Expression, crystallization and preliminary diffraction studies of the Pseudomonas putida cytochrome P450cam operon repressor CamR.
The Pseudomonas putida cam repressor (CamR) is a homodimeric protein that binds to the camO DNA operator to inhibit the transcription of the cytochrome P450cam operon camDCAB. CamR has two functional domains: a regulatory domain and a DNA-binding domain. The binding of the inducer D-camphor to the regulatory domain renders the DNA-binding domain unable to bind camO. Native CamR and its selenomethionyl derivative have been overproduced in Escherichia coli and purified. Native CamR was crystallized under the following conditions: (i) 12-14% PEG 4000, 50 mM Na PIPES, 0.1 M KCl, 1% glycerol pH 7.3 at 288 K with and without camphor and (ii) 1.6 M P(i), 50 mM Na PIPES, 2 mM camphor pH 6.7 at 278 K. The selenomethionyl derivative CamR did not crystallize under either of these conditions, but did crystallize using 12.5% PEG MME 550, 25 mM Na PIPES, 2.5 mM MgCl2 pH 7.3 at 298 K. Preliminary X-ray diffraction studies revealed the space group to be orthorhombic (P2(1)2(1)2), with unit-cell parameters a = 48.0, b = 73.3, c = 105.7 A. Native and selenomethionyl derivative data sets were collected to 3 A resolution at SPring-8 and the Photon Factory.
Maenaka
Katsumi
K
Structural Biology Center, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-8540, Japan.
Fukushi
Kouji
K
Aramaki
Hironori
H
Shirakihara
Yasuo
Y
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D019475:Camphor 5-Monooxygenase / Q000235:genetics*; D003460:Crystallization; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D009876:Operon / Q000235:genetics*; D016958:Pseudomonas putida / Q000737:chemistry*; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism*; D014961:X-Ray Diffraction
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D019475:Camphor 5-Monooxygenase