Activities, Binds to and Inhibits Dihydroorotase, a Key Enzyme in Pyrimidine Biosynthesis

“…From a biochemical point of view, DHOase [10,40] is a member of the cyclic amidohydrolase family [38,41], which also includes DHPase [42][43][44][45][46], ALLase [47][48][49], hydantoinase (HYDase) [50,51], and imidase [52][53][54]. These metal-dependent enzymes catalyze the hydrolysis of the cyclic amide bond of each substrate, in either five-or six-membered rings, in the metabolism of purines and pyrimidines.…”

A fundamental catalytic difference between zinc and manganese dependent enzymes revealed in a bacterial isatin hydrolase

“…The AHS members are often categorised as both Zn 2+ and Mn 2+ dependent hydrolases. However, amidohydrolases involved with purines degradation in both bacteria and plants are mainly Mn 2+ dependent allantoinases 12 , 13 , even though Zn 2+ dependent allantoinase activity has been reported 14 .…”

Structural Basis for pH-Dependent Oligomerization of Dihydropyrimidinase fromPseudomonas aeruginosaPAO1

“…The overall structure and architecture of the active site of P. aeruginosa dihydropyrimidinase are similar to those of other dihydropyrimidinases ( Figure 3 ) and other members of the amidohydrolase family of enzymes, such as hydantoinases, dihydroorotases, and allantoinases ( Figure 3 ). The active sites of these enzymes contain four histidines, one aspartate, and one carboxylated lysine residue, which are required for metal binding and catalytic activity [ 12 , 14 , 15 , 19 , 20 , 34 , 35 ].…”