Crystal Structure of Allantoinase from Escherichia coli BL21: A Molecular Insight into a Role of the Active Site Loops in Catalysis

Abstract

Allantoinase (ALLase; EC 3.5.2.5) possesses a binuclear metal center in which two metal ions are bridged by a posttranslationally carbamylated lysine. ALLase acts as a key enzyme for the biogenesis and degradation of ureides by catalyzing the conversion of allantoin into allantoate. Biochemically, ALLase belongs to the cyclic amidohydrolase family, which also includes dihydropyrimidinase, dihydroorotase, hydantoinase (HYDase), and imidase. Previously, the crystal structure of ALLase from Escherichia coli K-12 (EcALLase-K12) was reported; however, the two active site loops crucial for substrate binding were not determined. This situation would limit further docking and protein engineering experiments. Here, we solved the crystal structure of E. coli BL21 ALLase (EcALLase-BL21) at a resolution of 2.07 Å (PDB ID 8HFD) to obtain more information for structural analyses. The structure has a classic TIM barrel fold. As compared with the previous work, the two missed active site loops in EcALLase-K12 were clearly determined in our structure of EcALLase-BL21. EcALLase-BL21 shared active site similarity with HYDase, an important biocatalyst for industrial production of semisynthetic penicillin and cephalosporins. Based on this structural comparison, we discussed the functional role of the two active site loops in EcALLase-BL21 to better understand the substrate/inhibitor binding mechanism for further biotechnological and pharmaceutical applications.

Binding Pattern and Structural Interactome of the Anticancer Drug 5-Fluorouracil: A Critical Review

“…Dihydropyrimidinase [ 61 ] is ubiquitously found in living organisms, playing a crucial role in catalyzing a key step in the hydrolysis of dihydrouracil to N-carbamoyl-β-alanine during pyrimidine degradation [ 62 , 63 ]. As a member of the cyclic amidohydrolase family, which includes, dihydroorotase [ 64 , 65 ], and allantoinase [ 66 , 67 , 68 ] with similar active sites, dihydropyrimidinase features an unusual post-translational carbamylated modification of the Lys residue (Kcx) within its active site. Notably, dihydropyrimidinase exhibits the capacity to bind 5-FU [ 69 ] and 5-aminouracil [ 70 ].…”

Hydantoanabaenopeptins from Lake Kinneret Microcystis Bloom, Isolation, and Structure Elucidation of the Possible Intermediates in the Anabaenopeptins Biosynthesis

“…The fourth possible route D suggests that both the cyclization to the hydantoin and its hydrolysis to the ureido moiety occur during the condensation process of lysine to the N-carboxy-amino acid. The hydantoin formation and opening might be catalyzed by a single enzyme of the cyclic amidohydrolase family (allantoinase, dihydropyrimidinase, dihydrooratase, hydantoinase, urease, and imidase) [25] that can reversibly catalyze the concession of N-carboxy-peptide to the hydantoin and its stereoselective hydrolysis to the ureido-bridge-containing peptide [26]. Alternatively, such conversion might be spontaneous in a similar fashion to the nonenzymatic isomerization of Asp in protein under physiological conditions.…”