Engineering Thermostable CYP2D Enzymes for Biocatalysis Using Combinatorial Libraries of Ancestors for Directed Evolution (CLADE)

Abstract

The structure of metabolites of drug candidates must frequently be characterised during drug discovery and development. However, synthesising metabolites with the correct stereoselective modifications can be challenging for chemically complex parent compounds. Biocatalysis using human drug‐metabolising enzymes, such as cytochrome P450 2D6 (CYP2D6) is an alternative to chemical synthesis. However, most natural enzymes are unstable and have poor efficiency, limiting yields in preparative biotransformations. The aim of this study was to develop a library of robust mutant CYP2D enzymes for biocatalysis. The CLADE (combinatorial libraries of ancestors for directed evolution) approach increased the stability of CYP2D mutants obtained by DNA shuffling using three extant CYP2D forms. The resulting mutants showed divergent profiles of activity towards typical CYP2D substrates and included thermostable forms that may be useful for the further evolution of biocatalysts for specific applications.

Resurrection and characterization of ancestral CYP11A1 enzymes

“…In contrast to these P450s, CYP11A_N1 was not likely to have existed in a hot environment where thermal robustness was required, given that it is expected to have evolved around the time vertebrates emerged on earth and where temperatures were similar to those presently [9]. However, such high thermal robustness is in line with other ancestral vertebrate P450s previously reconstructed and characterized [27][28][29]. We propose that the observed high level of thermostability seen in these ancestors is a carryover from older P450 enzymes from primordial organisms which may have existed in hot, marine environments on the early Earth.…”

“…Microsomal P450 ancestors were shown in previous studies to possess substantially increased thermostability over their descendants [27][28][29]. For this reason, we tested the thermostability properties of the purified ancestral CYP11A1 enzymes via thermal unfolding studies.…”

“…CYP11A_N1 was of special interest since it showed only 55.0% sequence identity to the extant CYP11A1 form, suggesting potentially altered product or substrate selectivity which could provide novel insights into ancient steroid hormone biosynthesis. Furthermore, recent studies have revealed that microsomal ancestral P450s of similar age display a significant increase (up to 30 °C) in thermostability [27,28]. Thus, CYP11A_N1 was of additional interest to test whether the stabilization of the P450 fold via ASR also applies to mitochondrial P450 enzymes.…”

“…Just recently, the promising bioinformatic approach of ancestral sequence reconstruction (ASR) was successfully applied for the resurrection of protein ancestors of selected microsomal P450s, including the drug‐metabolizing mammalian enzymes CYP3A4 [27], CYP2D6 [28], and CYP1B1 [29]. The ASR process can in general be described in three steps: firstly known amino acid sequences of the respective phylogenetic group are collated and aligned to produce a multiple sequence alignment (MSA); secondly, the MSA is used to build a phylogenetic tree, which provides a visual and computational representation of the evolutionary relationships; and thirdly, both the phylogenetic tree and the MSA are used in tandem with an amino acid substitution rate matrix to infer the most likely ancestral state at each position in the alignment [30].…”

“…The ASR of microsomal P450 enzymes resurrected to date has provided intriguing insights into changes that translated to exceptional enzyme features such as increased thermostability, altered product selectivity, or improved catalytic activity [28,29]. Despite the recent success in resurrection of microsomal P450s, ASR has not been applied yet to mitochondrial cytochromes P450.…”