Novel Metabolic Transformation Pathway for Cyclic Imides in Blastobacter sp. Strain A17p-4

Abstract

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

Distribution of cyclic imide-transforming activity in microorganisms

“…2. Microorganisms were cultivated in minimum liquid medium as described previously [1] containing 20 mM succinimide, at pH 7.0 for bacteria and at pH 6.0 for yeasts and molds. Cultivation was carried out at 28³C with shaking for 24 days for bacteria and yeasts, and 4^7 days for molds.…”

“…The successive cyclic imide-transforming activities, i.e. succinimide-, succinamic acid-, and succinatetransforming activities [1] were investigated in microorganisms growing vigorously in minimum liquid medium with succinimide as a sole carbon source. The reaction conditions and analytical methods were the same as described above, except that suc-cinimide, succinamic acid and succinate were used as the substrates.…”

“…Since Blastobacter sp. A17p-4 utilizes succinimide as a sole carbon source [1], we examined whether this activity also exists in other microorganisms. The strains listed in Fig.…”

“…1A) pathway was ¢rst reported in a bacterium Blastobacter sp. A17p-4 [1]. This transformation involves ring-opening hydrolysis of cyclic imides catalyzed by a novel enzyme, imidase [2], successive amidohydrolysis of half-amide, and further transformation through TCA cycle-like reactions.…”

Crystallization and preliminary X-ray diffraction analysis of cyclic imide hydrolase (CIH) fromPseudomonas putidaYZ-26

“…The imidase participates in the metabolism of simple imides: it is involved in ring-opening hydrolysis of cyclic imides to half-amides and the resulting half-amide is hydrolyzed by halfamidase to a dicarboxylate, which then undergoes further transformation through tricarboxylic acid (TCA) cyclic reactions (Ogawa et al, 1996(Ogawa et al, , 1997. According to these reports, the imidase is a metal-dependent enzyme and is a homogenous trimer with a subunit molecular weight of 35 kDa (Ogawa et al, 1996). Another CIH from pig liver is a homogenous hexamer with a subunit molecular weight of 51 kDa and is also a metal-dependent enzyme (Yang et al, 1993).…”