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Directed evolution of the tryptophan synthase β-subunit for stand-alone function recapitulates allosteric activation

Buller, Andrew R. and Brinkmann-Chen, Sabine and Romney, David K. and Herger, Michael and Murciano-Calles, Javier and Arnold, Frances H. (2015) Directed evolution of the tryptophan synthase β-subunit for stand-alone function recapitulates allosteric activation. Proceedings of the National Academy of Sciences of the United States of America, 112 (47). pp. 14599-14604. ISSN 0027-8424. PMCID PMC4664345. https://resolver.caltech.edu/CaltechAUTHORS:20151112-080107171

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Abstract

Enzymes in heteromeric, allosterically regulated complexes catalyze a rich array of chemical reactions. Separating the subunits of such complexes, however, often severely attenuates their catalytic activities, because they can no longer be activated by their protein partners. We used directed evolution to explore allosteric regulation as a source of latent catalytic potential using the β-subunit of tryptophan synthase from Pyrococcus furiosus (PfTrpB). As part of its native αββα complex, TrpB efficiently produces tryptophan and tryptophan analogs; activity drops considerably when it is used as a stand-alone catalyst without the α-subunit. Kinetic, spectroscopic, and X-ray crystallographic data show that this lost activity can be recovered by mutations that reproduce the effects of complexation with the α-subunit. The engineered PfTrpB is a powerful platform for production of Trp analogs and for further directed evolution to expand substrate and reaction scope.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1073/pnas.1516401112DOIArticle
http://www.pnas.org/content/112/47/14599PublisherArticle
http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1516401112/-/DCSupplementalPublisherSupporting Information
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4664345/PubMed CentralArticle
ORCID:
AuthorORCID
Buller, Andrew R.0000-0002-9635-4844
Brinkmann-Chen, Sabine0000-0002-5419-4192
Romney, David K.0000-0003-0498-7597
Arnold, Frances H.0000-0002-4027-364X
Additional Information:© 2015 National Academy of Sciences. Edited by Alan R. Fersht, Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom, and approved October 16, 2015 (received for review August 17, 2015). Published online before print November 9, 2015. The authors thank Jackson Cahn, Dr. Robert Trachman, Dr. Mike Chen, and Belinda Wenke for helpful discussions and comments on the manuscript. We thank the staff of the Caltech Molecular Observatory, Dr. Jens Kaiser, Dr. Julie Hoy, and Pavle Nikolovski for crystallographic support. The Molecular Observatory is supported by the Gordon and Betty Moore Foundation, the Beckman Institute, and the Sanofi-Aventis Bioengineering Research Program at Caltech. This work was funded through the Jacobs Institute for Molecular Engineering for Medicine; Ruth Kirschstein NIH Postdoctoral Fellowship F32GM110851 (to A.R.B.); and the AlfonsoMartín Escudero Foundation (J.M.C.). Author contributions: A.R.B., S.B.-C., D.K.R., M.H., and J.M.-C. designed research; A.R.B., S.B.-C., D.K.R., M.H., and J.M.-C. performed research; A.R.B., S.B.-C., D.K.R., M.H., J.M.-C., and F.H.A. analyzed data; and A.R.B., S.B.-C., D.K.R., and F.H.A. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 5DVZ, 5DW0, 5DW3, and 5E0K). This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1516401112/-/DCSupplemental.
Group:Jacobs Institute for Molecular Engineering for Medicine
Funders:
Funding AgencyGrant Number
Gordon and Betty Moore FoundationUNSPECIFIED
Caltech Beckman InstituteUNSPECIFIED
Caltech Sanofi-Aventis Bioengineering Research ProgramUNSPECIFIED
Jacobs Institute for Molecular Engineering for MedicineUNSPECIFIED
NIHF32GM110851
AlfonsoMartín Escudero FoundationUNSPECIFIED
Subject Keywords:protein engineering; allostery; noncanonical amino acid; PLP
Issue or Number:47
PubMed Central ID:PMC4664345
Record Number:CaltechAUTHORS:20151112-080107171
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20151112-080107171
Official Citation:Andrew R. Buller, Sabine Brinkmann-Chen, David K. Romney, Michael Herger, Javier Murciano-Calles, and Frances H. Arnold Directed evolution of the tryptophan synthase β-subunit for stand-alone function recapitulates allosteric activation PNAS 2015 112 (47) 14599-14604; published ahead of print November 9, 2015, doi:10.1073/pnas.1516401112
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:62067
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:18 Nov 2015 00:59
Last Modified:14 Oct 2019 18:08

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