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 (kcat) 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.

Synergistic catalysis in an artificial enzyme by simultaneous action of two abiological catalytic sites

“…1,2 A key challenge in the design of such artificial enzymes is the creation of the active site. An important approach involves the introduction of abiological catalytically active moieties, which could be transition metal complexes or organocatalytic groups, [3][4][5][6][7] in stable protein scaffolds that give rise to basal level of activities, that can then be improved by fine-tuning the protein environment by mutagenesis. 8,9 Current efforts towards the creation of artificial enzymes have focused exclusively on introducing a single abiological catalytic moiety.…”

“…stop codon suppression. [22][23][24] Recently we have reported on the application of this methodology for the creation of a designer enzyme featuring a unnatural catalytic paminophenylalanine (pAF) residue, 4,5 Here, the aniline side chain of pAF was used as nucleophilic catalyst for formation of hydrazones from aldehydes ( Figure 1-c). The reaction involves the transient formation of an iminium ion intermediate, which is a common activation strategy in many organocatalytic reactions.…”

Artificial Metalloenzymes: From Selective Chemical Transformations to Biochemical Applications

“…A related oxime formation reaction was also tested and LmrR(V15pAF) exhibited satisfactory performance, highlighting its proficient activity as a general nucleophilic catalyst. Mayer, Roelfes, and coworkers applied directed evolution to LmrR(V15pAF) for hydrazine formation [ 93 ]. They targeted 13 residues forming the hydrophobic pore of LmrR(V15pAF) and conducted several rounds of evolution to identify beneficial mutations.…”

Break the template boundary: Test of different protein scaffolds by genetic code expansion resulted in NADPH-dependent secondary amine catalysis

“…Genetic code expansion has the advantage of fewer restrictions on the choice of protein scaffolds and the site therein. [27][28][29][30][31] Previously, an aniline motif has been added to the multidrug binding protein LmrR by incorporating unnatural amino acid p-azidophenylalanine followed by chemical reduction. [27][28][29][30] Whilst being less nucleophilic than pyrrolidine, 8,10 the aniline in LmrR was able to catalyze various carbonheteroatom and carbon-carbon ligation reactions.…”

“…[27][28][29][30] Whilst being less nucleophilic than pyrrolidine, 8,10 the aniline in LmrR was able to catalyze various carbonheteroatom and carbon-carbon ligation reactions. [27][28][29][30] Another example is the introduction of a N-methyl histidine residue into the computationally designed protein BH32; 31 subsequent laboratory evolution afforded a catalyst capable of hydrolyzing uorescein esters through formation of a covalent intermediate. Previously, secondary amines have been incorporated into protein scaffolds (e.g., βgalactosidase) as pyrrolysine analogues, in which proline and its derivatives were attached to lysine through isopeptide bonds.…”