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J. Biol. Chem., Vol. 278, Issue 51, 51307-51315, December 19, 2003

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Characterization of HdnoR, the Transcriptional Repressor of the 6-Hydroxy-D-nicotine Oxidase Gene of Arthrobacter nicotinovorans pAO1, and its DNA-binding Activity in Response to L- and D-Nicotine Derivatives^*

Cristinel Sandu, Calin B. Chiribau, and Roderich Brandsch

From the Institute of Biochemistry and Molecular Biology, University of Freiburg, 79104 Freiburg, Germany

Utilization of L-nicotine as growth substrate by Arthrobacter nicotinovorans pAO1 starts with hydroxylation of the pyridine ring at C6. Next, the pyrrolidine ring is oxidized by 6-hydroxy-L-nicotine oxidase, which acts strictly stereo-specific on the L-enantiomer. Surprisingly, L-nicotine also induces the synthesis of a 6-hydroxy-D-nicotine-specific oxidase in the bacteria. Genes of nicotine-degrading enzymes are located on the catabolic plasmid pAO1. The pAO1 sequence revealed that the 6-hydroxy-D-nicotine oxidase gene is flanked by two open reading frames with a similarity to amino acid permeases and a divergently transcribed open reading frame with a similarity to proteins of the tetracycline repressor TetR family. Reverse transcription PCR and primer extension analysis of RNA transcripts isolated from A. nicotinovorans pAO1 indicated that the 6-hydroxy-D-nicotine oxidase gene represents a transcriptional unit. DNA electromobility shift assays established that the purified TetR-similar protein represents the 6-hydroxy-D-nicotine oxidase gene repressor HdnoR and binds to the 6-hydroxy-D-nicotine oxidase gene operator with a K_d of 21 nM. The enantiomers 6-hydroxy-D- and 6-hydroxy-L-nicotine acted in vitro as inducers. In vivo analysis of 6-hydroxy-D-nicotine oxidase gene transcripts from bacteria grown with L- and D-nicotine confirmed this conclusion. The poor discrimination by HdnoR between the 6-hydroxy-L- and 6-hydroxy-D-nicotine enantiomers explains the presence of the 6-hydroxy-D-nicotine-specific enzyme in bacteria grown on L-nicotine.

Received for publication, July 18, 2003 , and in revised form, October 1, 2003.

^* This work was supported by a grant of the Deutsche Forschungsgemeinschaft (to R. B.) The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Molecular Biophysics, Rockefeller University, New York, NY.

To whom correspondence should be addressed: Inst. of Biochemistry and Molecular Biology, Hermann-Herder-Str. 7, 79104 Freiburg, Germany. Tel.: 49-761-2035231; Fax: 49-761-2035253; E-mail: roderich.brandsch{at}biochemie.uni-freiburg.de.

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