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

Journal name:
Nature Chemistry
Volume:
8,
Pages:
419–425
Year published:
DOI:
doi:10.1038/nchem.2474
Received
Accepted
Published online

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.

At a glance

Figures

  1. Dynamic range of the TxtE F/G loop.
    Figure 1: Dynamic range of the TxtE F/G loop.

    a, The missing density between residues 175 and 184 of the wild-type crystal structure (PDB ID: 4TPO) indicates a disordered F/G loop (left panel), which was rebuilt through homology modelling (right panel). b, Subsequent large-scale MD simulations show that the F/G loop transitions between a set of disordered open-lid (left panel) and a set of structured closed-lid conformations in which the structurally unresolved His176 engages in a direct interaction with the substrate L-Trp (right panel). Hydrogen atoms are omitted for the sake of clarity. The substrate and haem are in a black-stick rendition and the F/G loop is shown in orange. A PyMOL session file of b is available as part of the Supplementary Information.

  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 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.

    a, In the closed-lid state, the F/G loop (orange) promotes a set of productive and tightly packed active-site conformations that support a small number of enclosed structural water molecules (W1–W3). These form a hydrogen-bond network with the substrate's amino acid moiety, whereas the hydrophobic substrate indole ring is ‘de-wetted’ and rests proximal to the ferric peroxynitrite species. b, The open-lid state enables substrate binding and product release. It also renders the active site accessible to the bulk solvent in the presence of the substrate, which can interfere with its productive alignment in the active site. c, The productive (C1–C4) and non-productive (O1–O4) active-site states are connected by a single conformational TS. The Supplementary Information contains additional detail on the diagram. d, Analysis of the TS reveals that the Gly58–Tyr175–Tyr89 contacts in a are replaced by a set of His176–Tyr89 interactions, which identifies His176 as a mutational target. The substrate and haem are in black and represent the geometric mean of the corresponding state. Water molecules (red spheres) are drawn from 20 random structures across the corresponding state.

  3. Discovery of a nitration regioselectivity switch at His176.
    Figure 3: Discovery of a nitration regioselectivity switch at His176.

    a, Schematic of the regiospecific nitration catalysed by wild-type TxtE and the TxtE His176Phe/Tyr/Trp variants. b, LC-MS chromatograms of the nitrated products synthesized by wild-type TxtE and the TxtE His176Phe/Tyr/Trp variants. c, Diode array detector (DAD) UV–visible absorption spectra of the 5NT standard and the nitrated products produced by the TxtE His176Phe/Tyr/Trp variants.

  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 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.

    ad, Compared with the wild-type His176 (a), the increased steric demands of Phe176 (b), Tyr176 (c) and Trp176 (d) cause the substrate indole moiety to adopt a shifted binding orientation that is packed more tightly against the back of the active site, which further determines the orientation of the haem-Fe-bound peroxynitrite (Fig. 5). The substrate indole responds first by adopting a retreated conformation (compare a with b) and then by assuming an increasingly parallel orientation relative to the plane of the haem cofactor (compare b with c and d). The F/G loop is shown in orange.

  5. Mutation of His176 to Phe fine tunes the substrate-to-peroxynitrite alignment and active-site water network.
    Figure 5: Mutation of His176 to Phe fine tunes the substrate-to-peroxynitrite alignment and active-site water network.

    a,b, TxtE wild-type simulations predominantly show the C4-carbon of L-Trp proximal to the peroxynitrite nitrogen (a), indicated by a yellow distance marker, and simulations of the His176Phe mutant predominantly show the C5-carbon closest to the peroxynitrite N (b). The two active sites support a distinct set of structured water molecules (highlighted by yellow circles).

  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 6: Crystal structures of His176Phe/Tyr variants have a resolved closed-lid F/G loop that aligns with the predicted closed-lid MD geometries.

    a, His176Phe (crystal structure in white and orange, MD geometries in blue and green). b, His176Tyr (crystal structure in white and orange, MD geometries in blue and green). The crystal structures (white) in a and b show the F/G loop (orange) in the closed-lid state and further show an active-site arrangement that supports the predicted closed-lid MD geometries—for both, the flipped (blue) and unflipped (green) indole-group ensembles. The range of dynamics is indicated through the overlaid hair lines, which correspond to 20 structures randomly drawn from each of the MD states.

  7. Identification of naturally occurring TxtE homologues that catalyse the production of 5NT.
    Figure 7: Identification of naturally occurring TxtE homologues that catalyse the production of 5NT.

    a, Sequence subalignment of the F/G loops of TxtE and seven homologues (sequence identity, 67–77%) reveals the natural occurrence of Trp176 in several homologues. b, LC-MS chromatograms of the nitrated products synthesized by the Trp176-containing TxtE homologues. c, DAD UV–visible absorption spectra of the 5NT standard and the nitrated products produced by the Trp176-containing TxtE homologues from S. virginiae, S. sp. Mg1, S. lavendulae and S. marina XMU15.

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Author information

Affiliations

  1. Division of Chemistry and Chemical Engineering 210-41, California Institute of Technology, California 91125, USA

    • Sheel C. Dodani,
    • Jackson K. B. Cahn,
    • Ye Su &
    • Frances H. Arnold
  2. Department of Chemistry, SIMBIOS NIH Center for Biomedical Computation, and Center for Molecular Analysis and Design, Stanford University, 318 Campus Drive, Stanford, California 94305, USA

    • Gert Kiss &
    • Vijay S. Pande

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.

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The authors declare no competing financial interests.

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