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Directed evolution mimics allosteric activation by stepwise tuning of the conformational ensemble

Buller, Andrew R. and van Roye, Paul and Cahn, Jackson K. B. and Scheele, Remkes A. and Herger, Michael and Arnold, Frances H. (2018) Directed evolution mimics allosteric activation by stepwise tuning of the conformational ensemble. Journal of the American Chemical Society, 140 (23). pp. 7256-7266. ISSN 0002-7863. PMCID PMC5999571. http://resolver.caltech.edu/CaltechAUTHORS:20180501-112938311

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Abstract

Allosteric enzymes contain a wealth of catalytic diversity that remains distinctly underutilized for biocatalysis. Tryptophan synthase is a model allosteric system and a valuable enzyme for the synthesis of noncanonical amino acids (ncAA). Previously, we evolved the β-subunit from Pyrococcus furiosus, PfTrpB, for ncAA synthase activity in the absence of its native partner protein PfTrpA. However, the precise mechanism by which mutation activated TrpB to afford a stand-alone catalyst remained enigmatic. Here, we show that directed evolution caused a gradual change in the rate-limiting step of the catalytic cycle. Concomitantly, the steady-state distribution of the intermediates shifts to favor covalently bound Trp adducts, which have increased thermodynamic stability. The biochemical properties of these evolved, stand-alone TrpBs converge on those induced in the native system by allosteric activation. High-resolution crystal structures of the wild-type enzyme, an intermediate in the lineage, and the final variant, encompassing five distinct chemical states, show that activating mutations have only minor structural effects on their immediate environment. Instead, mutation stabilizes the large-scale motion of a subdomain to favor an otherwise transiently populated closed conformational state. This increase in stability enabled the first structural description of Trp covalently bound in a catalytically active TrpB, confirming key features of catalysis. These data combine to show that sophisticated models of allostery are not a prerequisite to recapitulating its complex effects via directed evolution, opening the way to engineering stand-alone versions of diverse allosteric enzymes.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://dx.doi.org/10.1021/jacs.8b03490DOIArticle
https://pubs.acs.org/doi/suppl/10.1021/jacs.8b03490PublisherSupporting Information
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5999571/PubMed CentralArticle
ORCID:
AuthorORCID
Buller, Andrew R.0000-0002-9635-4844
Arnold, Frances H.0000-0002-4027-364X
Additional Information:© 2018 American Chemical Society. Received: March 30, 2018; Published: April 30, 2018. We thank Sabine Brinkmann-Chen, David K. Romney, and Christina E. Boville for many helpful discussions, assistance during protein purifications, and their comments on this manuscript. The Caltech Molecular Observatory is supported by the Gordon and Betty Moore Foundation, the Beckman Institute, and the Sanofi-Aventis Bioengineering Research Program at Caltech. A.R.B. was supported by a Ruth Kirschstein National Institutes of Health Postdoctoral Fellowship (F32G110851). R.A.S. was supported by the Backer Foundation. The authors declare no competing financial interest.
Funders:
Funding AgencyGrant Number
Gordon and Betty Moore FoundationUNSPECIFIED
Caltech Beckman InstituteUNSPECIFIED
Sanofi-Aventis Bioengineering Research ProgramUNSPECIFIED
NIH Postdoctoral FellowshipF32G110851
Backer FoundationUNSPECIFIED
Subject Keywords:biocatalysis, enzymology, allostery, pyridoxal phosphate, non-canonical amino acid
PubMed Central ID:PMC5999571
Record Number:CaltechAUTHORS:20180501-112938311
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20180501-112938311
Official Citation:Directed Evolution Mimics Allosteric Activation by Stepwise Tuning of the Conformational Ensemble. Andrew R. Buller, Paul van Roye, Jackson K. B. Cahn, Remkes A. Scheele, Michael Herger, and Frances H. Arnold. Journal of the American Chemical Society 2018 140 (23), 7256-7266. DOI: 10.1021/jacs.8b03490
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:86158
Collection:CaltechAUTHORS
Deposited By: George Porter
Deposited On:01 May 2018 19:42
Last Modified:20 Aug 2018 17:14

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