title: 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.
authors.affiliation: Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037;
authors.forename: Han
authors.initials: H
authors.lastname: Xiao
authors.affiliation: Departments of Biological Sciences and Chemistry, Bridge Institute, University of Southern California, Los Angeles, CA 90089.
authors.forename: Fariborz
authors.initials: F
authors.lastname: Nasertorabi
authors.affiliation: Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037;
authors.forename: Sei-Hyun
authors.initials: SH
authors.lastname: Choi
authors.affiliation: Departments of Biological Sciences and Chemistry, Bridge Institute, University of Southern California, Los Angeles, CA 90089.
authors.forename: Gye Won
authors.initials: GW
authors.lastname: Han
authors.affiliation: Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037;
authors.forename: Sean A
authors.initials: SA
authors.lastname: Reed
authors.affiliation: Departments of Biological Sciences and Chemistry, Bridge Institute, University of Southern California, Los Angeles, CA 90089 schultz@scripps.edu stevens@usc.edu.
authors.forename: Raymond C
authors.initials: RC
authors.lastname: Stevens
authors.affiliation: Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037; schultz@scripps.edu stevens@usc.edu.
authors.forename: Peter G
authors.initials: PG
authors.lastname: Schultz
mesh_terms: D000596:Amino Acids / Q000235:genetics*; D002384:Catalysis; D002506:Cephalexin / Q000378:metabolism; D003001:Cloning, Molecular; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D019143:Evolution, Molecular; D005815:Genetic Code / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D015394:Molecular Structure; D011487:Protein Conformation; D014961:X-Ray Diffraction; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D001618:beta-Lactamases; D002506:Cephalexin
keywords: beta-lactamase; catalytic activity; conformational effects; evolutionary advantage; noncanonical amino acid