Imidase, a Dihydropyrimidinase‐Like Enzyme Involved in the Metabolism of Cyclic Imides

Ogawa, Jun; Soong, Chee-Leong; Honda, Michinari; Shlmizu, Sakayu

doi:10.1111/j.1432-1033.1997.0322a.x

Cited by 37 publications

(24 citation statements)

References 23 publications

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“…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%

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

2000

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“…The relative molecular mass of the native enzyme and subunit were determined by high-performance liquid chromatography (HPLC) on a TSK G-3000SW column (0.75 by 60 cm; Tosoh, Tokyo, Japan) and SDS-PAGE, respectively, as described previously (10). The relative molecular masses of the native enzyme and the subunit were determined from the relative mobility of the marker proteins purchased from Oriental Yeast Co. and Daiich Chemical Co. (molecular weight marker III), respectively.…”

Section: Methodsmentioning

confidence: 99%

Purification, Characterization, and Gene Cloning of Purine Nucleosidase from Ochrobactrum anthropi

Ogawa

Takeda

Xie

et al. 2001

Appl Environ Microbiol

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A bacterium, Ochrobactrum anthropi, produced a large amount of a nucleosidase when cultivated with purine nucleosides. The nucleosidase was purified to homogeneity. The enzyme has a molecular weight of about 170,000 and consists of four identical subunits. It specifically catalyzes the irreversible N-riboside hydrolysis of purine nucleosides, the K m values being 11.8 to 56.3 M. The optimal activity temperature and pH were 50°C and pH 4.5 to 6.5, respectively. Pyrimidine nucleosides, purine and pyrimidine nucleotides, NAD, NADP, and nicotinamide mononucleotide are not hydrolyzed by the enzyme. The purine nucleoside hydrolyzing activity of the enzyme was inhibited (mixed inhibition) by pyrimidine nucleosides, with K i and K i values of 0.455 to 11.2 M. Metal ion chelators inhibited activity, and the addition of Zn 2؉ or Co 2؉ restored activity. A 1.5-kb DNA fragment, which contains the open reading frame encoding the nucleosidase, was cloned, sequenced, and expressed in Escherichia coli. The deduced 363-amino-acid sequence including a 22-residue leader peptide is in agreement with the enzyme molecular mass and the amino acid sequences of NH 2 -terminal and internal peptides, and the enzyme is homologous to known nucleosidases from protozoan parasites. The amino acid residues forming the catalytic site and involved in binding with metal ions are well conserved in these nucleosidases.Recently, nucleosides and a variety of chemically synthesized nucleoside analogs have attracted a great deal of interest, as they have antibiotic, antiviral, and antitumoral effects (9). In light of this trend, we conducted studies on the microbial metabolism of nucleosides (5). In this study, we found that a bacterium, Ochrobactrum anthropi, shows a high level of activity in the N-riboside cleavage of purine nucleosides. This reaction is important in the decomposition of purine nucleosides in foodstuffs which cause hyperuricemia, an increasingly common disease in adults (3).The enzymatic N-riboside cleavage of nucleosides is a common reaction in various organisms (1,20). This reaction seems to participate in a salvage or assimilation pathway for nucleosides. Two kinds of enzymes, nucleoside phosphorylases (EC 2.4.2.-) and nucleosidases (nucleoside hydrolase; EC 3.2.2.-), are known to catalyze this reaction. Nucleoside phosphorylases catalyze the phosphorolytic cleavage of nucleosides and show ribosyl transferase activity (7). These enzymes play roles mainly in the salvage pathway. Nucleoside phosphorylases have been well studied. In addition, they have been purified from various sources and used as catalysts for the synthesis of nucleoside analogs through base exchange reactions (7, 21). In contrast, nucleosidases catalyze the irreversible hydrolysis of nucleosides and participate mainly in the assimilation pathway.There have been few studies on microbial nucleosidases acting on purine and pyrimidine nucleosides and no reports of homogenously purified bacterial purine and pyrimidine nucleosidases (8,18,19).In this study, we report the...

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“…Imidase activity was assayed as described previously (23). One unit of half-amidase and imidase was defined as the amount of enzyme that catalyzed consumption of the substrate or formation of the product at a rate of 1 mol/min under the assay conditions described above.…”

Section: Methodsmentioning

confidence: 99%

“…Microbial transformation of cyclic imides found in the bacterium Blastobacter sp. strain A17p-4 (22) involves ring opening of cyclic imide to monoamidated dicarboxylate (half-amide) catalyzed by imidase (23), half-amide hydrolysis to dicarboxylate catalyzed by amidase, and subsequent trichloroacetic acid (TCA) cycle-like reactions (Fig. 1).…”

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A Novel Amidase (Half-Amidase) for Half-Amide Hydrolysis Involved in the Bacterial Metabolism of Cyclic Imides

Soong

Ogawa

Shimizu

2000

Appl Environ Microbiol

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A novel amidase involved in bacterial cyclic imide metabolism was purified from Blastobacter sp. strain A17p-4. The enzyme physiologically functions in the second step of cyclic imide degradation, i.e., the hydrolysis of monoamidated dicarboxylates (half-amides) to dicarboxylates and ammonia. Enzyme production was enhanced by cyclic imides such as succinimide and glutarimide but not by amide compounds which are conventional substrates and inducers of known amidases. The purified amidase showed high catalytic efficiency toward half-amides such as succinamic acid (K m ‫؍‬ 6.2 mM; k cat ‫؍‬ 5.76 s ؊1) and glutaramic acid (K m ‫؍‬ 2.8 mM; k cat ‫؍‬ 2.23 s ؊1 ). However, the substrates of known amidases such as short-chain (C 2 to C 4 ) aliphatic amides, long-chain (above C 16 ) aliphatic amides, amino acid amides, aliphatic diamides, ␣-keto acid amides, N-carbamoyl amino acids, and aliphatic ureides were not substrates for the enzyme. Based on its high specificity toward half-amides, the enzyme was named half-amidase. This half-amidase exists as a monomer with an M r of 48,000 and was strongly inhibited by heavy metal ions and sulfhydryl reagents.A variety of cyclic amide-metabolizing systems occur in nature and play important roles in pyrimidine and purine metabolism, amino acid metabolism (histidine degradation), antibiotic metabolism (␤-lactam decomposition), creatinine degradation, etc.Cyclic imide is a kind of cyclic amide, and the metabolism of cyclic imides has been studied in relation to the detoxification of the antiepileptic agents ethotoin and phensuximide in mammals (3,36). During the course of a study on cyclic amide transformation for hydantoin from the practical viewpoint of industrial D-amino acid production, we recently found that microorganisms also transform cyclic imides (21,(24)(25)(26)(27). Microbial transformation of cyclic imides found in the bacterium Blastobacter sp. strain A17p-4 (22) involves ring opening of cyclic imide to monoamidated dicarboxylate (half-amide) catalyzed by imidase (23), half-amide hydrolysis to dicarboxylate catalyzed by amidase, and subsequent trichloroacetic acid (TCA) cycle-like reactions (Fig. 1). The reactions and enzymes (imidase and amidase) involved in the metabolism have practical potential for production of organic acids from cyclic imides or their metabolites and for fine enzymatic synthesis of useful compounds. For example, pyruvate, an effective precursor in the synthesis of various drugs and agrochemicals, was produced from succinimide or its metabolites (especially fumarate, a cheap material) through cyclic imide-transforming pathway ( Fig. 1A and B) (28). Imidase was applied for the regiospecific synthesis of useful half-amide (3-carbamoyl-␣-picolinic acid, an intermediate for herbicide) from a cyclic imide (2,3-pyridinedicarboximide) (J. Ogawa, M. Ito, T. Segawa, C.-L. Soong, and S. Shimizu, Abstr. Annu. Meet. '99 Soc. Biosci. Bioeng., abstr. 182, 1999 [in Japanese]). Amidase also has a potential for the chiral resolution of dicarboxylates through the ...

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Imidase, a Dihydropyrimidinase‐Like Enzyme Involved in the Metabolism of Cyclic Imides

Cited by 37 publications

References 23 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

Purification, Characterization, and Gene Cloning of Purine Nucleosidase from Ochrobactrum anthropi

A Novel Amidase (Half-Amidase) for Half-Amide Hydrolysis Involved in the Bacterial Metabolism of Cyclic Imides

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