Evaluation of alkoxyresorufins as fluorescent substrates for cytochrome P450 BM3 and site-directed mutants
Section snippets
Materials
The alkoxyresorufins were purchased from Sigma–Aldrich. The plasmid pT1-P450BM3 containing the BM3 cDNA under control of the temperature-inducible PRPL promoter was a gift from R. D. Schmid (Institute of Technical Biochemistry, University of Stuttgart, Germany). The bacteriophage U3 was a gift from M. Kranendonk (Department of Genetics, Faculty of Medical Sciences, University of Lisbon, Portugal). All other chemicals were of analytical grade and were obtained from standard suppliers.
Site-directed mutagenesis
Five
Expression of BM3
All five mutants were expressed successfully in E. coli DH5α cells, and the triple mutant was also expressed in DH5α LPSd cells. All expression levels were comparable to those of wild-type BM3. For all of the expressed enzymes, a typical culture yielded 300 to 400 nM of enzyme, and no P420—the inactive form of P450—could be detected (Fig. 2). This shows that the presence of the mutations did not influence the enzyme expression levels or the stability. After the isolation procedure, the cytosolic
Discussion
The aim of this study was to develop a fluorescent high-throughput screening method for BM3 that can be used for inhibition screening. Such an assay would be of great value to identify novel ligands that are able to bind BM3 or BM3 mutants. Because of their favorable fluorescence properties and their structural similarity to drug-like molecules, alkoxyresorufins were selected as potential substrates for BM3. Of the four alkoxyresorufins that were tested on wild-type and mutant forms BM3,
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