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

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.

Structural insights into oxidation of medium-chain fatty acids and flavanone by myxobacterial cytochrome P450 CYP267B1

“…According to a structural alignment performed against the whole PDB archive, the structure of CYP267B1 shows the highest match to "closed" crystal structures of PikC, another P450 accepting macrolides as substrates [59], ( Figure 4A, Supplementary Table 2). Similar to PikC, the inner active site cavity of CYP267B1 is very spacious, consistent with its ability to accept a great variety of substrate sizes, ranging from very small ones like α-ionone [22] to large macrocyclic compounds such as epothilone D [24]. Towards the heme the pocket narrows, restricting access to the heme-iron, in particular due to the presence of residue Leu92 located in the substrate recognition site 1 (SRS1) ( Figure 4B).…”

“…Both enzymes convert epothilone D, but a superposition with the structure of epothilone D-bound EpoK reveals that this substrate cannot adopt the same binding mode in the active site pocket of CYP267B1, due to a clash with His90 ( Figure 4B). This difference may be related to previous observations that these enzymes convert epothilone D with quite different regioselectivities: while EpoK converts epothilone D exclusively to epothilone B, CYP267B1 oxidizes epothilone D to 5 different products [24]. The large pocket of CYP267B1 likely allows epothilone D to adopt several alternate binding modes resulting in the broad regioselectivity.…”

“…A recently characterized bacterial P450 with an exceptionally broad substrate scope is CYP267B1 from the myxobacterium Sorangium cellulosum So ce56 [20], one of the 21 P450 monooxygenases identified in this bacterium [21]. Without the need for any protein engineering, the enzyme was shown to readily accept different classes of substrates, from small carotenoid-derived aroma compounds or sesquiterpenes [22,23] to sterically challenging molecules like epothilone D [24]. CYP267B1 was also shown to oxidize several structurally diverse drugs, such as diclofenac, ibuprofen, oxymetazoline or repaglinide, as well as antidepressant or antipsychotic drugs like amitriptyline, chlorpromazine, imipramine or promethazine [25,26].…”

An indole‐deficient Escherichia coli strain improves screening of cytochromes P450 for biotechnological applications

“…Inspired by these reports, we have been engaged in studying the CYPome of the myxobacterium Sorangium cellulosum So ce56, which contains 21 CYP encoding genes . Within our studies, we were also able to reveal the potential of some myxobacterial CYPs as drug metabolizers, especially for antidepressant, antipsychotic, and anticancer drugs, in which an Escherichia coli based whole‐cell biocatalyst has been employed for the bioconversion . Aside from the screening of orphan CYPs, enzyme improvement by molecular evolution is of general interest whether to enhance poor turnover numbers, find new nonnatural reactions, or switch stereoselectivities .…”

Determinants of thermostability in the cytochrome P450 fold

“…The most thermostable Fd that has been identified to interact with multiple class I P450s is the C-terminal domain of an electron transfer protein (Etp1) from the fission yeast, Schizosaccharomyces pombe, Etp1 fd [122][123][124][125][126]. Etp1 fd is homologous to vertebrate-type Fds [122] and has been characterized with respect to its thermal stability as the full length (Etp1 fd (505-631)) and a truncated form (Etp1 fd (516-618)) created for crystallization [127].…”