Purification and characterization of theD-hydantoinase from bacillus circulans

Lukša, Virginijus; Starkuviene, Vytaute; StarkuvienE, B.; Dagys, Rimantas

doi:10.1007/bf02787998

Cited by 9 publications

(4 citation statements)

References 16 publications

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“…The cross-linking experiments with sulfosuccinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride also supported the prediction that the quaternary structure of both enzymes was tetrameric. The molecular mass and oligomeric structure of AllB and HyuA were found to be similar to those of cyclic amidohydrolases from a variety of other sources (3,6,17,21,22,46).…”

Section: Identification Of the Two Cyclic Amidohydrolases From E Colimentioning

confidence: 72%

“…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.…”

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confidence: 99%

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

2000

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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...

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Section: Identification Of the Two Cyclic Amidohydrolases From E Colimentioning

confidence: 72%

mentioning

confidence: 99%

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

2000

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“…Other hydantoinases from B. stearothermophilus SD-1 and Bacillus circulans have been reported to have preference for Mn ions for their activity. 19,20 The metal ions in the hydrolases are required to increase the nucleophilicity of a water molecule present between the two metal ions. In the urease family of hydrolases, the actual nucleophile is a hydroxide ion (water molecule), and the metal and other ligand residues (from the enzyme) facilitate an increase in the nucleophilicity of the water molecule.…”

Section: Discussionmentioning

confidence: 99%

Molecular Structure of d-Hydantoinase from Bacillus sp. AR9: Evidence for Mercury Inhibition

Kishan

Vohra

Ganesan

et al. 2005

Journal of Molecular Biology

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“…IP-I 671 (Runser and Ohleyer 1990), and several Bacillus spp. (Lee et al 1997;Luksa et al 1997;Sharma and Vohra 1997;Park et al 1998). Although HYD is frequently used as synonym for prokaryotic DHP, an HYD from Agrobacterium species was proved to have no DHP activity (Runser and Meyer 1993).…”

Section: Introductionmentioning

confidence: 98%

Phylogenetic Analysis and Biochemical Characterization of a Thermostable Dihydropyrimidinase from Alkaliphilic Bacillus sp. TS-23

Lin

Hsu

et al. 2005

Antonie Van Leeuwenhoek

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Two degenerate primers established from the alignment of highly conserved amino acid sequences of bacterial dihydropyrimidinases (DHPs) were used to amplify a 330-bp gene fragment from the genomic DNA of Bacillus sp. TS-23 and the amplified DNA was successfully used as a probe to clone a dhp gene from the strain. The open reading frame of the gene consisted of 1422 bp and was deduced to contain 472 amino acids with a molecular mass of 52 kDa. The deduced amino acid sequence exhibited greater than 45% identity with that of prokaryotic D-hydantoinases and eukaryotic DHPs. Phylogenetic analysis showed that Bacillus sp. TS-23 DHP is grouped together with Bacillus stearothermophilus D-hydantoinase and related to dihydroorotases and allantoinases from various organisms. His6-tagged DHP was over-expressed in Escherichia coli and purified by immobilized metal affinity chromatography to a specific activity of 3.46 U mg(-1) protein. The optimal pH and temperature for the purified enzyme were 8.0 and 60 degrees C, respectively. The half-life of His6-tagged DHP was 25 days at 50 degrees C. The enzyme activity was stimulated by Co2+ and Mn2+ ions. His6-tagged DHP was most active toward dihydrouracil followed by hydantoin derivatives. The catalytic efficiencies (kcat/Km) of the enzyme for dihydrouracil and hydantoin were 2.58 and 0.61 s(-1) mM(-1), respectively.

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Purification and characterization of theD-hydantoinase from bacillus circulans

Cited by 9 publications

References 16 publications

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

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

Molecular Structure of d-Hydantoinase from Bacillus sp. AR9: Evidence for Mercury Inhibition

Phylogenetic Analysis and Biochemical Characterization of a Thermostable Dihydropyrimidinase from Alkaliphilic Bacillus sp. TS-23

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