N-acetylanthranilate amidase from Arthrobacter nitroguajacolicus Rü61a, an alpha/beta-hydrolase-fold protein active towards aryl-acylamides and -esters, and properties of its cysteine-deficient variant

Abstract

N-acetylanthranilate amidase (Amq), a 32.8-kDa monomeric amide hydrolase, is involved in quinaldine degradation by Arthrobacter nitroguajacolicus Rü61a. Sequence analysis and secondary structure predictions indicated that Amq is related to carboxylesterases and belongs to the alpha/beta-hydrolase-fold superfamily of enzymes; inactivation of (His(6)-tagged) Amq by phenylmethanesulfonyl fluoride and diethyl pyrocarbonate and replacement of conserved residues suggested a catalytic triad consisting of S155, E235, and H266. Amq is most active towards aryl-acetylamides and aryl-acetylesters. Remarkably, its preference for ring-substituted analogues was different for amides and esters. Among the esters tested, phenylacetate was hydrolyzed with highest catalytic efficiency (k(cat)/K(m) = 208 mM(-1) s(-1)), while among the aryl-acetylamides, o-carboxy- or o-nitro-substituted analogues were preferred over p-substituted or unsubstituted compounds. Hydrolysis by His(6)Amq of primary amides, lactams, N-acetylated amino acids, azocoll, tributyrin, and the acylanilide and urethane pesticides propachlor, propham, carbaryl, and isocarb was not observed; propanil was hydrolyzed with 1% N-acetylanthranilate amidase activity. The catalytic properties of the cysteine-deficient variant His(6)AmqC22A/C63A markedly differed from those of His(6)Amq. The replacements effected some changes in K(m)s of the enzyme and increased k(cat)s for most aryl-acetylesters and some aryl-acetylamides by factors of about three to eight while decreasing k(cat) for the formyl analogue N-formylanthranilate by several orders of magnitude. Circular dichroism studies indicated that the cysteine-to-alanine replacements resulted in significant change of the overall fold, especially an increase in alpha-helicity of the cysteine-deficient protein. The conformational changes may also affect the active site and may account for the observed changes in kinetic properties.

FIG. 1.

Conversion of quinaldine (2-methylquinoline) to anthranilic acid by A. nitroguajacolicus Rü61a (19, 34). I, quinaldine 4-oxidase; II, 1H-4-oxoquinaldine-3-monooxygenase; III, 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase; IV, N-acetylanthranilate amidase (Amq). For details, see the text.

FIG. 2.

Multiple alignment of Amq (EMBL accession no. CAD61042) and related proteins, performed with the Clustal W algorithm (2). COest, carboxylesterase from Xanthomonas axonopodis pv. citri strain 306 (15) (AAM37245); KFase, kynurenine formamidase from Mus musculus (50) (AAM44406); RAest, hypothetical esterase from Ralstonia eutropha JMP134 (46) (AAZ64529). Residues conserved throughout are marked with an asterisk, while residues marked with a colon and dot indicate conserved and semiconserved substitutions, respectively. Residues of the putative catalytic triad are highlighted in gray and are marked with a diamond. The conserved motif (G-X-S-X-G-G/A) surrounding the catalytic nucleophile of the α/β hydrolase-fold enzymes is enclosed in a box. α-Helical regions and β-strands predicted for Amq by the program PredictProtein (61) are marked with wavy shaded boxes and large white arrows, respectively. Amino acid sequences of Amq that are underlined with continuous and broken lines indicate α-helices and β-strands, respectively, as shown in the three-dimensional model of the core region of Amq (aa 71 to 175, framed by vertical arrows), calculated with ProModII 3.70 (64).

FIG. 3.

Purification of His₆Amq from E. coli M15(pREP4)(pWFAAM) and electrophoretic properties of His₆Amq and His₆AmqC22A/C63A. (A) SDS-PAGE and (B) corresponding Western blot of proteins after each purification step. Lane M, protein standard; lane 1, crude extract from E. coli M15(pREP4)(pWFAAM) (30 μg protein); lane 2, His₆Amq after Ni²⁺ chelate affinity chromatography (5 μg protein); lane 3, His₆Amq after anion exchange chromatography (5 μg protein). (C) Western blot of His₆Amq after Ni²⁺ chelate affinity chromatography in the absence (lane 4) and presence (lane 5) of DTT (20 μg protein in each lane). (D) SDS-PAGE of His₆AmqC22A/C63A (lane 6, 8 μg protein) and His₆Amq (lane 7, 10 μg protein) in the absence of DTT; the arrow indicates dimers of His₆Amq.

FIG. 4.

Effects of pH (A) and temperature (B) on His₆Amq activity towards the physiological substrate N-acetylanthranilate.

FIG. 5.

CD spectra of His₆Amq and His₆AmqC22A/C63A. The spectra were recorded in a 0.1-mm cuvette. deg, degree.

FIG. 6.

Presumed hydrogen bonding in o-nitroacetanilide (A) and N-acetylanthranilate (B).