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Directed Evolution of an Allosteric Tryptophan Synthase to Create a Platform for Synthesis of Noncanonical Amino Acids

Murciano-Calles, Javier and Buller, Andrew R. and Arnold, Frances H. (2017) Directed Evolution of an Allosteric Tryptophan Synthase to Create a Platform for Synthesis of Noncanonical Amino Acids. In: Directed Enzyme Evolution: Advances and Applications. Springer , Cham, Switzerland, pp. 1-16. ISBN 978-3-319-50411-7.

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Tryptophan and its derivatives are important natural products and have many biochemical and synthetic applications. However, the more elaborate these derivatives are, the more complex the synthesis becomes. In this chapter, we summarize the development of an engineered enzymatic platform for synthesis of diverse tryptophan analogs. This endeavor utilizes the tryptophan synthase (TrpS) enzyme, an α_2β_2 heterodimeric protein complex that catalyzes the last two steps in the biosynthetic pathway of tryptophan. Although the synthetically useful reaction (indole + Ser = Trp) takes place in the β-subunit (TrpB), the exquisite allosteric regulation of this enzyme impedes the use of isolated TrpB due to its dramatically decreased activity in the absence of the α-subunit (TrpA). This chapter discusses our efforts to engineer TrpB to serve as a general platform for the synthesis of noncanonical amino acids. We used directed evolution to enhance the activity of TrpB from Pyrococcus furiosus (PfTrpB), so that it can act as a stand-alone biocatalyst. Remarkably, we found that mutational activation mimics the allosteric activation induced by binding of TrpA. Toward our goal of expanding the substrate scope of this reaction, we activated other homologs with the same mutations discovered for PfTrpB. We found improved catalysts for the synthesis of 5-substituted tryptophans, an important biological motif. Finally, we performed directed evolution of TrpB for synthesis of β-branched amino acids, a group of products whose chemical syntheses are particularly challenging.

Item Type:Book Section
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URLURL TypeDescription
Buller, Andrew R.0000-0002-9635-4844
Arnold, Frances H.0000-0002-4027-364X
Additional Information:© 2017 Springer International Publishing AG. First Online: 15 February 2017. We gratefully thank Sabine Brinkmann-Chen for a critical reading of this chapter. We also thank David K. Romney for his helpful discussions during the elaboration of the chapter. Javier Murciano-Calles acknowledges financial support from the Alfonso Martín Escudero Foundation. This work was funded through the Jacobs Institute for Molecular Engineering for Medicine and Ruth Kirschstein NIH Postdoctoral Fellowship F32GM110851 (to Andrew R. Buller).
Funding AgencyGrant Number
Alfonso Martín Escudero FoundationUNSPECIFIED
Jacobs Institute for Molecular Engineering for MedicineUNSPECIFIED
NIH Postdoctoral FellowshipF32GM110851
Subject Keywords:Catalytic Efficiency; Comm Domain; Allosteric Regulation; Substrate Scope; Chemical Shift Perturbation
Record Number:CaltechAUTHORS:20180702-074326733
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:87502
Deposited By: Tony Diaz
Deposited On:02 Jul 2018 18:00
Last Modified:15 Nov 2021 20:48

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