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Volume 272, Number 7, Issue of February 14, 1997 pp. 3986-3992
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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Effector Specificity Mutants of the Transcriptional Activator NahR of Naphthalene Degrading Pseudomonas Define Protein Sites Involved in Binding of Aromatic Inducers

(Received for publication, September 6, 1996, and in revised form, October 28, 1996)

From the Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Campus de Cantoblanco, 28049 Madrid, Spain

This work reports a genetic analysis of the interactions between NahR, the LysR-type regulator of the NAH operons for biodegradation of naphthalene in Pseudomonas, and its aromatic effectors. Six mutants encoding NahR variants responsive to salicylate analogs such as benzoate, which is not an inducer for the wild type regulator, were isolated with a polymerase chain reaction-based saturation mutagenesis protocol. Most mutants displaying a specific change of effector profile bore single amino acid substitutions within a short protein segment of 60 residues located at the central portion of the NahR sequence. Some of the protein variants exhibited an increased affinity for salicylate and also for otherwise suboptimal effectors, with apparent K_s values 5-100-fold lower than those of the wild type NahR protein. In addition, all mutants were activated by inducers bearing novel substituents at positions 1 or 2 of the aromatic ring and displayed also an enhanced tolerance to changes at positions 3 and 4. Correlation between mutations in NahR and the structures of the new effectors suggested that protein sites Met¹¹⁶, Arg¹³², Asn¹⁶⁹, and Arg²⁴⁸ are involved in effector recognition and binding during the earlier steps of the process leading to transcriptional activation of cognate NAH promoters.

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