P450-Catalyzed Regio- and Diastereoselective Steroid Hydroxylation: Efficient Directed Evolution Enabled by Mutability Landscaping

Abstract

Cytochrome P450 monooxygenases play a crucial role in the biosynthesis of many natural products and in the human metabolism of numerous pharmaceuticals. This has inspired synthetic organic and medicinal chemists to exploit them as catalysts in regio-and stereoselective CH-activating oxidation of structurally simple and complex organic compounds such as steroids. However, levels of regio-and stereoselectivity as well as activity are not routinely high enough for real applications. Protein engineering using rational design or directed evolution has helped in many respects, but simultaneous engineering of multiple catalytic traits such as activity, regioselectivity, and stereoselectivity, while overcoming tradeoffs and diminishing returns, remains a challenge. Here we show that the exploitation of information derived from mutability landscapes and molecular dynamics simulations for rationally designing iterative saturation mutagenesis constitutes a viable directed evolution strategy. This combined approach is illustrated by the evolution of P450 BM3 mutants which enable nearly perfect regio-and diastereoselective hydroxylation of five different steroids specifically at the C16-position with unusually high activity, while avoiding activity−selectivity trade-offs as well as keeping the screening effort relatively low. The C16 alcohols are of practical interest as components of biologically active glucocorticoids.

Two‐step Screening for Identification of Drug‐metabolizing Bacterial Cytochromes P450 with Diversified Selectivity

“…P450 BM3 wild type does not accept testosterone, but variants thereof carrying up to 20 mutations were reported to perform stereo‐ and regioselective oxidations of steroids like testosterone at position 2β, 15β and 16β …”

“…Although significant advances have been achieved to reduce screening efforts among P450 BM3 variants, the herein described P450s might represent valuable alternatives and new targets for protein engineering as they naturally convert steroids with quite high activity and moderate selectivity.…”

Directed Evolution of P450 Fatty Acid Decarboxylases via High‐Throughput Screening towards Improved Catalytic Activity

“…In all cases, a fast and accurate screening method for a specific catalytic trait is the crucial prerequisite of obtaining desired enzymes from numerous variants . While notable progress has been made in improving library quality, lack of efficient high‐throughput screening (HTS) methods can still hamper the application of DE, particularly, in large library screening of the reactions where substrates, products, or cofactors are not readily detectable unless using highly specified and sophisticated approaches such as GC or HPLC …”

One‐shot optimization of multiple enzyme parameters: Tailoring glucose oxidase for pH and electron mediators

“…The detailed methodology and a demonstration of its ability to identify mutants of four proteins with improved fitness have been reported elsewhere, although only one property was tested for each protein (Cadet et al, 2018a;Cadet et al, 2018b). Here, we applied innov'SAR to the problem of multiple parameter optimization, which has been tackled by other researchers, for example, to simultaneously optimize activity and stability (Giver, Gershenson, Freskgard, & Arnold, 1998), enantioselectivity and thermostability (Li, Zhang, Sun, Liu, & Reetz, 2016), thermostability, activity, and stereoselectivity (Acevedo-Rocha et al, 2018), activity and stereoselectivity (Hall, Sarkar, Lee, Munday, & Bell, 2016), or activity, thermostability, and pH range (Zheng et al, 2017). These earlier studies used classical experimental approaches such as iterative saturation mutagenesis (ISM) CASTing, random mutagenesis or recombination, screening, which did not include a machine learning component for fast and efficient screening and selection.…”