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

Academic Article
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Overview

related to degree

  • Xiao, Han, Ph.D. in Chemical Biology, Scripps Research 2010 - 2015

authors

  • Xiao, Han
  • Nasertorabi, F.
  • Choi, S. H.
  • Han, G. W.
  • Reed, S. A.
  • Stevens, Raymond
  • Schultz, Peter

publication date

  • June 2015

journal

  • Proceedings of the National Academy of Sciences of the United States of America  Journal

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 ?-lactamase. A screen for growth on the ?-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.
  • 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.

subject areas

  • Amino Acids
  • Catalysis
  • Cephalexin
  • Cloning, Molecular
  • Crystallization
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli
  • Evolution, Molecular
  • Genetic Code
  • Genetic Vectors
  • Kinetics
  • Models, Molecular
  • Molecular Structure
  • Protein Conformation
  • X-Ray Diffraction
  • beta-Lactamases
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Research

keywords

  • beta-lactamase
  • catalytic activity
  • conformational effects
  • evolutionary advantage
  • noncanonical amino acid
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Identity

PubMed Central ID

  • PMC4460475

International Standard Serial Number (ISSN)

  • 0027-8424

Digital Object Identifier (DOI)

  • 10.1073/pnas.1507741112

PubMed ID

  • 26038548
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Additional Document Info

start page

  • 6961

end page

  • 6966

volume

  • 112

issue

  • 22

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