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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The metabolic transformation pathway for cyclic imides in microorganisms was studied in Blastobacter sp. strain A17p-4. This novel pathway involves, in turn, hydrolytic ring opening of a cyclic imide to yield a monoamidated dicarboxylate, hydrolytic deamidation of the monoamidated dicarboxylate to yield a dicarboxylate, and dicarboxylate transformation similar to that in the tricarboxylic acid cycle. The initial step is catalyzed by a novel enzyme, imidase. Imidase and subsequent enzymes involved in this metabolic pathway are induced by some cyclic imides, such as succinimide and glutarimide. Induced cells metabolize various cyclic imides. Two cyclic-ureide transformation pathways have been well characterized. One is dihydropyrimidine transformation (Fig. 1A) (11, 15), and the other is hydantoin transformation (Fig. 1B) (13). Cyclic imides have structures similar to those of cyclic ureides and are known to be hydrolyzed by mammalian dihydropyrimidinase, which functions in pyrimidine metabolism (3-5). However, there have been no reports on the microbial transformation of cyclic imides. The microbial transformation of cyclic ureides involves reactions useful for the production of optically active compounds. The best-known example is optically active amino acid production by hydantoin-transforming microorganisms (16). Blastobacter sp. strain A17p-4, which is a gram-negative, nonmotile, non-spore-forming, obligatorily aerobic, nonfermentative rod, was isolated by us as a competent strain for D-amino acid production from 5-monosubstituted hydantoin (6). The strain is good material for the analysis of enzymes involved in cyclic-ureide transformation because of its high activity. During the course of enzymatic analysis of hydantoin transformation in this bacterium (8), we found the transformation of not only diverse cyclic ureides but also cyclic imides in the bacterium (8). That was the first report on the microbial transformation of cyclic imides, and we discovered that the initial step of the transformation is catalyzed by a novel enzyme, imidase, which is different from known cyclic-ureide-transforming enzymes (7). Imidase catalyzes the hydrolysis of a cyclic imide to a monoamidated dicarboxylate. We investigated the metabolic fate of cyclic imides in Blastobacter sp. strain A17p-4 and found that monoamidated dicarboxylates were further transformed. In this report, we describe the presence of a novel metabolic transformation pathway for cyclic imides in this bacterium. MATERIALS AND METHODS Chemicals. 2-Methylsuccinimide was synthesized from methylsuccinimide and aqueous ammonia (1). All of the other chemicals used were of analytical grade and are commercially available. Microorganism and media. Blastobacter sp. strain A17p-4 (6) was used for all experiments. The nutrient medium used comprised 1.5 g of uracil, 1 g of KH 2 PO 4 , 1 g of K 2 HPO 4 , 0.3 g of MgSO 4 ⅐ 7H 2 O, 0.1 g of FeSO 4 ⅐ 7H 2 O, 3 g of yeast extract, 3 g of meat extract, 2 g of peptone, and 10 g of glycerol in 1 liter

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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.

An FAD-dependent glucose dehydrogenase (GDH) from Aspergillus terreus was purified and crystallized at 293 K using the sitting-drop vapour-diffusion method. A data set was collected to a resolution of 1.6 Å from a single crystal at 100 K using a rotating-anode X-ray source. The crystal belonged to space group P21, with unit-cell parameters a = 56.56, b = 135.74, c = 74.13 Å, β = 90.37°. The asymmetric unit contained two molecules of GDH. The Matthews coefficient was calculated to be 2.2 Å(3) Da(-1) and the solvent content was estimated to be 44%.