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

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. In this paper, 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 acyl-enzyme intermediate—that lower the free energy of activation of the substrate transacylation reaction. Finally, 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 geneticmore » code may offer novel solutions to proteins as they evolve new activities.« less
 [1] ;  [2] ;  [1] ;  [2] ;  [1] ;  [2] ;  [1]
  1. The Scripps Research Institute, La Jolla, CA (United States)
  2. Univ. of Southern California, Los Angeles, CA (United States)
Publication Date:
Grant/Contract Number:
Published Article
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Volume: 112; Journal Issue: 22; Journal ID: ISSN 0027-8424
National Academy of Sciences, Washington, DC (United States)
Research Org:
Scripps Research Inst., La Jolla, CA (United States)
Sponsoring Org:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
Country of Publication:
United States
59 BASIC BIOLOGICAL SCIENCES; noncanonical amino acid; beta-lactamase; catalytic activity; evolutionary advantage; conformational effects
OSTI Identifier:
Alternate Identifier(s):
OSTI ID: 1347595