Cyclic ureide and imide metabolism in microorganisms producing a d-hydantoinase useful for d-amino acid production

Soong, Chee-Leong; Ogawa, Jun; Shimizu, Sakayu

doi:10.1016/s1381-1177(00)00204-6

Cited by 10 publications

(5 citation statements)

References 27 publications

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“…This metabolic route, especially the hydrolysis of dihydro-derivatives catalyzed by dihydropyrimidinase, has attracted much attention, because it is a potential target for drug therapy in the treatment of cancer (9, 10), and it also has been used for the industrial production of optically active amino acids (5,11,12). In contrast, oxidative pyrimidine metabolism has been scarcely investigated, and the references available so far are limited to the early studies performed by three groups of scientists (6 -8).…”

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confidence: 99%

Barbiturase, a Novel Zinc-containing Amidohydrolase Involved in Oxidative Pyrimidine Metabolism

Soong¹,

Ogawa

Sakuradani³

et al. 2002

Journal of Biological Chemistry

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Barbiturase, which catalyzes the reversible amidohydrolysis of barbituric acid to ureidomalonic acid in the second step of oxidative pyrimidine degradation, was purified to homogeneity from Rhodococcus erythropolis JCM 3132. The characteristics and gene organization of barbiturase suggested that it is a novel zinc-containing amidohydrolase that should be grouped into a new family of the amidohydrolases superfamily. The amino acid sequence of barbiturase exhibited 48% identity with that of herbicide atrazine-decomposing cyanuric acid amidohydrolase but exhibited no significant homology to other proteins, indicating that cyanuric acid amidohydrolase may have evolved from barbiturase. A putative uracil phosphoribosyltransferase gene was found upstream of the barbiturase gene, suggesting mutual interaction between pyrimidine biosynthesis and oxidative degradation. Metal analysis with an inductively coupled radiofrequency plasma spectrophotometer revealed that barbiturase contains ϳ4.4 mol of zinc per mol of enzyme. The homotetrameric enzyme had K m and V max values of 1.0 mM and 2.5 mol/min/mg of protein, respectively, for barbituric acid. The enzyme specifically acted on barbituric acid, and dihydro-L-orotate, alloxan, and cyanuric acid competitively inhibited its activity. The full-length gene encoding the barbiturase (bar) was cloned and overexpressed in Escherichia coli. The kinetic parameters and physicochemical properties of the cloned enzyme were apparently similar to those of the wild-type.In a biological system, pyrimidines are metabolized through either a reductive or an oxidative pathway (1, 2). It is well recognized that mammals, plants, and microorganisms utilize the reductive pathway for pyrimidine degradation (3-5), whereas some microorganisms use the oxidative pathway (6 -8).In reductive pyrimidine metabolism, uracil, or thymine is first reduced to its dihydro-derivative, which in turn is hydrolyzed to an N-carbamoyl-␤-amino acid and finally decarbamoylated to a ␤-amino acid. This metabolic route, especially the hydrolysis of dihydro-derivatives catalyzed by dihydropyrimidinase, has attracted much attention, because it is a potential target for drug therapy in the treatment of cancer (9, 10), and it also has been used for the industrial production of optically active amino acids (5,11,12). In contrast, oxidative pyrimidine metabolism has been scarcely investigated, and the references available so far are limited to the early studies performed by three groups of scientists (6 -8). These reports showed that pyrimidine bases are first oxidized to barbituric acid derivatives, and then the barbituric acid derivatives are further hydrolyzed by barbiturase (EC 3.5.2.1) to urea and malonate derivatives. However, these studies were carried out with crude enzyme preparations, and the results presented were inadequate for confirming the enzymatic conversion of barbituric acid to urea and malonate.We have elucidated the enzymes involved in the oxidative pathway, and clarified their physiological functions, in a ...

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Barbiturase, a Novel Zinc-containing Amidohydrolase Involved in Oxidative Pyrimidine Metabolism

Soong¹,

Ogawa

Sakuradani³

et al. 2002

Journal of Biological Chemistry

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“…1). This metabolic route, especially the hydrolysis of dihydro-derivatives catalyzed by dihydropyrimidinase, has been studied extensively because of the biotechnological attraction for the industrial production of optically active amino acids (3,7,8).The oxidative pathway of pyrimidine degradation was first reported in 1952, almost concurrently by three groups of scientists, in bacteria, Mycobacterium, Corynebacterium, and Norcardia, isolated from soil (4 -6, 9). In this pathway, uracil or thymine is first oxidized to barbituric acid or 5-methylbarbituric acid, respectively.…”

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Novel Amidohydrolytic Reactions in Oxidative Pyrimidine Metabolism: Analysis of the Barbiturase Reaction and Discovery of a Novel Enzyme, Ureidomalonase

Soong

Ogawa

Shimizu

2001

Biochemical and Biophysical Research Communications

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“…The recently found microbial cyclic imide transformation (22,23,32,33) is attracting increasing attention as a novel process for production of useful organic acids and as a new tool for fine enzymatic synthesis of chiral and regioisomeric compounds (34,35). The half-amidase plays an important role in this transformation, linking cyclic imide metabolism to the TCA cycle.…”

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confidence: 99%

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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Cyclic ureide and imide metabolism in microorganisms producing a d-hydantoinase useful for d-amino acid production

Cited by 10 publications

References 27 publications

Barbiturase, a Novel Zinc-containing Amidohydrolase Involved in Oxidative Pyrimidine Metabolism

Barbiturase, a Novel Zinc-containing Amidohydrolase Involved in Oxidative Pyrimidine Metabolism

Novel Amidohydrolytic Reactions in Oxidative Pyrimidine Metabolism: Analysis of the Barbiturase Reaction and Discovery of a Novel Enzyme, Ureidomalonase

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

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