Purification and characterization of aryl acylamidase from Nocardia globerula

Abstract

Aryl acylamidase was purified from an extract of N-acetyl-o-toluidine-induced cells of Nocardiu globerulu I F 0 13510 in ten steps. The purified enzyme appeared to be homogeneous from analysis by polyacrylamide gel electrophoresis. The enzyme has a molecular mass of approximately 126 kDa and consists of two subunits which are identical in molecular mass. The purified enzyme catalyzed the hydrolysis of N-acetyl-o-toluidine to o-toluidine and acetic acid at a rate of 47.7 pmol . min-' . mg-' at 35°C. It also catalyzed the hydrolysis of various anilide derivatives and esters, as well as the transfer of an acetyl group to aniline as an acetyl acceptor. The purified enzyme was sensitive to thiol reagents such as HgClz and p-chloromercuribenzoate. The amino-terminal sequence (28 amino acid residues) of the enzyme was determined. Based on the substrate specificity of this enzyme, the pathway intermediates involved in the conversion of n-acetyl-o-toluidine to 4-hydroxy-N-acetyl-o-toluidine are discussed Microbial hydroxylation of aromatic compounds catalyzes position-specific and stereospecific reactions efficiently. This reaction has been utilized in efforts to produce useful compounds in the field of medicine and for chemical industries 6wc0cH3 -t 1/2 0, -no &c0cH3 ( 1 1 Recently, we surveyed the hydroxylation of N-acetyl-otoluidine to 4'-hydroxy-N-acetyl-o-toluidine by various bacteria and actinomycetes. Nocardia globerula I F 0 13510 was selected as the best strain to accumulate 4-hydroxy-N-acetylo-toluidine [lo]. As soon as N-acetyl-o-toluidine was added to culture broth of N. globerulu, o-toluidine was formed. Subsequently, in accordance with the decrease in o-toluidine, an accumulation of 4'-hydroxy-N-acetyl-o-toluidine in the culture broth was observed. Therefore, we presumed that the first step may be the hydrolysis of N-acetyl-o-toluidine to o-toluidine by aryl acylamidase, being associated with the conversion of N-acetyl-o-toluidine into 4-hydroxy-N-acetylo-toluidine.In the present study, in order to elucidate the accumulation mechanism of 4-hydroxy-N-acetyl-o-toluidine, we attemptedCorrespondence to

N-Acetylanthranilate Amidase fromArthrobacter nitroguajacolicusRü61a, an α/β-Hydrolase-Fold Protein Active towards Aryl-Acylamides and -Esters, and Properties of Its Cysteine-Deficient Variant

“…Activity towards aryl-acylesters has been reported for other aryl-acylamidases; however, the specificities again are different for enzymes from different sources. Activity of the Nocardia enzyme, for example, towards phenylacetate is about 76% of its activity towards the amide analogue, acetanilide (70). Aryl-acylamidase from strain AE1 converts p-nitrophenylacetate with higher levels of activity than phenylacetate; its catalytic efficiency with phenylacetate and acetanilide is very similar (6).…”

“…However, substrate specificities reported for other bacterial aryl-acylamidases indicate that preference for ortho-substituted anilides which can form such hydrogen-bonded species is not a general feature of these enzymes. Aryl-acylamidase from P. acidovorans AE1 actually hydrolyzes acetanilide, p-nitroacetanilide, and o-nitroacetanilide with similar activity (6); the activity of aryl-acylamidase from Nocardia globerula IFO 13510 towards acetanilide and p-nitroacetanilide also is similar, but this enzyme barely converts o-nitroacetanilide, presumably due to steric hindrance of ortho substituents (70). The amino acid sequences of the amidases from P. acidovorans (a 57-kDa monomer) and N. globerula (a 126-kDa homodimer) are not known.…”

“…Biochemical data have been published on enzymes from Pseudomonas striata (37), Bacillus sphaericus ATCC 12123 (18), P. acidovorans AE1 (6,30), P. fluorescens ATCC 39004 (26), Rhodococcus erythropolis NCIB 12273 (68), P. pickettii (31), and Nocardia globerula IFO 13510 (70). Recently, a urethane hydrolase was isolated from Rhodococcus equi TB-60, which besides catalyzing cleavage of urethane bonds to release amines effectively hydrolyzes p-nitrophenylacetate and acetanilides (3).…”

Purification and characterization of a novel enzyme, arylalkyl acylamidase, from Pseudomonas putida Sc2

“…However, enzyme activity was not affected by EDTA. The enzymes of Pseudonzonas acidovoruns AE1 [6] and N. globerula [25] catalyze transacetylation, but the present enzyme did not exhibit transacetylation activity. On the other hand, acetylation with CH3COONa and 2-phenylethylamine as substrates was detected at a very low rate compared with the rate of hydrolysis of N-acetyl-2-phenylethylamine.…”

“…In comparison with aryl acylarmdases, the present enzyme shows many differences. The molecular masses of common aryl acylamidases are 50-80 kDa [2,4,6,8,10, 111, they were found to be monomers, except for the Nncardia globerulu enzyme [25], whose moelcular mass is 126 kDa and which is composed of two subunits. In comparison, the present enzyme has a much hgher molecular mass (150 kDa) and is composed of four subunits.…”