Exploring the potential impact of an expanded genetic code on protein function

Abstract

With few exceptions, all living organisms encode the same 20 canonical amino acids; however, it remains an open question whether organisms with additional amino acids beyond the common 20 might have an evolutionary advantage. Here, we begin to test that notion by making a large library of mutant enzymes in which 10 structurally distinct noncanonical amino acids were substituted at single sites randomly throughout TEM-1 β-lactamase. A screen for growth on the β-lactam antibiotic cephalexin afforded a unique p-acrylamido-phenylalanine (AcrF) mutation at Val-216 that leads to an increase in catalytic efficiency by increasing k cat , but not significantly affecting K M . To understand the structural basis for this enhanced activity, we solved the X-ray crystal structures of the ligand-free mutant enzyme and of the deacylation-defective wild-type and mutant cephalexin acyl-enzyme intermediates. These structures show that the Val-216-AcrF mutation leads to conformational changes in key active site residues-both in the free enzyme and upon formation of the acylenzyme intermediate-that lower the free energy of activation of the substrate transacylation reaction. The functional changes induced by this mutation could not be reproduced by substitution of any of the 20 canonical amino acids for Val-216, indicating that an expanded genetic code may offer novel solutions to proteins as they evolve new activities.noncanonical amino acid | beta-lactamase | catalytic activity | evolutionary advantage | conformational effects

Playing with the Molecules of Life

“…One noncanonical amino acid substitution led to an enzyme with increased catalytic efficiency; x-ray crystallographic analysis suggested that the ncAA functioned by restricting the conformation of the active site to more efficiently stabilize the rate-limiting transition state. 298, 299 Similarly, a metA variant was isolated from a random ncAA library using a temperature dependent selection scheme that was stabilized by a keto-containing amino acid by a remarkable 23 °C, and likely involves formation of a ketone adduct to a nucleophilic side chain at the homodimer interface. Similar in vitro evolution experiments have demonstrated that ncAAs with long chain thiols can form extended disulfide crosslinks that lead to significant protein stabilization.…”

The use of unnatural amino acids to study and engineer protein function

“…For example, replacing valine-216 in b-lactamase with p-acrylamido-phenylalanine (13) improved its catalytic efficiency eightfold [47]. None of the twenty natural amino acids conferred a similar enhancement, underlining the unique potential of UAAs in protein engineering and directed evolution.…”

Enhancing protein stability with extended disulfide bonds

“…For example, it has been recently shown that a unique noncanonical amino acid mutation in TEM-1 β-lactamase significantly increases the enzyme's catalytic activity for the substrate cephalexin, a result that cannot be recapitulated by substitution of canonical amino acids at this site (1). This same enzyme has been reengineered to be dependent on a noncanonical active site residue for activity, a dependency that was maintained for hundreds of generations without escape (2).…”