7-Benzyloxyquinoline oxidation by P450eryF A245T: finding of a new fluorescent substrate probe.
The main objective of the present study was to find a fluorescent substrate probe for cytochrome P450eryF (P450eryF). P450eryF is a bacterial P450 that catalyzes the hydroxylation of 6-deoxyerythronolide B at the 6S position, a necessary step in the biosynthesis of erythromycin. The lack of a conserved threonine residue in the I-helix, in contrast to other P450s, makes P450eryF unable to oxidize other substrates. A recent study [Xiang et al. (2000) J. Biol. Chem. 275, 35999-36006] has shown that the substitution of Ala-245 by threonine confers on P450eryF significant testosterone hydroxylase activity. Therefore, we investigated various known fluorescent P450 substrates with P450eryF wild-type as well as two mutants, A245S and A245T. Among the various fluorescent compounds tested, 7-benzyloxyquinoline (7-BQ) was found to be the most suitable probe for P450eryF A245T, with rates of oxidation being lower for A245S and wild-type enzyme. The steady-state kinetics of 7-BQ oxidation by A245T are sigmoidal (V(max) = 0.71 nmol/min/nmol, n = 2.18, and S(50) = 132 microM). alpha-Naphthoflavone (alpha-NF), a well-known activator of CYP3A4, did not stimulate 7-BQ oxidation by A245T, although the S(50) value for alpha-NF binding to wild-type P450eryF was similar to P450 3A4. Interestingly, spectral binding studies of wild-type P450eryF and A245T with ketoconazole and miconazole showed differential binding behaviors. Titration of wild-type with ketoconazole and miconazole and of A245T with miconazole showed the expected type-II binding. However, titration of A245T with ketoconazole produced a spectrum similar to type-I. Inhibition studies showed that both ketoconazole and miconazole are able to inhibit 7-BQ oxidation by A245T, although miconazole showed a slightly higher potency. In brief, the present study reports the discovery of 7-BQ as the first fluorescent and only the second unnatural substrate, and of miconazole as an effective P450eryF inhibitor.
Khan
Kishore K
KK
Department of Pharmacology and Toxicology, University of Texas Medical Branch, 301 University Boulevard, Galveston 77555-1031, USA. kkkhan@utmb.edu
Halpert
James R
JR
D000736:Androstenes / Q000378:metabolism; D001426:Bacterial Proteins; D002478:Cells, Cultured; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D005822:Genetic Vectors / Q000138:chemical synthesis; D007654:Ketoconazole / Q000378:metabolism; D008024:Ligands; D008825:Miconazole / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D010616:Phenanthrenes / Q000378:metabolism; D011804:Quinolines / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012044:Regression Analysis; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013997:Time Factors
D000736:Androstenes; D001426:Bacterial Proteins; D065607:Cytochrome P-450 Enzyme Inhibitors; D005456:Fluorescent Dyes; D008024:Ligands; D010616:Phenanthrenes; D011804:Quinolines; D011994:Recombinant Proteins; C065956:7-benzyloxyquinoline; C041014:9-aminophenanthrene; D008825:Miconazole; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C054444:eryF protein, Saccharopolyspora erythraea; D007654:Ketoconazole; D010100:Oxygen