Directed evolution of enantioselective hybrid catalysts: a novel concept in asymmetric catalysis

The concept of directed evolution of enantioselective hybrid catalysts was proposed in 2001/2002 and implemented experimentally for the first time in a proof-of-concept study in 2006. The idea is based on directed evolution, which comprises repeating cycles of random gene mutagenesis/expression/screening in a Darwinistic sense for the purpose of improving the catalytic profile of enzymes. In the case of hybrid catalysts, mutagenesis/expression of a protein is first performed with formation of a library of mutants, which are then modified chemically en masse with the introduction of an appropriate achiral ligand system harboring a transition metal. Screening these mutant hybrid catalysts in a given transition metal-catalyzed reaction then leads to an improved catalyst, so that the corresponding gene can be used to start another evolutionary cycle. This process can be repeated as often as needed until the desired catalytic profile has been reached, e.g., enhanced enantioselectivity.     

Directed evolution of enzymes for product chemistry

This review discusses the achievements of directed evolution of enzymes relevant to natural and unnatural product chemistry focusing on the period 1998-2004, citing 156 references. A few selected papers published before 1998 are also cited for their fundamental relevance, together with additional reviews covering other aspects of directed evolution applied to closely related topics. An overview of the latest methods applied in directed evolution is given in the introduction, together with a range of approaches for the selection of variants of enzymes with desired catalytic properties. All classes of enzymes are systematically covered.     

Computer simulations of enantioselective ester hydrolyses catalyzed by Pseudomonas cepacia lipase

On the basis of the X-ray crystal structure of the lipase from Pseudomonas cepacia (PcL)-an enzyme representative for a whole family of Pseudomonas lipases (lipase PS, SAM-2, AK 10, and others with a high degree of homology with PcL)-a computational study was performed to rationalize both the enantioselectivity and substrate specificity (tolerance) displayed by this lipase in the enantioselective hydrolysis of racemic esters 1a-12a from various secondary aromatic alcohols. The major goal of this project was the development of a binding model for PcL which is able to rationalize the experimental findings to predict "a priori the enantioselective behavior of PcL toward a wider range of substrates. A two-step modeling procedure, namely, docking experiments followed by construction of tetrahedral intermediates, was used for the simulation of the involved enzyme-substrate recognition/hydrolysis processes. The study of the recognition process (docking experiments) led to unambiguous identification of the binding geometry for the two enantiomeric series of substrates, but did not suggest a definitive interpretation of the behavior of PcL. Taking into consideration the stereoelectronic requirements of the enzymatic hydrolysis reaction, both the enantioselectivity and tolerance of the enzyme were then explained through the study of the tetrahedral intermediates, in turn constructed from the calculated docking geometries of 1a-12a.     

Directed evolution of the fatty-acid hydroxylase P450 BM-3 into an indole-hydroxylating catalyst

The self-sufficient cytochrome P450 BM-3 enzyme from Bacillus megaterium catalyzes subterminal hydroxylation of saturated long-chain fatty acids and structurally related compounds. Since the primary structure of P450 BM-3 is homologous to that of mammalian P450 type II, it represents an excellent model for this family of enzymes. During studies on the directed evolution of P450 BM-3 into a medium-chain fatty-acid hydroxylase, several mutants, in particular the triple mutant Phe87Val, Leu188Gln, Ala74Gly, were observed to hydroxylate indole, producing indigo and indirubin at a catalytic efficiency of 1365 M(-1)s(-1) (kcat=2.73 s(-1) and Km=2.0 mM). Both products were unequivocally characterized by NMR and MS analysis. Wild-type P450 BM-3 is incapable to hydroxylate indole. These results demonstrate that an enzyme can be engineered to catalyze the transformation of substrates with structures widely divergent from those of its native substrate.     

Directed evolution and axial chirality: optimization of the enantioselectivity of Pseudomonas aeruginosa lipase towards the kinetic resolution of a racemic allene

Directed evolution of Pseudomonas aeruginosa lipase by the use of combinatorial active site saturation test (CAST) criteria provided a highly enantioselective mutant (Leu162Phe) for kinetic resolution of an axially chiral allene, p-nitrophenyl 4-cyclohexyl-2-methylbuta-2,3-dienoate (E=111); the high enantioselectivity of the Leu162Phe mutant was rationalized by pi-pi stacking.     

Kinetic resolution of cyanohydrins via enantioselective acylation catalyzed by lipase PS-30

By using lipase PS-30 as catalyst, the kinetic resolution of a series of racemic cyanohydrins has been achieved via enantioselective acylation. The values of kinetic enantiomeric ratio (E) reached up to 314. Substituent effect is also briefly discussed.     

Reversible activity changes of lipid-coated lipase for enantioselective esterification in supercritical fluoroform

Enzymatic activity of a lipid-coated lipase for enantioselective esterification of (R)-1-phenylethanol in supercritical fluoroform can be reversibly controlled by changing pressure or temperature that reflects changes of dielectric constant.     

Isolation of lipase producer and its performance in enantioselective hydrolysis of glycidyl butyrate

A racemic glycidyl butyrate resolving strain, preliminarily identified as a Rhizopus sp., had been isolated from soil. Its extracellular lipase was found to enantioselectively hydrolyze the (S)-enantiomer of the chiral ester, with optimal activities at pH 5.3 and 42°C. Higher en antioselectivity of theenzyme was observed at lower temperatures, while the best anantioselectivity was obtained at pH 5.5–6.0, with an, E value (enantiomeric ratio) of 57.     

Directed evolution of cyclic peptides for inhibition of autophagy

In recent decades it has become increasingly clear that induction of autophagy plays an important role in the development of treatment resistance and dormancy in many cancer types. Unfortunately, chloroquine (CQ) and hydroxychloroquine (HCQ), two autophagy inhibitors in clinical trials, suffer from poor pharmacokinetics and high toxicity at therapeutic dosages. This has prompted intense interest in the development of targeted autophagy inhibitors to re-sensitize disease to treatment with minimal impact on normal tissue. We utilized Scanning Unnatural Protease Resistant (SUPR) mRNA display to develop macrocyclic peptides targeting the autophagy protein LC3. The resulting peptides bound LC3A and LC3B—two essential components of the autophagosome maturation machinery—with mid-nanomolar affinities and disrupted protein–protein interactions (PPIs) between LC3 and its binding partners in vitro. The most promising LC3-binding SUPR peptide accessed the cytosol at low micromolar concentrations as measured by chloroalkane penetration assay (CAPA) and inhibited starvation-mediated GFP-LC3 puncta formation in a concentration-dependent manner. LC3-binding SUPR peptides re-sensitized platinum-resistant ovarian cancer cells to cisplatin treatment and triggered accumulation of the adapter protein p62 suggesting decreased autophagic flux through successful disruption of LC3 PPIs in cell culture. In mouse models of metastatic ovarian cancer, treatment with LC3-binding SUPR peptides and carboplatin resulted in almost complete inhibition of tumor growth after four weeks of treatment. These results indicate that SUPR peptide mRNA display can be used to develop cell-penetrating macrocyclic peptides that target and disrupt the autophagic machinery in vitro and in vivo.

SUPR peptide mRNA display was used to evolve a cell-permeable, macrocyclic peptide for autophagy inhibition.     

Directed evolution of streptavidin variants using in vitro compartmentalization

We have developed and implemented an in vitro compartmentalization (IVC) selection scheme for the identification of streptavidin (SA) variants with altered specificities for the biotin analog desthiobiotin. Wild-type SA and selected variants bind desthiobiotin with similar affinities ( approximately 10(-13) M), but the variants have off rates almost 50 times slower and a half-life for dissociation of 24 hr at 25 degrees C. The utility of streptavidin variants with altered specificities and kinetic properties was shown by constructing protein microarrays that could be used to differentially organize and immobilize DNAs bearing these ligands. The methods we have developed should prove to be generally useful for generating a variety of novel SA reagents and for evolving other extremely high-affinity protein:ligand couples.     

Directed evolution of hybrid enzymes: Evolving enantioselectivity of an achiral Rh-complex anchored to a protein

The concept of utilizing the methods of directed evolution for tuning the enantioselectivity of synthetic achiral metal-ligand centers anchored to proteins has been implemented experimentally for the first time.     

Directed evolution of high-affinity antibody mimics using mRNA display

We constructed a library of >10(12) unique, covalently coupled mRNA-protein molecules by randomizing three exposed loops of an immunoglobulin-like protein, the tenth fibronectin type III domain (10Fn3). The antibody mimics that bound TNF-alpha were isolated from the library using mRNA display. Ten rounds of selection produced 10Fn3 variants that bound TNF-alpha with dissociation constants (K(d)) between 1 and 24 nM. After affinity maturation, the lowest K(d) measured was 20 pM. Selected antibody mimics were shown to capture TNF-alpha when immobilized in a protein microarray. 10Fn3-based scaffold libraries and mRNA-display allow the isolation of high-affinity, specific antigen binding proteins; potential applications of such binding proteins include diagnostic protein microarrays and protein therapeutics.     

Catalytic enantioselective stereoablative reactions: an unexploited approach to enantioselective catalysis

Approaches to the preparation of enantioenriched materials via catalytic methods that destroy stereogenic elements of a molecule are discussed. Although these processes often decrease overall molecular complexity, there are several notable advantages including material recycling, enantiodivergence and convergence, and increased substrate scope. Examples are accompanied by discussion of the critical design elements required for the success of these methods.     

Directed evolution and substrate specificity profile of homing endonuclease I-SceI

The laboratory evolution of enzymes with tailor-made DNA cleavage specificities would represent new tools for manipulating genomes and may enhance our understanding of sequence-specific DNA recognition by nucleases. Below we describe the development and successful application of an efficient in vivo positive and negative selection system that applies evolutionary pressure either to favor the cleavage of a desired target sequence or to disfavor the cleavage of nontarget sequences. We also applied a previously described in vitro selection method to reveal the comprehensive substrate specificity profile of the wild-type I-SceI homing endonuclease. Together these tools were used to successfully evolve mutant I-SceI homing endonucleases with altered DNA cleavage specificities. The most highly evolved enzyme cleaves the target mutant DNA sequence with a selectivity that is comparable to wild-type I-SceI's preference for its cognate substrate.     

Directed evolution of enzymes: new biocatalysts for asymmetric synthesis

Directed evolution has been employed to generate new enzymes for the deracemisation of chiral amines.