title: Function of a glutamine synthetase-like protein in bacterial aniline oxidation via γ-glutamylanilide.
abstract: Acinetobacter sp. strain YAA has five genes (atdA1 to atdA5) involved in aniline oxidation as a part of the aniline degradation gene cluster. From sequence analysis, the five genes were expected to encode a glutamine synthetase (GS)-like protein (AtdA1), a glutamine amidotransferase-like protein (AtdA2), and an aromatic compound dioxygenase (AtdA3, AtdA4, and AtdA5) (M. Takeo, T. Fujii, and Y. Maeda, J. Ferment. Bioeng. 85:17-24, 1998). A recombinant Pseudomonas strain harboring these five genes quantitatively converted aniline into catechol, demonstrating that catechol is the major oxidation product from aniline. To elucidate the function of the GS-like protein AtdA1 in aniline oxidation, we purified it from recombinant Escherichia coli harboring atdA1. The purified AtdA1 protein produced gamma-glutamylanilide (γ-GA) quantitatively from aniline and l-glutamate in the presence of ATP and MgCl2. This reaction was identical to glutamine synthesis by GS, except for the use of aniline instead of ammonia as the substrate. Recombinant Pseudomonas strains harboring the dioxygenase genes (atdA3 to atdA5) were unable to degrade aniline but converted γ-GA into catechol, indicating that γ-GA is an intermediate to catechol and a direct substrate for the dioxygenase. Unexpectedly, a recombinant Pseudomonas strain harboring only atdA2 hydrolyzed γ-GA into aniline, reversing the γ-GA formation by AtdA1. Deletion of atdA2 from atdA1 to atdA5 caused γ-GA accumulation from aniline in recombinant Pseudomonas cells and inhibited the growth of a recombinant Acinetobacter strain on aniline, suggesting that AtdA2 prevents γ-GA accumulation that is harmful to the host cell.
authors.affiliation: Department of Materials Science and Chemistry, Graduate School of Engineering, University of Hyogo, Himeji, Hyogo, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Takeo
authors.forename: Akira
authors.initials: A
authors.lastname: Ohara
authors.forename: Shinji
authors.initials: S
authors.lastname: Sakae
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Okamoto
authors.forename: Chitoshi
authors.initials: C
authors.lastname: Kitamura
authors.forename: Dai-ichiro
authors.initials: D
authors.lastname: Kato
authors.forename: Seiji
authors.initials: S
authors.lastname: Negoro
mesh_terms: D000150:Acinetobacter / Q000378:metabolism; D000814:Aniline Compounds / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D005974:Glutamate-Ammonia Ligase / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism; D010084:Oxidation-Reduction; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D016958:Pseudomonas putida / Q000378:metabolism*
chemical_list: D000814:Aniline Compounds; D001426:Bacterial Proteins; D018698:Glutamic Acid; D005974:Glutamate-Ammonia Ligase; C023650:aniline
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