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Structural, Functional, and Spectroscopic Characterization of the Substrate Scope of the Novel Nitrating Cytochrome P450 TxtE

Dodani, Sheel C. and Cahn, Jackson K. B. and Heinisch, Tillmann and Brinkmann-Chen, Sabine and McIntosh, John A. and Arnold, Frances H. (2014) Structural, Functional, and Spectroscopic Characterization of the Substrate Scope of the Novel Nitrating Cytochrome P450 TxtE. ChemBioChem, 15 (15). pp. 2259-2267. ISSN 1439-4227. PMCID PMC4260628. http://resolver.caltech.edu/CaltechAUTHORS:20140908-101026220

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Abstract

A novel cytochrome P450 enzyme, TxtE, was recently shown to catalyze the direct aromatic nitration of L-tryptophan. This unique chemistry inspired us to ask whether TxtE could serve as a platform for engineering new nitration biocatalysts to replace current harsh synthetic methods. As a first step toward this goal, and to better understand the wild-type enzyme, we obtained high-resolution structures of TxtE in its substrate-free and substrate-bound forms. We also screened a library of substrate analogues for spectroscopic indicators of binding and for production of nitrated products. From these results, we found that the wild-type enzyme accepts moderate decoration of the indole ring, but the amino acid moiety is crucial for binding and correct positioning of the substrate and therefore less amenable to modification. A nitrogen atom is essential for catalysis, and a carbonyl must be present to recruit the αB′1 helix of the protein to seal the binding pocket.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1002/cbic.201402241DOIArticle
http://onlinelibrary.wiley.com/doi/10.1002/cbic.201402241/abstractPublisherArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4260628PubMed CentralArticle
ORCID:
AuthorORCID
Arnold, Frances H.0000-0002-4027-364X
Additional Information:© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. Received: May 15, 2014. Published online on September 2, 2014. We thank Dr. Jens Kaiser and Dr. Pavle Nikolovski of the Beckman Molecular Observatory (Caltech) for assistance with crystallography and Dr. Scott Virgil and the 3CS Center for Catalysis and Chemical Synthesis (Caltech) for assistance with LC-MS analyses. The authors thank the Resnick Sustainability Institute (Caltech) (support to J.K.B.C.) and Dow Chemical Company for support through the Dow-Resnick innovation program. S.C.D. and J.A.M. are supported by Ruth L. Kirschstein NRSA postdoctoral fellowships from the National Institutes of Health (5F32M106618 and 5F32M101792, respectively). T.H. is supported by a postdoctoral fellowship from the Swiss National Science Foundation (Fellowship PBBSP2_146809). The Beckman Molecular Observatory is supported by the Gordon and Betty Moore Foundation, the Beckman Institute, and the Sanofi-Aventis Bioengineering Research Program (Caltech). The authors thank Dr. Fei Sun for sharing of the XL1-Blue E. coli strain and helpful discussions. The content of this paper is solely the responsibility of the authors and does not represent the official views of any of the funding agencies.
Group:Resnick Sustainability Institute
Funders:
Funding AgencyGrant Number
Resnick Sustainability InstituteUNSPECIFIED
Dow Chemical CompanyUNSPECIFIED
NIH Postdoctoral Fellowship5F32M106618
NIH Postdoctoral Fellowship5F32M101792
Swiss National Science Foundation (SNSF)PBBSP2_146809
Subject Keywords:aromatic nitration; crystal-structure determination; cytochromes; enzyme catalysis
PubMed Central ID:PMC4260628
Record Number:CaltechAUTHORS:20140908-101026220
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20140908-101026220
Official Citation:Dodani, S. C., Cahn, J. K. B., Heinisch, T., Brinkmann-Chen, S., McIntosh, J. A. and Arnold, F. H. (2014), Structural, Functional, and Spectroscopic Characterization of the Substrate Scope of the Novel Nitrating Cytochrome P450 TxtE. ChemBioChem, 15: 2259–2267. doi: 10.1002/cbic.201402241
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:49332
Collection:CaltechAUTHORS
Deposited By: Jason Perez
Deposited On:08 Sep 2014 23:34
Last Modified:11 Sep 2017 16:45

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