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doi: 10.1038/srep14881.

Highly Efficient CYP167A1 (EpoK) dependent Epothilone B Formation and Production of 7-Ketone Epothilone D as a New Epothilone Derivative

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Highly Efficient CYP167A1 (EpoK) dependent Epothilone B Formation and Production of 7-Ketone Epothilone D as a New Epothilone Derivative

Fredy Kern et al. Sci Rep..

Abstract

Since their discovery in the soil bacterium Sorangium cellulosum, epothilones have emerged as a valuable substance class with promising anti-tumor activity. Because of their benefits in the treatment of cancer and neurodegenerative diseases, epothilones are targets for drug design and pharmaceutical research. The final step of their biosynthesis - a cytochrome P450 mediated epoxidation of epothilone C/D to A/B by CYP167A1 (EpoK) - needs significant improvement, in particular regarding the efficiency of its redox partners. Therefore, we have investigated the ability of various hetero- and homologous redox partners to transfer electrons to EpoK. Hereby, a new hybrid system was established with conversion rates eleven times higher and Vmax of more than seven orders of magnitudes higher as compared with the previously described spinach redox chain. This hybrid system is the most efficient redox chain for EpoK described to date. Furthermore, P450s from So ce56 were identified which are able to convert epothilone D to 14-OH, 21-OH, 26-OH epothilone D and 7-ketone epothilone D. The latter one represents a novel epothilone derivative and is a suitable candidate for pharmacological tests. The results revealed myxobacterial P450s from S. cellulosum So ce56 as promising candidates for protein engineering for biotechnological production of epothilone derivatives.

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Figures

Figure 1
Figure 1. Conversion of epothilone D to B catalyzed by EpoK inS. cellulosum So ce90; electron transfer partners are unknown.
Figure 2
Figure 2
(A) Epothilone D conversions by EpoK with different electron transfer partners (spinach: ratio 1:100:0.025U; spinach*: ratio 1:100:0.125U for P450:Fdx:FdR; reductase in units); (B) Epothilone D conversions with EpoK, ferredoxin SynFdx (Fd) fromSynechocystis and selected reductases (ratio 1:10:1 in grey bars and ratio 1:20:3 in black bars); bars designated as “Literature” were calculated from published data using spinach redox system: dashed and black bar
Figure 3
Figure 3. Kinetic studies on epothilone B formation supported by EpoK/SynFdx/FNR hybrid system (ratio 1:20:3 for EpoK:SynFdx:FNR).
The Hill model with nH = 2 resulted in a sigmoidal fit for EpoK kinetics with a coefficient of determination (R2) of 0.99. The inset shows the Lineweaver-Burk plot of the data with R2 = 0.99.
Figure 4
Figure 4
(A) Phylogenetic tree of EpoK and CYPome of So ce56. The tree was constructed by using protein sequences and the “One Click” mode of the online phylogenetic analysis tool from Information Génomique et Structurale, Marseille, France. The bar in the left corner indicates 0.4 amino acid substitutions per amino acid for the branch length. (B) HPLC chromatogram of epothilone D (dashed line) and its conversion by CYP267B1/Fdx8/FdR_B (solid line).
Figure 5
Figure 5. Proposed collision-induced dissociation spectrum of 7-ketone epothilone D (m/z 490).
Loss of water at position C-3 results in a fragment withm/z 472. The fragment ion atm/z 402 appears after the loss of CO2 and C2H4O. Step-wise fragmentation of the precursor ion is proposed and presented counterclockwise.
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