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Unlocking reactivity of TrpB: a general biocatalytic platform for synthesis of tryptophan analogs

Romney, David K. and Murciano-Calles, Javier and Wehrmüller, Jöri Elias and Arnold, Frances H. (2017) Unlocking reactivity of TrpB: a general biocatalytic platform for synthesis of tryptophan analogs. Journal of the American Chemical Society, 131 (39). pp. 10769-10776. ISSN 0002-7863. PMCID PMC5589443. doi:10.1021/jacs.7b05007. https://resolver.caltech.edu/CaltechAUTHORS:20170717-101626008

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Abstract

Derivatives of the amino acid tryptophan (Trp) serve as precursors for the chemical and biological synthesis of complex molecules with a wide range of biological properties. Trp analogues are also valuable as building blocks for medicinal chemistry and as tools for chemical biology. While the enantioselective synthesis of Trp analogues is often lengthy and requires the use of protecting groups, enzymes have the potential to synthesize such products in fewer steps and with the pristine chemo- and stereoselectivity that is a hallmark of biocatalysis. The enzyme TrpB is especially attractive because it can form Trp analogues directly from serine (Ser) and the corresponding indole analogue. However, many potentially useful substrates, including bulky or electron-deficient indoles, are poorly accepted. We have applied directed evolution to TrpB from Pyrococcus furiosus and Thermotoga maritima to generate a suite of catalysts for the synthesis of previously intractable Trp analogues. For the most challenging substrates, such as nitroindoles, the key to improving activity lay in the mutation of a universally conserved and mechanistically important residue, E104. The new catalysts express at high levels (>200 mg/L of Escherichia coli culture) and can be purified by heat treatment; they can operate up to 75 °C (where solubility is enhanced) and can synthesize enantiopure Trp analogues substituted at the 4-, 5-, 6-, and 7-positions, using Ser and readily available indole analogues as starting materials. Spectroscopic analysis shows that many of the activating mutations suppress the decomposition of the active electrophilic intermediate, an amino-acrylate, which aids in unlocking the synthetic potential of TrpB.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1021/jacs.7b05007DOIArticle
http://pubs.acs.org/doi/abs/10.1021/jacs.7b05007PublisherArticle
http://pubs.acs.org/doi/suppl/10.1021/jacs.7b05007PublisherSupporting Information
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5589443PubMed CentralArticle
ORCID:
AuthorORCID
Romney, David K.0000-0003-0498-7597
Arnold, Frances H.0000-0002-4027-364X
Additional Information:© 2017 American Chemical Society. Received: May 15, 2017; Published: July 14, 2017. This work was funded by the Jacobs Institute for Molecular Medicine (Caltech) and the Gordon and Betty Moore Foundation (through the Caltech Programmable Molecular Technology Initiative). D.K.R. was supported by a Ruth Kirschstein NIH Postdoctoral Fellowship (F32GM117635), and J.M.-C. was supported by a fellowship from the Alfonso Martı́n Escudero Foundation. The authors thank Dr. Sabine Brinkmann-Chen for advice on developing the screening assay, Dr. Andrew Buller for helpful discussions and comments on the manuscript, and Elliot Mackrell for assistance in screening the catalyst libraries. The authors declare the following competing financial interest(s): The contents of this paper are the subject of a patent application submitted by Caltech, and some authors are entitled to a royalty on revenues arising from that patent.
Funders:
Funding AgencyGrant Number
Jacobs Institute for Molecular Engineering for MedicineUNSPECIFIED
Gordon and Betty Moore FoundationUNSPECIFIED
NIH Postdoctoral FellowshipF32GM117635
Alfonso Martín Escudero FoundationUNSPECIFIED
Issue or Number:39
PubMed Central ID:PMC5589443
DOI:10.1021/jacs.7b05007
Record Number:CaltechAUTHORS:20170717-101626008
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20170717-101626008
Official Citation:Unlocking Reactivity of TrpB: A General Biocatalytic Platform for Synthesis of Tryptophan Analogues David K. Romney, Javier Murciano-Calles, Jöri E. Wehrmüller, and Frances H. Arnold Journal of the American Chemical Society 2017 139 (31), 10769-10776 DOI: 10.1021/jacs.7b05007
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:79127
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:17 Jul 2017 19:22
Last Modified:15 Nov 2021 17:45

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