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 kcat, but not significantly affecting KM. 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 acyl-enzyme 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.

Keywords: beta-lactamase; catalytic activity; conformational effects; evolutionary advantage; noncanonical amino acid.

Fig. 1.

ncAA mutagenesis and a growth-based screen. (A) The TAG-scanned library was transformed into cells expressing a polyspecific amber suppressor tRNA/aaRS pair. The growth-based screen was carried out with various ncAAs, and the mutated residues within the target gene were determined by sequence analysis. (B) The structures of ncAAs used in this study. Suppression efficiencies of tRNA_CUA^Tyr/PolyRS pair in the absence and presence of various amino acids were evaluated by using a GFP-based assay.

Fig. 2.

Characterization of wild-type and mutant β-lactamases. (A) Structures of β-lactam antibiotics used in this study. (B) MICs of cephalexin for wild-type and V216X mutant enzymes. (C) Kinetic parameters for hydrolysis of cephalexin by wild-type and V216X mutant enzymes.

Fig. 3.

Crystal structures of wild-type and Val-216–AcrF mutant β-lactamases. (A) X-ray crystal structure of ligand-free wild-type enzyme (PDB ID code 1BTL). (B) X-ray crystal structure of benzylpenicillin acyl-enzyme intermediate for wild-type enzyme (PDB ID code 1FQG). (C) X-ray crystal structure of cephalexin acyl-enzyme intermediate for wild-type enzyme. (D) X-ray crystal structure of ligand-free Val-216–AcrF mutant enzyme. (E) X-ray crystal structure of cephalexin acyl-enzyme intermediate for the Val-216–AcrF mutant enzyme. (F) Overlay of active-site residues of cephalexin-bound and ligand-free Val-216–AcrF mutant enzymes. (G) X-ray crystal structure of ligand-free mutant enzyme with Val-216–AcrF substitution displayed as cyan spheres. (H) Packing interactions between cephalexin and side chains of mutant enzyme with Val-216–AcrF substitution. (I) Overlay of active site residues as spheres of cephalexin-bound and ligand-free mutant enzymes with Val-216–AcrF substitution.