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

Abstract

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.

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Figure 1: Dynamic range of the TxtE F/G loop.
Figure 2: Productive and non-productive active-site arrangements are separated by a single conformational transition state (TS) and depend on whether the F/G loop is closed or open.
Figure 3: Discovery of a nitration regioselectivity switch at His176.
Figure 4: MD simulations show the C5-selective variants in similar edge-to-face interactions with the L-Trp substrate indole moiety with that observed for wild-type TxtE.
Figure 5: Mutation of His176 to Phe fine tunes the substrate-to-peroxynitrite alignment and active-site water network.
Figure 6: Crystal structures of His176Phe/Tyr variants have a resolved closed-lid F/G loop that aligns with the predicted closed-lid MD geometries.
Figure 7: Identification of naturally occurring TxtE homologues that catalyse the production of 5NT.

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Acknowledgements

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.

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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.

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Correspondence to Vijay S. Pande or Frances H. Arnold.

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Dodani, S., Kiss, G., Cahn, J. et al. Discovery of a regioselectivity switch in nitrating P450s guided by molecular dynamics simulations and Markov models. Nature Chem 8, 419–425 (2016). https://doi.org/10.1038/nchem.2474

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