Controlling the enantioselectivity of enzymes by directed evolution: practical and theoretical ramifications

Abstract

A fundamentally new approach to asymmetric catalysis in organic chemistry is described based on the in vitro evolution of enantioselective enzymes. It comprises the appropriate combination of gene mutagenesis and expression coupled with an efficient high-throughput screening system for evaluating enantioselectivity (enantiomeric excess assay). Several such cycles lead to a "Darwinistic" process, which is independent of any knowledge concerning the structure or the mechanism of the enzyme being evolved. The challenge is to choose the optimal mutagenesis methods to navigate efficiently in protein sequence space. As a first example, the combination of error-prone mutagenesis, saturation mutagenesis, and DNA-shuffling led to a dramatic enhancement of enantioselectivity of a lipase acting as a catalyst in the kinetic resolution of a chiral ester. Mutations at positions remote from the catalytically active center were identified, a surprising finding, which was explained on the basis of a novel relay mechanism. The scope and limitations of the method are discussed, including the prospect of directed evolution of stereoselective hybrid catalysts composed of robust protein hosts in which transition metal centers have been implanted.

Fig. 1.

Directed evolution of an enantioselective enzyme (11, 12, 41).

Fig. 2.

General concept of assembly of designed nucleotides (26); two strategies for the linking of fragments are possible (cases I and II). In case I the two genes, A and B, that are to be virtually shuffled are aligned, the different colored stars refer to information that encodes different amino acids, whereas oligonucleotide fragments with both colored stars in the same position of the parent gene denote the synthetic oligonucleotide fragment with degenerate nucleotides. The gray blocks denote conserved regions of sequence that can be used as the linking part with homologous recombination. Case II shows no homology between flanking oligos, which can be assembled by ligation between single-stranded DNA with unknown terminal sequences.

Fig. 3.

Increasing the E values of the lipase-catalyzed hydrolysis of the chiral ester 1 by cumulative mutations caused by epPCR (10, 12).

Fig. 4.

Extended combinatorial multiple-cassette mutagenesis in the evolution of an (S)-selective lipase variant (green star, position 20; purple star, position 161; yellow star, position 234; red circle, position 53; orange circle, position 180; blue circle, position 272) (37).

Fig. 5.

Schematic summary of the directed evolution of enantioselective enzymes (lipase variants) catalyzing the hydrolytic kinetic resolution of ester 1 (37). CMCM, combinatorial multiple-cassette mutagenesis.

Fig. 6.

Mechanism of lipase-catalyzed hydrolysis of esters (7, 9).

Fig. 7.

Crystal structure of the wild-type lipase from P. aeruginosa (40) with the active center (blue) and six mutations (yellow) of variant J (46).

Fig. 8.

Comparison between the wild-type (Left) and the double mutant S53P+L162G (Right) (46).

Fig. 9.

Concept of directed evolution of hybrid catalysts (, –68).