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.
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 Kd 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.
Sandu
Cristinel
C
Institute of Biochemistry and Molecular Biology, University of Freiburg, 79104 Freiburg, Germany.
Chiribau
Calin B
CB
Brandsch
Roderich
R
D000595:Amino Acid Sequence; D001173:Arthrobacter / Q000235:genetics*; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D005810:Multigene Family; D009538:Nicotine / Q000031:analogs & derivatives / Q000494:pharmacology*; D010088:Oxidoreductases / Q000235:genetics*; D011485:Protein Binding / Q000187:drug effects; D012097:Repressor Proteins; D013237:Stereoisomerism; D014158:Transcription, Genetic / Q000187:drug effects*
D012097:Repressor Proteins; D009538:Nicotine; D004247:DNA; D010088:Oxidoreductases; C021413:6-hydroxy-D-nicotine oxidase