EpoK, a cytochrome P450 involved in biosynthesis of the anticancer agents epothilones A and B. Substrate-mediated rescue of a P450 enzyme.
The epothilones are a new class of highly promising anticancer agents with a mode of action akin to that of paclitaxel but with distinct advantages over that drug. The principal natural compounds are epothilones A and B, which have an epoxide in the macrocyclic lactone ring, and C and D, which have a double bond instead of the epoxide group. The epoxidation of epothilones C and D to A and B, respectively, is mediated by EpoK, a cytochrome P450 enzyme encoded in the epothilone gene cluster. Here we report high-yield expression of EpoK, characterization of the protein, demonstration that the natural substrate can prevent-and even reverse-denaturation of the protein, identification of ligands and surrogate substrates, development of a high-throughput fluorescence activity assay based on the H(2)O(2)-dependent oxidation of 7-ethoxy-4-trifluoromethylcoumarin, and identification of effective inhibitors of the enzyme. These results will facilitate improvements in the yields of epothilones C and D and the engineering of EpoK to prepare novel epothilone analogues. Furthermore, the finding that the denatured enzyme is rescued by the substrate offers a potential paradigm for control of the P450 catalytic function.
Ogura
Hiroshi
H
Department of Pharmaceutical Chemistry, University of California, 600 16th Street, San Francisco, California 94143-2280, USA.
Nishida
Clinton R
CR
Hoch
Ute R
UR
Perera
Roshan
R
Dawson
John H
JH
Ortiz de Montellano
Paul R
PR
D000970:Antineoplastic Agents / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D001665:Binding Sites; D002942:Circular Dichroism; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000302:isolation & purification; D004791:Enzyme Inhibitors / Q000737:chemistry; D034261:Epothilones / Q000096:biosynthesis*; D004852:Epoxy Compounds / Q000737:chemistry; D007093:Imidazoles / Q000737:chemistry; D007700:Kinetics; D008024:Ligands; D009231:Myxococcales / Q000201:enzymology; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D011485:Protein Binding; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
D000970:Antineoplastic Agents; D001426:Bacterial Proteins; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D034261:Epothilones; D004852:Epoxy Compounds; D007093:Imidazoles; D008024:Ligands; D011994:Recombinant Proteins; C093787:epothilone A; C033308:sulconazole; D003577:Cytochrome P-450 Enzyme System; C403885:EpoK protein, Polyangium cellulosum; D010088:Oxidoreductases; C093788:epothilone B