Bacterial cytochrome P450 enzymes: Semi-rational design and screening of mutant libraries in recombinant Escherichia coli cells.

Bacterial cytochromes P450 (P450s) have been recognized as attractive targets for biocatalysis and protein engineering. They are soluble cytosolic enzymes that demonstrate higher stability and activity than their membrane-associated eukaryotic counterparts. Many bacterial P450s possess broad substrate spectra and can be produced in well-known expression hosts like Escherichia coli at high levels, which enables quick and convenient mutant libraries construction. However, the majority of bacterial P450s interacts with two auxiliary redox partner proteins, which significantly increase screening efforts. We have established recombinant E. coli cells for screening of P450 variants that rely on two separate redox partners. In this chapter, a case study on construction of a selective P450 to synthesize a precursor of several chemotherapeutics, (-)-podophyllotoxin, is described. The procedure includes co-expression of P450 and redox partner genes in E. coli with subsequent whole-cell conversion of the substrate (-)-deoxypodophyllotoxin in 96-deep-well plates. By omitting the chromatographic separation while measuring mass-to-charge ratios specific for the substrate and product via MS in so-called multiple injections in a single experimental run (MISER) LC/MS, the analysis time could be drastically reduced to roughly 1 min per sample. Screening results were verified by using isolated P450 variants and purified redox partners.

Molecular evolution of a cytochrome P450 for the synthesis of potential antidepressant (2R,6R)-hydroxynorketamine.

Saturation mutagenesis at seven first-sphere residues of the cytochrome P450 monooxygenase 154E1 (CYP154E1) from Thermobifida fusca YX was applied to construct a variant with only three substitutions that enabled the effective two-step synthesis of the potential antidepressant (2R,6R)-hydroxynorketamine. A recombinant E. coli whole-cell system was essential for GC/MS based medium-throughput screening and at the same time facilitated the oxidation of the substrate (R)-ketamine at a higher scale for product isolation and subsequent NMR analysis.

Chemoenzymatic Route to Oxyfunctionalized Cembranoids Facilitated by Substrate and Protein Engineering.

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.

Simulation-Guided Design of Cytochrome P450 for Chemo- and Regioselective Macrocyclic Oxidation.

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 ß-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.

Design and improvement of artificial redox modules by molecular fusion of flavodoxin and flavodoxin reductase from Escherichia coli.

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.