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Discovery of a regioselectivity switch in nitrating P450s guided by molecular dynamics simulations and Markov models

Dodani, Sheel C. and Kiss, Gert and Cahn, Jackson K. B. and Su, Ye and Pande, Vijay S. and Arnold, Frances H. (2016) Discovery of a regioselectivity switch in nitrating P450s guided by molecular dynamics simulations and Markov models. Nature Chemistry, 8 (5). pp. 419-425. ISSN 1755-4330. PMCID PMC4843824. doi:10.1038/NCHEM.2474.

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The dynamic motions of protein structural elements, particularly flexible loops, are intimately linked with diverse aspects of enzyme catalysis. Engineering of these loop regions can alter protein stability, substrate binding and even dramatically impact enzyme function. When these flexible regions are unresolvable structurally, computational reconstruction in combination with large-scale molecular dynamics simulations can be used to guide the engineering strategy. Here we present a collaborative approach that consists of both experiment and computation and led to the discovery of a single mutation in the F/G loop of the nitrating cytochrome P450 TxtE that simultaneously controls loop dynamics and completely shifts the enzyme's regioselectivity from the C4 to the C5 position of L-tryptophan. Furthermore, we find that this loop mutation is naturally present in a subset of homologous nitrating P450s and confirm that these uncharacterized enzymes exclusively produce 5-nitro-L-tryptophan, a previously unknown biosynthetic intermediate.

Item Type:Article
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URLURL TypeDescription ReadCube access CentralArticle
Dodani, Sheel C.0000-0003-0271-6080
Cahn, Jackson K. B.0000-0001-8849-4516
Pande, Vijay S.0000-0003-2774-1178
Arnold, Frances H.0000-0002-4027-364X
Alternate Title:Discovery of a regioselectivity switch in nitrating P450s guided by MD simulations and Markov models
Additional Information:© 2016 Macmillan Publishers Limited. Received 15 September 2015; Accepted 03 February 2016; Published online 21 March 2016. We thank J. Kaiser and P. Nikolovski of the Beckman Molecular Observatory (Caltech) for assistance with crystallography and S. Virgil and the 3CS Center for Catalysis and Chemical Synthesis (Caltech) for assistance with LC-MS analyses. This work was funded by the Gordon and Betty Moore Foundation through grant GBMF2809 to the Caltech Programmable Molecular Technology Initiative (to F.H.A.). S.C.D. is supported by a Ruth L. Kirschstein National Research Service Award postdoctoral fellowship from the National Institutes of Health (NIH) (5F32GM106618). G.K. acknowledges support from the Lawrence Scholars Program, the NIH Simbios Program (U54 GM072970) and the Center for Molecular Analysis and Design (Stanford). J.K.B.C. acknowledges the support of the Resnick Sustainability Institute (Caltech). 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 S. Brinkmann-Chen, T. K. Hyster, J. A. McIntosh, C. K. Prier, R. T. McGibbon and M. M. Sultan for helpful discussions. This research is part of the Blue Waters sustained-petascale computing project, which is supported by the National Science Foundation (awards OCI-0725070 and ACI-1238993) and the state of Illinois. 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. Author contributions: S.C.D. and G.K. contributed equally to this work. S.C.D. and G.K. designed the research. S.C.D., G.K., J.K.B.C. and Y.S. performed the research. F.H.A. and V.S.P. supervised and provided advice. S.C.D., G.K. and J.K.B.C. analysed the data. S.C.D., G.K., J.K.B.C. and F.H.A. wrote the text and conceived the figures with input from all of the authors. Competing financial interests: The authors declare no competing financial interests.
Group:Resnick Sustainability Institute
Funding AgencyGrant Number
Gordon and Betty Moore FoundationGBMF2809
NIH Postdoctoral Fellowship5F32GM106618
Lawrence Scholars ProgramUNSPECIFIED
NIHU54 GM072970
Stanford UniversityUNSPECIFIED
Resnick Sustainability InstituteUNSPECIFIED
Caltech Beckman InstituteUNSPECIFIED
Caltech Sanofi-Aventis Bioengineering Research ProgramUNSPECIFIED
State of IllinoisUNSPECIFIED
Issue or Number:5
PubMed Central ID:PMC4843824
Record Number:CaltechAUTHORS:20160323-124510465
Persistent URL:
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:65625
Deposited By: Tony Diaz
Deposited On:25 Mar 2016 02:30
Last Modified:05 May 2022 17:43

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