EpoK, a Cytochrome P450 Involved in Biosynthesis of the Anticancer Agents Epothilones A and B. Substrate-Mediated Rescue of a P450 Enzyme

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.

Substrate Preferences and Catalytic Parameters Determined by Structural Characteristics of Sterol 14α-Demethylase (CYP51) from Leishmania infantum

“…This is the first observation of P450 recovery for a CYP51, although a reversible conversion of P420 into P450 has been seen before in other CYPs (e.g. EpoK (35) and CYP142 (36)), suggesting substratedependent structural rearrangements that lead to deprotonation of the heme-coordinating cysteine so that the thiolate (P450) instead of the neutral thiol (P420) (37) remains the proximal ligand upon reduction of the heme iron. More detailed studies are required to clarify the mechanism of this phenomenon in L. infantum CYP51.…”

Genome Mining in Sorangium cellulosum So ce56

“…The potent anticancer agent epothilone, for example, was discovered from S. cellulosum So ce90 (13,14). Epothilone is one of so far seven known myxobacterial compounds, the biosynthesis of which involves cytochromes P450 (15). Besides the epothilones, these are the antifungal leupyrrins (16) and the cytotoxic spirangienes (17) (also from S. cellulosum), the antibiotic myxovirescin from Myxococcus (18), the electron transport inhibitor stigmatellin (19) and the antibiotic aurafuron (20) from Stigmatella aurantiaca, and the antifungal ajudazols from Chondromyces crocatus (21).…”

Spectroscopic characterization of the ferric states of Amphitrite ornata dehaloperoxidase and Notomastus lobatus chloroperoxidase: His-ligated peroxidases with globin-like proximal and distal properties

“…HRP and Mb represent the limiting cases of homogenous five-coordinate His ligation and six-coordinate water/His ligation, respectively. MCD spectroscopy is a powerful technique for identifying axial ligands in structurally uncharacterized heme proteins by comparing their spectral properties with those of structurally defined heme proteins and model complexes [31][32][33][34][35][36].…”