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

Abstract

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 ...

Cloning and Heterologous Expression of an Enantioselective Amidase from Rhodococcus erythropolis Strain MP50

“…The chemo-and regioselectivities of amidases are utilized for the production of antibiotics (penicillin acylase), the hydrolysis of C-terminal amide groups in peptides (peptide amidase), the analysis of glycoproteins [peptide-N 4 -(N-acetyl-␤-D-glucosaminyl)asparagine amidase F], or the transformation of cyclic imides (halfamidase, imidase) (5,25,50,55,57,59). Enantioselective amidases are used for the production of optical active D-or L-␣-amino acids, hydroxycarboxylic acids, or ␣-methylarylacetic and ␣-methoxyarylacetic acids.…”

Comparative genomic analysis of iprodione‐degrading Paenarthrobacter strains reveals the iprodione catabolic molecular mechanism in Paenarthrobacter sp. strain YJN‐5

“…(ii) The subsequent hydrolysis of compound II, a hydantoin derivative, to (3,5‐dichlorophenylurea)acetic acid (compound III) should be catalysed by a hydrolase in the iprodione‐metabolic pathway. Little is known regarding the degradation of compound II, although the degradation of hydantoin and cyclic imide is well documented (Lapointe et al ., 1994; Soong et al ., 2000; Engel et al ., 2012). As per current understanding, the hydantoin ring cleavage reaction can be catalysed by hydantoinase (Watabe et al ., 1992).…”

Screening and Industrial Application of Unique Microbial Reactions Involved in Nucleic Acid and Lipid Metabolisms

“…and found to be specific toward half-amides. 46) These enzyme activities are widely distributed among bacteria, yeast, and molds. 47) Based on these findings, potential imidases that are applicable to the regiospecific hydrolysis of 2,3-pyridinedicarboxyimide to 3-carbamoyl--picolinic acid were screened (Fig.…”