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. 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 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. 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 genetic code maymore »
- Authors:
-
- Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037,
- Departments of Biological Sciences and Chemistry, Bridge Institute, University of Southern California, Los Angeles, CA 90089
- Publication Date:
- Research Org.:
- Scripps Research Inst., La Jolla, CA (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC), Basic Energy Sciences (BES)
- OSTI Identifier:
- 1235105
- Alternate Identifier(s):
- OSTI ID: 1347595
- Grant/Contract Number:
- SC0011787
- Resource Type:
- Published Article
- Journal Name:
- Proceedings of the National Academy of Sciences of the United States of America
- Additional Journal Information:
- Journal Name: Proceedings of the National Academy of Sciences of the United States of America Journal Volume: 112 Journal Issue: 22; Journal ID: ISSN 0027-8424
- Publisher:
- Proceedings of the National Academy of Sciences
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; noncanonical amino acid; beta-lactamase; catalytic activity; evolutionary advantage; conformational effects
Citation Formats
Xiao, Han, Nasertorabi, Fariborz, Choi, Sei-hyun, Han, Gye Won, Reed, Sean A., Stevens, Raymond C., and Schultz, Peter G. Exploring the potential impact of an expanded genetic code on protein function. United States: N. p., 2015.
Web. doi:10.1073/pnas.1507741112.
Xiao, Han, Nasertorabi, Fariborz, Choi, Sei-hyun, Han, Gye Won, Reed, Sean A., Stevens, Raymond C., & Schultz, Peter G. Exploring the potential impact of an expanded genetic code on protein function. United States. https://doi.org/10.1073/pnas.1507741112
Xiao, Han, Nasertorabi, Fariborz, Choi, Sei-hyun, Han, Gye Won, Reed, Sean A., Stevens, Raymond C., and Schultz, Peter G. Mon .
"Exploring the potential impact of an expanded genetic code on protein function". United States. https://doi.org/10.1073/pnas.1507741112.
@article{osti_1235105,
title = {Exploring the potential impact of an expanded genetic code on protein function},
author = {Xiao, Han and Nasertorabi, Fariborz and Choi, Sei-hyun and Han, Gye Won and Reed, Sean A. and Stevens, Raymond C. and Schultz, Peter G.},
abstractNote = {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 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. 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 genetic code may offer novel solutions to proteins as they evolve new activities.},
doi = {10.1073/pnas.1507741112},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
number = 22,
volume = 112,
place = {United States},
year = {Mon May 18 00:00:00 EDT 2015},
month = {Mon May 18 00:00:00 EDT 2015}
}
https://doi.org/10.1073/pnas.1507741112
Web of Science
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