Engineering high chemo-, regio-, and stereoselectivity is a prerequisite for enzyme usage in organic synthesis. Cytochromes P450 can oxidize a broad range of substrates, including macrocycles, which are becoming popular scaffolds for therapeutic agents. However, a large conformational space explored by macrocycles not only reduces the selectivity of oxidation but also impairs computational enzyme design strategies based on docking and molecular dynamics (MD) simulations. We present a novel design workflow that uses enhanced-sampling Hamiltonian replica exchange (HREX) MD and focuses on quantifying the substrate binding for suggesting the mutations to be made. This computational approach is applied to P450 BM3 with the aim to shift regioselectively toward one of the numerous possible positions during β-cembrenediol oxidation. The predictions are experimentally tested and the resulting product distributions validate our design strategy, as single mutations led up to 5-fold regioselectivity increases. We thus conclude that the HREX-MD-based workflow is a promising tool for the identification of positions for mutagenesis aiming at P450 enzymes with improved regioselectivity.
A variety of fusion proteins between the versatile redox partners flavodoxin (FldA) and flavodoxin reductase (Fpr) from Escherichia coli was constructed with the aim to improve the electron transfer properties. The order in which FldA and Fpr were fused and the linker region between them was varied in a systematic manner. A simple molecular tool, designated “DuaLinX”, was developed that facilitated the parallel introduction of flexible glycine-rich and rigid proline-rich linkers between the fusion partners in a single cloning event. The fusion constructs were tested for their ability to transfer electrons to cytochrome c and cytochrome P450 109B1 from Bacillus subtilis. With CYP109B1, the performance of the constructs showed, independent of the domain order, a strong dependency on linker length, whereas with cytochrome c this phenomenon was less pronounced. Constructs carrying linkers of ≥15 residues effectively supported the CYP109B1-catalysed hydroxylation of myristic acid. Constructs carrying proline-rich linkers generally outperformed their glycine-rich counterparts. The best construct, FldA-Fpr carrying linker ([E/L]PPPP)4, supported CYP109B1 activity equally well as equivalent amounts of the non-fused redox partners, while cytochrome c reductase activity was ~2.7-fold improved. Thus, to functionally connect redox partners, rigid proline-rich linkers may be attractive alternatives to the commonly used flexible glycine-rich linkers.
Recently,
the anesthetic (S)-ketamine has been
approved as a rapid-acting and long-lasting antidepressant. Its metabolite,
(2S,6S)-hydroxynorketamine, has
been found to have a similar antidepressant effect but with less undesirable
side effects, which make this compound an interesting target for synthesis.
Using the first-sphere mutagenesis of the cytochrome P450 154E1 from Thermobifida fusca YX, we constructed a triple mutant
that enables the effective production of (2S,6S)-hydroxynorketamine from (S)-ketamine.
This engineered P450 monooxygenase catalyzes the consecutive oxidative
N-demethylation and highly regio- and stereoselective C6-hydroxylation
reactions leading directly to the desired product with 85% product
selectivity. The integration of this selective monooxygenase into
an Escherichia coli whole-cell biocatalyst
allowed the production of (2S,6S)-hydroxynorketamine at a semipreparative scale. The metabolite was
purified and its structure was confirmed by NMR spectroscopy.
Cembranoids constitute a large family of 14-membered oxygenated macrocyclic diterpenoids with potential as therapeutic agents. Selective late-stage oxidations of cembranoid scaffolds remain a challenge for chemical catalysts but can be accomplished by enzymes. Here, a new chemoenzymatic route to oxyfunctionalized 14-membered macrocycles including cembranoids is described. This route combines a metal-catalyzed ring-closing metathesis with a subsequent P450 BM3-catalyzed hydroxylation and delivers cembranoid-like analogues. Systematic substrate probing with a set of synthetic 14-membered macrocycles revealed that the regioselectivity of a P450 BM3-based biocatalyst increased with increasing ring rigidity as well as size and polarity of the exocyclic substituents. Enzyme regioselectivity could further be improved by first-sphere active site mutagenesis. The V78A/F87A variant catalyzed hydroxylation of cembranoid-ol (9S/R)-3 d with 90 % regioselectivity for C5 position. Extensive NMR analysis of Mosher esters and single crystal X-ray structure determination revealed a remarkable diastereoselectivity of this P450 BM3 mutant depending on substrate stereochemistry, which led exclusively to the syn-cembranoid-diols (5S,9S)-4 and (5R,9R)-4.
Engineered cytochrome P450 monooxygenase CYP154E1 enables the effective synthesis of the potential antidepressant (2R,6R)-hydroxynorketamine via N-demethylation and regio- and stereoselective hydroxylation of (R)-ketamine.
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