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}
}
Free Publicly Available Full Text
Publisher's Version of Record
https://doi.org/10.1073/pnas.1507741112
https://doi.org/10.1073/pnas.1507741112
Other availability
Search WorldCat to find libraries that may hold this journal
Cited by: 55 works
Citation information provided by
Web of Science
Web of Science
Save to My Library
You must Sign In or Create an Account in order to save documents to your library.
Works referenced in this record:
Bacteriophages use an expanded genetic code on evolutionary paths to higher fitness
journal, February 2014
- Hammerling, Michael J.; Ellefson, Jared W.; Boutz, Daniel R.
- Nature Chemical Biology, Vol. 10, Issue 3
Hidden alternative structures of proline isomerase essential for catalysis
journal, December 2009
- Fraser, James S.; Clarkson, Michael W.; Degnan, Sheena C.
- Nature, Vol. 462, Issue 7273
Evolution of cyclic peptide protease inhibitors
journal, June 2011
- Young, T. S.; Young, D. D.; Ahmad, I.
- Proceedings of the National Academy of Sciences, Vol. 108, Issue 27
Multiplex Iterative Plasmid Engineering for Combinatorial Optimization of Metabolic Pathways and Diversification of Protein Coding Sequences
journal, August 2013
- Li, Yifan; Gu, Qun; Lin, Zhenquan
- ACS Synthetic Biology, Vol. 2, Issue 11
Refinement of Macromolecular Structures by the Maximum-Likelihood Method
journal, May 1997
- Murshudov, G. N.; Vagin, A. A.; Dodson, E. J.
- Acta Crystallographica Section D Biological Crystallography, Vol. 53, Issue 3
Overview of the CCP 4 suite and current developments
journal, March 2011
- Winn, Martyn D.; Ballard, Charles C.; Cowtan, Kevin D.
- Acta Crystallographica Section D Biological Crystallography, Vol. 67, Issue 4
The energy landscapes and motions of proteins
journal, December 1991
- Frauenfelder, H.; Sligar, S.; Wolynes, P.
- Science, Vol. 254, Issue 5038
Evolving Methods for Macromolecular Crystallography
book, January 2007
- Read, Randy J.; Sussman, Joel L.
- NATO Science Series II: Mathematics, Physics and Chemistry
Structural and Biochemical Evidence That a TEM-1 β-Lactamase N170G Active Site Mutant Acts via Substrate-assisted Catalysis
journal, October 2009
- Brown, Nicholas G.; Shanker, Sreejesh; Prasad, B. V. Venkataram
- Journal of Biological Chemistry, Vol. 284, Issue 48
Programming cells by multiplex genome engineering and accelerated evolution
journal, July 2009
- Wang, Harris H.; Isaacs, Farren J.; Carr, Peter A.
- Nature, Vol. 460, Issue 7257, p. 894-898
More extended-spectrum beta-lactamases.
journal, September 1991
- Jacoby, G. A.; Medeiros, A. A.
- Antimicrobial Agents and Chemotherapy, Vol. 35, Issue 9
The role of dynamic conformational ensembles in biomolecular recognition
journal, October 2009
- Boehr, David D.; Nussinov, Ruth; Wright, Peter E.
- Nature Chemical Biology, Vol. 5, Issue 11
Genetic Incorporation of Histidine Derivatives Using an Engineered Pyrrolysyl-tRNA Synthetase
journal, March 2014
- Xiao, Han; Peters, Francis B.; Yang, Peng-Yu
- ACS Chemical Biology, Vol. 9, Issue 5
The role of distant mutations and allosteric regulation on LovD active site dynamics
journal, April 2014
- Jiménez-Osés, Gonzalo; Osuna, Sílvia; Gao, Xue
- Nature Chemical Biology, Vol. 10, Issue 6
Effects of Asp-179 mutations in TEMpUC19 beta-lactamase on susceptibility to beta-lactams
journal, August 1995
- Vakulenko, S. B.; Toth, M.; Taibi, P.
- Antimicrobial Agents and Chemotherapy, Vol. 39, Issue 8
Inactivation of antibiotics and the dissemination of resistance genes
journal, April 1994
- Davies, J.
- Science, Vol. 264, Issue 5157
A potent new mode of β-lactamase inhibition revealed by the 1.7 Å X-ray crystallographic structure of the TEM-1–BLIP complex
journal, March 1996
- Strynadka, Natalie C. J.; Jensen, Susan E.; Alzari, Pedro M.
- Nature Structural Biology, Vol. 3, Issue 3
The Chemistry of the Antibody Molecule
journal, December 2002
- Schultz, Peter G.; Yin, Jun; Lerner, Richard A.
- Angewandte Chemie International Edition, Vol. 41, Issue 23
A Comparative Analysis of the Immunological Evolution of Antibody 28B4
journal, September 2001
- Yin, Jun; Mundorff, Emily C.; Yang, Priscilla L.
- Biochemistry, Vol. 40, Issue 36
Scaling and assessment of data quality
journal, December 2005
- Evans, Philip
- Acta Crystallographica Section D Biological Crystallography, Vol. 62, Issue 1, p. 72-82
Expanding the Genetic code
journal, June 2006
- Wang, Lei; Xie, Jianming; Schultz, Peter G.
- Annual Review of Biophysics and Biomolecular Structure, Vol. 35, Issue 1
A Dynamic Knockout Reveals That Conformational Fluctuations Influence the Chemical Step of Enzyme Catalysis
journal, April 2011
- Bhabha, G.; Lee, J.; Ekiert, D. C.
- Science, Vol. 332, Issue 6026
Exploring the Role of a Conserved Class A Residue in the Ω-Loop of KPC-2 β-Lactamase: A MECHANISM FOR CEFTAZIDIME HYDROLYSIS
journal, July 2012
- Levitt, Peter S.; Papp-Wallace, Krisztina M.; Taracila, Magdalena A.
- Journal of Biological Chemistry, Vol. 287, Issue 38
Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants
journal, December 1993
- Kabsch, W.
- Journal of Applied Crystallography, Vol. 26, Issue 6
Mass Spectral Kinetic Study of Acylation and Deacylation During the Hydrolysis of Penicillins and Cefotaxime by .beta.-Lactamase TEM-1 and the G238S Mutant
journal, September 1995
- Saves, Isabelle; Burlet-Schiltz, Odile; Maveyraud, Laurent
- Biochemistry, Vol. 34, Issue 37
A novel ceftazidime-hydrolysing extended-spectrum -lactamase, CTX-M-54, with a single amino acid substitution at position 167 in the omega loop
journal, May 2006
- Bae, I. K.
- Journal of Antimicrobial Chemotherapy, Vol. 58, Issue 2
Pre-steady state beta-lactamase kinetics. The trapping of a covalent intermediate and the interpretation of pH rate profiles.
journal, November 1983
- Anderson, E. G.; Pratt, R. F.
- Journal of Biological Chemistry, Vol. 258, Issue 21
Crystal structure ofEscherichia coli TEM1 β-lactamase at 1.8 Å resolution
journal, August 1993
- Jelsch, Christian; Mourey, Lionel; Masson, Jean-Michel
- Proteins: Structure, Function, and Genetics, Vol. 16, Issue 4
How conformational changes can affect catalysis, inhibition and drug resistance of enzymes with induced-fit binding mechanism such as the HIV-1 protease
journal, May 2013
- Weikl, Thomas R.; Hemmateenejad, Bahram
- Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, Vol. 1834, Issue 5
Structural Basis for Clinical Longevity of Carbapenem Antibiotics in the Face of Challenge by the Common Class A β-Lactamases from the Antibiotic-Resistant Bacteria
journal, September 1998
- Maveyraud, Laurent; Mourey, Lionel; Kotra, Lakshmi P.
- Journal of the American Chemical Society, Vol. 120, Issue 38
Protein Conformational Disorder and Enzyme Catalysis
book, January 2013
- Schulenburg, Cindy; Hilvert, Donald
- Dynamics in Enzyme Catalysis
Immunological Origins of Binding and Catalysis in a Diels-Alderase Antibody
journal, March 1998
- Romesberg, F. E.
- Science, Vol. 279, Issue 5358
Coot model-building tools for molecular graphics
journal, November 2004
- Emsley, Paul; Cowtan, Kevin
- Acta Crystallographica Section D Biological Crystallography, Vol. 60, Issue 12, p. 2126-2132
Relating Protein Motion to Catalysis
journal, June 2006
- Hammes-Schiffer, Sharon; Benkovic, Stephen J.
- Annual Review of Biochemistry, Vol. 75, Issue 1
Pyrrolysine Encoded by UAG in Archaea: Charging of a UAG-Decoding Specialized tRNA
journal, May 2002
- Srinivasan, G.
- Science, Vol. 296, Issue 5572
Molecular structure of the acyl-enzyme intermediate in β-lactam hydrolysis at 1.7 Å resolution
journal, October 1992
- Strynadka, Natalie C. J.; Adachi, Hiroyuki; Jensen, Susan E.
- Nature, Vol. 359, Issue 6397
Folding funnels, binding funnels, and protein function
journal, January 1999
- Tsai, Chung-Jung; Kumar, Sandeep; Ma, Buyong
- Protein Science, Vol. 8, Issue 6
The direct genetic encoding of pyrrolysine
journal, December 2005
- Krzycki, Joseph A.
- Current Opinion in Microbiology, Vol. 8, Issue 6
A General Method for Scanning Unnatural Amino Acid Mutagenesis
journal, January 2009
- Daggett, Kelly A.; Layer, Mark; Cropp, T. Ashton
- ACS Chemical Biology, Vol. 4, Issue 2
Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances
journal, January 2008
- Wiegand, Irith; Hilpert, Kai; Hancock, Robert E. W.
- Nature Protocols, Vol. 3, Issue 2
Adding New Chemistries to the Genetic Code
journal, June 2010
- Liu, Chang C.; Schultz, Peter G.
- Annual Review of Biochemistry, Vol. 79, Issue 1, p. 413-444
A semi-synthetic organism with an expanded genetic alphabet
journal, May 2014
- Malyshev, Denis A.; Dhami, Kirandeep; Lavergne, Thomas
- Nature, Vol. 509, Issue 7500, p. 385-388
Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7
journal, June 2008
- Langer, Gerrit; Cohen, Serge X.; Lamzin, Victor S.
- Nature Protocols, Vol. 3, Issue 7
The interplay between binding energy and catalysis in the evolution of a catalytic antibody
journal, September 1997
- Ulrich, Helle D.; Mundorff, Emily; Santarsiero, Bernard D.
- Nature, Vol. 389, Issue 6648
Site-saturation Mutagenesis of Tyr-105 Reveals Its Importance in Substrate Stabilization and Discrimination in TEM-1 β-Lactamase
journal, August 2004
- Doucet, Nicolas; De Wals, Pierre-Yves; Pelletier, Joelle N.
- Journal of Biological Chemistry, Vol. 279, Issue 44
Structural Insights into the Evolution of an Antibody Combining Site
journal, June 1997
- Wedemayer, G. J.
- Science, Vol. 276, Issue 5319
A Genetically Encoded aza-Michael Acceptor for Covalent Cross-Linking of Protein–Receptor Complexes
journal, May 2014
- Furman, Jennifer L.; Kang, Mingchao; Choi, Seihyun
- Journal of the American Chemical Society, Vol. 136, Issue 23
A Versatile Platform for Single- and Multiple-Unnatural Amino Acid Mutagenesis in Escherichia coli
journal, February 2013
- Chatterjee, Abhishek; Sun, Sophie B.; Furman, Jennifer L.
- Biochemistry, Vol. 52, Issue 10
A natural polymorphism in -lactamase is a global suppressor
journal, August 1997
- Huang, W.; Palzkill, T.
- Proceedings of the National Academy of Sciences, Vol. 94, Issue 16
Phaser crystallographic software
journal, July 2007
- McCoy, Airlie J.; Grosse-Kunstleve, Ralf W.; Adams, Paul D.
- Journal of Applied Crystallography, Vol. 40, Issue 4
Protein evolution with an expanded genetic code
journal, November 2008
- Liu, C. C.; Mack, A. V.; Tsao, M. -L.
- Proceedings of the National Academy of Sciences, Vol. 105, Issue 46
Structure-based design of a potent transition state analogue for TEM-1 β-lactamase
journal, August 1996
- Strynadka, Natalie C. J.; Martin, Richard; Jensen, S. E.
- Nature Structural & Molecular Biology, Vol. 3, Issue 8
Evolution of Iron(II)-Finger Peptides by Using a Bipyridyl Amino Acid
journal, March 2014
- Kang, Mingchao; Light, Kenneth; Ai, Hui-wang
- ChemBioChem, Vol. 15, Issue 6