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

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_mamide values (e.g., K_mamide = 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_mNH2OH = 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 be l-enantioselective towards α-hydroxy- and α-aminoamides.Many bacterial amidases (EC 3.5.1.4) have been described previously because of their amide hydrolysis activities. Wide-spectrum amidases from Rhodococcus sp. strain R312 (26) and Pseudomonas aeruginosa (1), which are very similar, hydrolyze only short-chain amides. These enzymes are made up of four and six identical subunits having molecular weights of about 45,000 and 35,000, respectively. Based on the results of experiments performed with inhibitors, they have been classified as belonging to a branch of sulfhydryl enzymes (1, 26). The other amidases, the enantioselective amidases from Pseudomonas chlororaphis B23 (5), Rhodococcus erythropolis MP50 (12, 27), Rhodococcus sp. strain R312 (20), Rhodococcus sp. strain N-774 (10), Rhodococcus sp. (21), and Rhodococcus rhodochrous J1 (14), belong to a group of amidases containing a GGSS signature in the amino acid sequence (4) and are made up of two (or eight) identical subunits. The corresponding genes are located in clusters containing genes encoding the two subunits of a nitrile hydratase (EC 4.2.1.84). These amidases were also previously classified as sulfhydryl enzymes (5, 15), but no active amino acid residue was identified in any of them. Recently, Kobayashi et al. (15) showed that the real active site residues of the amidase from R. rhodochrous J1 were Asp-191 and Ser-195 rather than the generally accepted Cys-203 residue. These authors showed that aspartic acid and serine residues of this enzyme were also present in the active site sequences of aspartic proteinases and suggested that there is an evolutionary relationship between amidases and aspartic proteinases.All of the different amidases also exhibit an acyl transfer activity in the presence of hydroxylamine: RCONH₂ + NH₂OH ↔ RCONHOH + NH₃. This kind of reaction was previously described for the wide-spectrum amidase from Rhodococcus sp. strain R312 (6), but there has been no detailed study examining the acyl transfer reaction of amidases belonging to the GGSS signature-containing group. The final reaction products (hydroxamic acids) are known to possess high chelating properties. Some of them (particularly α-aminohydroxamic acid derivatives) are potent inhibitors of matrix metalloproteases, a family of zinc endopeptidases involved in tissue remodelling (3). Some other hydroxamic acids (α-aminohydroxamic acids, synthetic siderophores, acetohydroxamic acid, etc.) have also been investigated as anti-human immunodeficiency virus agents or antimalarial agents or have been recommended for treatment of ureaplasma infections and anemia (2, 8, 13, 28). Moreover, some fatty hydroxamic acids have been studied as inhibitors of cylooxygenase and 5-lipooxygenase with potent antiinflammatory activity (9).Apart from these medical applications, some hydroxamic acids (particularly polymerizable unsaturated hydroxamic acids and mid-chain or long-chain hydroxamic acids) have also been extensively investigated in wastewater treatment and nuclear technology studies as a way to eliminate contaminating metal ions (11, 16, 18).In this paper we describe the formation of a wide range of hydroxamic acids with the enantioselective amidase (a 120,000-dalton homodimer) from Rhodococcus sp. strain R312, and we provide some additional information which enhanced our comprehension of the reaction mechanism of this amidase.