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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Imidase, which preferably hydrolyzed cyclic imides to monoamidated dicarboxylates, was purified to homogeneity from a cell-free extract of Blustohucter sp. A17p-4. Cyclic imides are known to be hydrolyzed by mammalian dihydropyrimidinases. However, imidase was quite different from known dihydropyrimidinases in structure and substrate specificity. The enzyme has a relative molecular mass of 105 000 and consists of three identical subunits. The purified enzyme showed higher activity and affinity toward cyclic imides, such as succinimide (K, = 0.94 mM; V,,,,, = 910 pmol . min-' . mg-I), glutarimide (Kn, = 4.5 mM; V,,,,, = 1000 pmol . min-' . mg-') and maleimide (K,,, = 0.34 mM; V,,, = 5800 pmol . min ' . mg-I), than toward cyclic ureides, which are the substrates of dihydropyrimidinases, such as dihydrouracil and hydantoin. Sulfur-containing cyclic imides, such as 2,4-thiazolidinedione and rhodanine, were also hydrolyzed. The enzyme catalyzed the reverse reaction, cyclization, but with much lower activity and affinity. The enzyme was non-competitively inhibited by succinate, which was found to be a key compound in cyclic-imide transformation in relation with the tricarboxylic acid cycle in this bacterium, suggesting that the role of imidase is to catalyze the initial step of cyclic-imide degradation.Keywords: imidase; dihydropyrimidinase; hydantoinase; imide; Blnstobucter.The hydrolysis of cyclic ureides is known to be involved in dihydropyrimidine metabolism (Fig. 1 A). Dihydropyrimidine metabolism occurs naturally and has an important function in the metabolism of pyrimidine. As for the hydrolysis of cyclic ureides, dihydropyrimidinase [ 11 and dihydroorotase [I, 21, which are involved i n the degradation and biosynthesis of pyrimidine, respcctively, have been studied.The microbial metabolism of cyclic-ureide compounds has attracted much attention because of the application of biocatalysis for the production of useful compounds. One example is hydantoin metabolism, which has been applied for the production of optically active amino acids [3, 41 (Fig. 1 B). Three types of enzymes, namely D-enantiomer-specific, L-enantiomer-specific and non-specific hydantoinases, are involved in hydantoin hydrolysis. This process is very similar to dihydropyrimidine metabolism. It has been proposed that hydantoin and dihydropyrimidine metabolisms are catalyzed by the same enzymes [5, 61, while Runser and Meyer reported the existence of a specific enzyme for hydantoin [7]. Therefore, the true features and the diversity of thcse cyclic-ureide-hydrolyzing enzymes remain unclear.An aerobic bacterium, Blnstobucter sp. A17p-4, isolated from soil, shows various dihydropyrimidine-metabolizing and hydantoin-metabolizing activities [8]. This finding prompted us to investigate the diversity of cyclic-ureide metabolism in this bacterium. During the course of our studies, we found an enzyme which actively hydrolyzed cyclic-imide compounds, such as succinimide, glutarimide and maleimide, more than the cyclic ureides of dihydrouracil and hyda...

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Bioprocesses, which involve biocatalysts for the production of useful compounds, are expected to become a leading player in green chemistry. The first step in bioprocess development is screening for useful biological reactions in the immense number of microorganisms with infinite diversity and versatility. This review introduces some examples of bioprocess development that started from process design stemming from the discovery of unique metabolic processes, reactions, and enzymes in microbial nucleic acid and lipid metabolisms.

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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The cyclic-imide-hydrolyzing activity of a prokaryotic cyclic-ureide-hydrolyzing enzyme, d-hydantoinase, was investigated. The enzyme hydrolyzed cyclic imides with bulky substituents such as 2-methylsuccinimide, 2-phenylsuccinimide, phthalimide, and 3,4-pyridine dicarboximide to the corresponding half-amides. However, simple cyclic imides without substituents, which are substrates of imidase (i.e., succinimide, glutarimide, and sulfur-containing cyclic imides such as 2,4-thiazolidinedione and rhodanine), were not hydrolyzed. The combined catalytic actions of bacterial d-hydantoinase and imidase can cover the function of a single mammalian enzyme, dihydropyrimidinase. Prokaryoticd-hydantoinase also catalyzed the dehydrative cyclization of the half-amide phthalamidic acid to the corresponding cyclic imide, phthalimide. The reversible hydrolysis of cyclic imides shown by prokaryoticd-hydantoinase suggested that, in addition to pyrimidine metabolism, it may also function in cyclic-imide metabolism.