Purification and characterization of a cam repressor (CamR) for the cytochrome P-450cam hydroxylase operon on the Pseudomonas putida CAM plasmid.
The cytochrome P-450cam hydroxylase operon of Pseudomonas putida PpG1 (ATCC 17543) encodes proteins responsible for early steps of the degradation of D-camphor. Transcription of this operon is negatively controlled by the cam repressor (CamR), and the expression of camR is autoregulated. CamR was purified from Escherichia coli harboring an overproducing plasmid. The repressor forms a homodimer with a molecular mass of 40 kDa, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and gel filtration. CamR protected a specific DNA region from attack by DNase I. This region contains a palindromic operator of the cytochrome P-450cam hydroxylase operon and of the camR gene. Protection was inhibited by the addition of 60 microM D-camphor and also by certain camphor analogs and degradation products, including D-3-bromocamphor, adamantane, 2-adamantanone, 5-exo-hydroxycamphor, and 2,5-diketocamphane. These analogs and degradation products induced cytochrome P-450cam hydroxylase operon expression in vivo.
Aramaki
H
H
Department of Microbiology, Daiichi College of Pharmaceutical Sciences, Fukuoka, Japan.
Sagara
Y
Y
Kabata
H
H
Shimamoto
N
N
Horiuchi
T
T
D001483:Base Sequence; D001665:Binding Sites; D002164:Camphor / Q000494:pharmacology; D019475:Camphor 5-Monooxygenase; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D006899:Mixed Function Oxygenases / Q000235:genetics*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D009875:Operator Regions, Genetic; D009876:Operon; D016958:Pseudomonas putida / Q000201:enzymology / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics* / Q000302:isolation & purification
D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D012097:Repressor Proteins; D002164:Camphor; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D019475:Camphor 5-Monooxygenase