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

Abstract

A superfamily of cyclic amidohydrolases, including dihydropyrimidinase, allantoinase, hydantoinase, and dihydroorotase, all of which are involved in the metabolism of purine and pyrimidine rings, was recently proposed based on the rigidly conserved structural domains in identical positions of the related enzymes. With these conserved domains, two putative cyclic amidohydrolase genes from Escherichia coli, flanked by related genes, were identified and characterized. From the genome sequence of E. coli, the allB gene and a putative open reading frame, tentatively designated as a hyuA (for hydantoin-utilizing enzyme) gene, were predicted to express hydrolases. In contrast to allB, high-level expression of hyuA in E. coli of a single protein was unsuccessful even under various induction conditions. We expressed HyuA as a maltose binding protein fusion protein and AllB in its native form and then purified each of them by conventional procedures. allB was found to encode a tetrameric allantoinase (453 amino acids) which specifically hydrolyzes the purine metabolite allantoin to allantoic acid. Another open reading frame, hyuA, located near 64.4 min on the physical map and known as a UUG start, coded for D-stereospecific phenylhydantoinase (465 amino acids) which is a homotetramer. As a novel enzyme belonging to a cyclic amidohydrolase superfamily, E. coli phenylhydantoinase exhibited a distinct activity toward the hydantoin derivative with an aromatic side chain at the 5 position but did not readily hydrolyze the simple cyclic ureides. 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). Dihydropyrimidinase and allantoinase catalyze the degradation of pyrimidines and purines, respectively, while hydantoinase, a microbial counterpart of dihydropyrimidinase, is also supposed to be involved in pyrimidine degradation. 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.Comparativ...

Logical Identification of an Allantoinase Analog (puuE) Recruited from Polysaccharide Deacetylases

“…2A, lower panel); once it had reached its maximum value, the spectrum decayed at the same rate as the spontaneous racemization of allantoin (34) (not shown), indicating that the enzyme has an absolute stereospecificity for the S enantiomer of allantoin. The formation of allantoic acid in the reaction catalyzed by PuuE could be unambiguously assessed by NMR spectroscopy using 13 C-labeled (S)-allantoin (Fig. 2B).…”

“…To obtain 13 C NMR spectra of the product of the PuuEcatalyzed reaction, a 1.1-ml solution of 0.1 M potassium phosphate, 80% D 2 O, pD 7.6, containing 15 N, 13 C-labeled (S)-allantoin (6.9 mM) was supplemented with PuuE (0.54 M); the solution was gently stirred for 1 min and then transferred in a 5-mm NMR tube to collect spectra at different times. The 13 C NMR spectra were proton decoupled and were collected at 25°C with a VARIAN Inova 600 instrument.…”

“…The reactions were conducted in 1 ml of 0.1 M potassium phosphate, pH 7.6. 15 N, 13 C-Labeled (S)-allantoin used in NMR assays was produced through the degradation of 15 N, 13 C-labeled uric acid (3) (6.9 mM) in the presence of 20 units of urate oxidase from C. utilis, 28 units of catalase from Corynebacterium glutamicum (Fluka), 3.7 g of zebrafish HIU hydrolase, and 25 g of zebrafish OHCU decarboxylase. The reactions were conducted in 1.1 ml of 0.1 M potassium phosphate, 80% D 2 O, pD 7.6.…”

“…By structural comparisons, these proteins were then included in a large superfamily (comprising urease, adenine deaminase, and other hydrolases) characterized by a common structural core consisting of an ellipsoidal (␤/␣) 8 barrel and a conserved metal-binding site (10). Genes coding for proteins with allantoinase activity have been documented in amphibians, insects, bacteria, and plants (11)(12)(13)(14); they are all homologous to DAL1 and constitute a monophyletic group in the amidohydrolase superfamily, thus making identification of allantoinase genes in sequence data bases straightforward. Surprisingly, however, no DAL1 sequences are found in organisms * This work was supported by the Universities of Parma and Padua.…”

Functional Characterization of Allantoinase Genes from Arabidopsis and a Nonureide-Type Legume Black Locust

“…In our previous EST analysis from the trunk wood of black locust (Yang et al, 2003), we found an EST that was homologous to other reported allantoinase genes such as those found in Escherichia coli (Kim et al, 2000), yeast (Buckholz and Cooper, 1991), and bullfrog (Hayashi et al, 1994). To obtain a full-length cDNA clone, we used 4-to 6-d-old seedlings to construct a cDNA library, and then screened them using the EST as a probe.…”