Open AccessExpression, crystallization and preliminary diffraction studies of the Pseudomonas putida cytochrome P450cam operon repressor CamR
Author(s) -
Maenaka Katsumi,
Fukushi Kouji,
Aramaki Hironori,
Shirakihara Yasuo
Publication year - 2005
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309105023225
Subject(s) - pseudomonas putida , operon , repressor , crystallization , cytochrome , chemistry , microbiology and biotechnology , physics , biology , crystallography , biochemistry , gene , organic chemistry , gene expression , mutant , enzyme
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 m M 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 m M Na PIPES, 2 m M 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 m M Na PIPES, 2.5 m M MgCl 2 pH 7.3 at 298 K. Preliminary X‐ray diffraction studies revealed the space group to be orthorhombic ( P 2 1 2 1 2), with unit‐cell parameters a = 48.0, b = 73.3, c = 105.7 Å. Native and selenomethionyl derivative data sets were collected to 3 Å resolution at SPring‐8 and the Photon Factory.