Directed Evolution of a Designer Enzyme Featuring an Unnatural Catalytic Amino Acid

Abstract

The impressive rate accelerations that enzymes display in nature often result from boosting the inherent catalytic activities of side chains by their precise positioning inside a protein binding pocket. Such fine-tuning is also possible for catalytic unnatural amino acids. Specifically, the directed evolution of a recently described designer enzyme, which utilizes an aniline side chain to promote a model hydrazone formation reaction, is reported. Consecutive rounds of directed evolution identified several mutations in the promiscuous binding pocket, in which the unnatural amino acid is embedded in the starting catalyst. When combined, these mutations boost the turnover frequency (k_cat ) of the designer enzyme by almost 100-fold. This results from strengthening the catalytic contribution of the unnatural amino acid, as the engineered designer enzymes outperform variants, in which the aniline side chain is replaced with a catalytically inactive tyrosine residue, by more than 200-fold.

Keywords: directed evolution; enzyme catalysis; enzyme design; hydrazones; organocatalysis.

Figure 1

A) Chemical structure of p‐aminophenylalanine. B) Formation of an iminium ion intermediate in the presence of anilines accelerates hydrazone (X=NH) and oxime (X=O) formation reactions (for clarity, the reversibility of these reactions is not shown). C) Crystal structure of the LmrR_pAF homodimer (PDB: 6I8N). Catalytic aniline side chains (red) and Trp96 (pink) are shown as sticks. The positions of the β‐carbons of 13 additional residues that line the binding pocket of LmrR_pAF are shown as spheres. A 3‐(N‐morpholino)propanesulfonic acid (MOPS) buffer molecule that was found to be sandwiched between the central tryptophans is omitted for clarity (see Supporting Information).

Figure 2

A) Reaction conditions for the model hydrazone formation between NBD‐H and 4‐HBA in cleared lysates. B) Close‐up of the hydrophobic pore in LmrR_pAF. Trp96s and pAF15s shown as sticks; β‐carbons of positions that gave rise to improved variants in round one and two are shown as spheres (color code as indicated). C) Evolutionary optimization of LmrR_pAF. Apparent catalytic efficiencies (k _cat/K _m)_app) of selected variants and their improvement with respect to LmrR_pAF (see Table S1 and Figure S3 in the Supporting Information for a more detailed analysis); errors are standard deviations of at least three independent experiments; color code as in Figure 2 B. D) Comparison of saturation kinetics at a 4‐HBA concentration of 5 mm for LmrR_pAF and the best variants obtained after two rounds of directed evolution. Errors are standard deviations of at least three independent experiments.