Acyl Transfer Activity of an Amidase from Rhodococcus sp. Strain R312: Formation of a Wide Range of Hydroxamic Acids

Abstract

The enantioselective amidase from Rhodococcus sp. strain R312 was produced in Escherichia coli and was purified in one chromatographic step. This enzyme was shown to catalyze the acyl transfer reaction to hydroxylamine from a wide range of amides. The optimum working pH values were 7 with neutral amides and 8 with α-aminoamides. The reaction occurred according to a Ping Pong Bi Bi mechanism. The kinetic constants demonstrated that the presence of a hydrophobic moiety in the carbon side chain considerably decreased the K m amide values (e.g.,K m amide = 0.1 mM for butyramide, isobutyramide, valeramide, pivalamide, hexanoamide, and benzamide). Moreover, very high turnover numbers (k cat) were obtained with linear aliphatic amides (e.g.,k cat = 333 s−1 with hexanoamide), whereas branched-side-chain-, aromatic cycle- or heterocycle-containing amides were sterically hindered. Carboxylic acids, α-amino acids, and methyl esters were not acyl donors or were very bad acyl donors. Only amides and hydroxamic acids, both of which contained amide bonds, were determined to be efficient acyl donors. On the other hand, the highest affinities of the acyl-enzyme complexes for hydroxylamine were obtained with short, polar or unsaturated amides as acyl donors (e.g.,K m NH2OH = 20, 25, and 5 mM for acetyl-, alanyl-, and acryloyl-enzyme complexes, respectively). No acyl acceptors except water and hydroxylamine were found. Finally, the purified amidase was shown to bel-enantioselective towards α-hydroxy- and α-aminoamides.

Nitrile‐converting enzymes: An eco‐friendly tool for industrial biocatalysis

“…In these structures, the active site is located at the bottom of this pocket. These structures also suggest that the residues located in the loops above this pocket play a role in determining specificity [106,141]. Other studies of the nitrilases of A. faecalis JM3 [112], P. stutzeri AK61 [137], and R. rhodochrous J1 [28] using far-UV circular dichroism, kinetic, and molecular mass analyses concluded that there was no significant structural conformational change upon substitution of the active cysteine with alanine or serine.…”

“…The ratio of acid to amide produced is variable and depends on the microbial species, substrate structure, incubation time, and the age of the culture batch [147]. An amidase model built according to the NitFhit structure showed that the catalytic residues were not only conserved but also aligned well with those of 1EMS [106]. Furthermore, equivalent distances between the catalytic residues from both structures were also observed ( Figs.…”

Transcriptional analysis of the nitrile‐degrading operon from Rhodococcus sp. ACV2 and high level production of recombinant amidase with an Escherichia coli – T7 expression system

“…This study was performed entirely with valeramide as substrate, but the amidase was also shown to transfer acyl groups of mid-chain amides or acids (4-6 carbon atoms) to hydroxylamine in the production of mid-chain hydroxamic acids or several a-aminohydroxamic acids that are particularly interesting for medical applications (Fournand et al 1998). The amidase can also be used for amide hydrolysis to produce the corresponding carboxylic acids.…”

“…After gene cloning, its nucleotidic sequence was shown to be identical to that of the enantio-selective amidase (unpublished results). Adipamidase transferred acyl groups of mid-chain amides (4-6 carbon atoms) or a-amino amides to hydroxylamine, leading to synthesis of mid-chain hydroxamic acids or a-aminohydroxamic acids (Fournand et al 1998).…”

Crystallization, diffraction data collection and preliminary crystallographic analysis of hexagonal crystals ofPseudomonas aeruginosaamidase

“…Although the detailed catalytic mechanism is still not totally understood (Findlater & Orsi, 1973;Woods et al, 1979;Novo et al, 2002), this enzyme has attracted great attention because of the variations in its substrate and inhibitor specificities that can be generated by single-point mutations (Tata et al, 1994;Karmali et al, 2000Karmali et al, , 2001Gregoriou & Brown, 1979, 1980. Microbial amidases with altered substrate specificities can be used in several industrial applications such as the detoxification of industrial effluents containing toxic amides and the production of hydroxamic acids and other organic acids (Fournand et al, 1998). Hydroxamic acids are known for their chelating properties (Vanjari & Pande, 2003) and some of them have been described as potent inhibitors of metalloproteases and have been investigated as anti-human immunodeficiency virus agents or antimalarial agents (Gao et al, 1995;Tsafack et al, 1996).…”