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Journal of Bacteriology, December 2006, p. 8430-8440, Vol. 188, No. 24
0021-9193/06/$08.00+0     doi:10.1128/JB.01085-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

N-Acetylanthranilate Amidase from Arthrobacter nitroguajacolicus Rü61a, an /ß-Hydrolase-Fold Protein Active towards Aryl-Acylamides and -Esters, and Properties of Its Cysteine-Deficient Variant ^,

Stephan Kolkenbrock,¹ Katja Parschat,¹ Bernd Beermann,² Hans-Jürgen Hinz,² and Susanne Fetzner^1*

Institut für Molekulare Mikrobiologie und Biotechnologie,¹ Institut für Physikalische Chemie, Westfälische Wilhelms-Universität Münster, D-48149 Münster, Germany²

Received 22 July 2006/ Accepted 1 October 2006

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 /ß-hydrolase-fold superfamily of enzymes; inactivation of (His₆-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 His₆Amq 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 His₆AmqC22A/C63A markedly differed from those of His₆Amq. The replacements effected some changes in K_ms of the enzyme and increased k_cats 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 -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.

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* Corresponding author. Mailing address: Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Corrensstraße 3, D-48149 Münster, Germany. Phone: 49 (0)251 83 39824. Fax: 49 (0)251 83 38388. E-mail: fetzner{at}uni-muenster.de.

Published ahead of print on 13 October 2006.

Supplemental material for this article may be found at http://jb.asm.org/.

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Journal of Bacteriology, December 2006, p. 8430-8440, Vol. 188, No. 24
0021-9193/06/$08.00+0     doi:10.1128/JB.01085-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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