Purification and characterization of a cam repressor (CamR) for the cytochrome P-450cam hydroxylase operon on the Pseudomonas putida CAM plasmid

Abstract

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 M 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.The cytochrome P-450cam hydroxylase operon (camDCAB) on the CAM plasmid of Pseudomonas putida PpG1 (ATCC 17453) is responsible for early steps of the D-camphor degradation pathway for catabolism of camphor to isobutyrate (17,18). D-Camphor degradation initiates oxidation to 5-exo-hydroxycamphor by a monooxygenase system which consists of three enzymes: NADH-putidaredoxin reductase (45 kDa; encoded by the camA gene), putidaredoxin (12 kDa; encoded by the camB gene), and cytochrome P-450cam (47 kDa; encoded by the camC gene) (7, 10). The second step is the conversion of alcohol to 2,5-diketocamphane, which is dehydrogenated by 5-exo-hydroxycamphor dehydrogenase (FdeH, 80 kDa; encoded by the camD gene) (14). Expression of the camDCAB operon and the camR gene is negatively regulated through interaction of the CamR protein with the single operator located in the overlapping promoter region between the camD-CAB operon and the camR gene. In the presence of D-camphor, these genes are divergently transcribed from the overlapping promoters (3, 12). When examining structural and functional properties of the CamR repressor, we constructed a heterologous expression system for overproduction of the CamR protein, with Escherichia coli harboring a plasmid for expression of CamR (3).We report here the purification and characterization of the CamR protein from an E. coli culture, in the presence of D-camphor. Both in vitro and in vivo, various camphor analogs inhibited binding of the CamR protein to the operator DNA. MATERIALS AND METHODSBacterial strains and plasmids. The bacterial strains used in this study were E. coli JM83 [FЈ ara ⌬(lac-proAB) rpsL(80lacZ⌬M15)] (21) and P. putida PpG277 (16). A CamR-overproducing plasmid, pHAOV1, was constructed as follows. The translational initiation codon of the camR gene was changed from GTG to ATG to increase translational efficiency, and then the camR gene was placed under the control of a bacteriophage PL promoter of the expression vector pUC-PL-cI (3, 19). Plasmid pHAOV1 wa...

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