Abstract
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.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Antineoplastic Agents / antagonists & inhibitors
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Antineoplastic Agents / chemistry*
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Antineoplastic Agents / isolation & purification
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Bacterial Proteins / antagonists & inhibitors
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Bacterial Proteins / chemistry*
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Bacterial Proteins / isolation & purification
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Binding Sites
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Circular Dichroism
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Cytochrome P-450 Enzyme Inhibitors
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Cytochrome P-450 Enzyme System / chemistry*
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Cytochrome P-450 Enzyme System / isolation & purification
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Enzyme Inhibitors / chemistry
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Epothilones / biosynthesis*
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Epoxy Compounds / chemistry
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Imidazoles / chemistry
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Kinetics
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Ligands
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Myxococcales / enzymology
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Oxidation-Reduction
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Oxidoreductases / antagonists & inhibitors
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Oxidoreductases / chemistry*
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Oxidoreductases / isolation & purification
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Protein Binding
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Recombinant Proteins / antagonists & inhibitors
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Recombinant Proteins / chemistry
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Recombinant Proteins / isolation & purification
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Spectrophotometry, Ultraviolet
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Substrate Specificity
Substances
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Antineoplastic Agents
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Bacterial Proteins
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Cytochrome P-450 Enzyme Inhibitors
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Enzyme Inhibitors
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Epothilones
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Epoxy Compounds
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Imidazoles
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Ligands
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Recombinant Proteins
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epothilone A
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sulconazole
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Cytochrome P-450 Enzyme System
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EpoK protein, Polyangium cellulosum
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Oxidoreductases
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epothilone B