Identification of the structural similarity in the functionally related amidohydrolases acting on the cyclic amide ring

Abstract

The functionally related amidohydrolases, including D-hydantoinases, dihydropyrimidinases, allantoinases and dihydro-orotases, share a similar catalytic function of acting on the cyclic amide ring. We aligned 16 amidohydrolases by taking account of the conservative substitution and found a number of highly conserved regions and invariant amino acid residues. Analyses of the secondary structure and hydropathy profile of the enzymes revealed a significant degree of similarity in the conserved regions. Among the regions, the long stretched region I is of particular interest, because it is mainly composed of invariant amino acid residues, showing a similarity of 69% for the enzymes. A search of the protein data bank using the sequence of the conserved region I identified a number of proteins possessing a similar catalytic property, providing a clue that this region might be linked with the catalytic function. As a particular sequence, one aspartic acid and four histidine residues are found to be rigidly conserved in the functionally related amidohydrolases. In order to investigate the significance of the conserved residues, site-directed mutagenesis was carried out typically for the D-hydantoinase gene cloned from Bacillus stearothermophilus SD1. These residues were found to be essential for metal binding as well as catalysis, strongly implying that these invariant residues play a critical role in other enzymes as well as in D-hydantoinase. On the basis of the similar catalytic function and existence of the rigidly conserved sequence, we propose a close evolutionary relationship among the functionally related amido hydrolases, including D-hydantoinase, dihydropyrimidinase, allantoinase and dihydroorotase.

Functional Expression and Characterization of the Two Cyclic Amidohydrolase Enzymes, Allantoinase and a Novel Phenylhydantoinase, from Escherichia coli

“…On the other hand, dihydroorotase participates in the de novo synthesis of pyrimidines (16). In a recent report, guanine deaminase from human kidney showed a common structural motif conserved in the family of cyclic amidohydrolases (46), expanding the family to more divergent enzymes acting on the purine and pyrimidine rings.Comparative analyses with the enzymes belonging to this superfamily revealed that the related enzymes possess very similar biochemical and structural properties in terms of the quaternary structure, oligomerization, metal dependency, and apparent reaction conditions, along with a striking similarity in the primary and secondary structures of the enzymes (19,21). In addition, the enzymes were found to have a number of conserved motifs, such as PGXIDXHXH, which is responsible for binding metal (19,21,46,47).…”

“…In addition, the enzymes were found to have a number of conserved motifs, such as PGXIDXHXH, which is responsible for binding metal (19,21,46,47). However, the approach undertaken to classify and predict the properties for the cyclic amidohydrolase family of enzymes has been mainly based on the substrate specificity and the sequence-based alignment using the protein database.…”

“…Other enzymes belonging to the superfamily exhibited the highest affinity for their own substrates and also showed a rather low level of activity with other substrates. Allantoinase, eukaryotic dihydropyrimidinase, microbial hydantoinase, and imidase were found to hydrolyze cyclic ureides such as allantoin, dihydropyrimidine, and hydantoin derivatives, while also showing rather low but distinct levels of activity towards other substrates (14,21,22,28,33). Therefore, to elucidate the functional role and substitution of the related enzymes in vivo, it is necessary to undertake genetic study in vivo.…”

“…The deduced amino acid sequence of the novel phenylhydantoinase shared a significant homology (>45%) with those of allantoinase and dihydropyrimidinase, but its functional role still remains to be elucidated. Despite the unclear physiological function of HyuA, its presence, along with the allantoin-utilizing AllB, strongly suggested that the cyclic ureides might be utilized as nutrient sources in E. coli.A superfamily of cyclic amidohydrolases (EC 3.5.2), including hydantoinase, dihydropyrimidinase, allantoinase, and dihydroorotase, has been recently proposed based on the functional and structural similarity of the related enzymes, providing evidence for an evolutionary common ancestor in related amidohydrolases (1,21,23). This family of enzymes is involved in the metabolism of pyrimidines and purines, sharing the property of hydrolyzing the cyclic amide bond of each substrate to the corresponding N-carbamyl amino acids (16,35).…”

“…A superfamily of cyclic amidohydrolases (EC 3.5.2), including hydantoinase, dihydropyrimidinase, allantoinase, and dihydroorotase, has been recently proposed based on the functional and structural similarity of the related enzymes, providing evidence for an evolutionary common ancestor in related amidohydrolases (1,21,23). This family of enzymes is involved in the metabolism of pyrimidines and purines, sharing the property of hydrolyzing the cyclic amide bond of each substrate to the corresponding N-carbamyl amino acids (16,35).…”