Jump to main content
Jump to site search

Issue 8, 2017
Previous Article Next Article

MD simulations and QM/MM calculations show that single-site mutations of cytochrome P450BM3 alter the active site’s complexity and the chemoselectivity of oxidation without changing the active species

Author affiliations

Abstract

It is a long-standing mechanistic consensus that the mutation of the proton-shuttle mediator Threonine (T) in Cytochrome P450 enzymes severs the water channel and thereby quenches the formation of the active species: the high-valent iron(IV)-oxo porphyrin π-cation radical species, compound I (Cpd I). Using MD simulations and hybrid QM/MM calculations of P450BM3 we demonstrate that this is not the case. Thus, while the original water channel is disrupted in the T268A mutant of the enzyme, a new channel is formed that generates Cpd I. With this new understanding, we address the puzzling regiochemical and kinetic-isotope effect (KIE) results (Volz et al., J. Am. Chem. Soc., 2002, 124, 9724–9725) on the sulfoxidation and N-dealkylation of dimethyl-(4-methylsulfanyl-phenyl)-amine by wild type (WT) P450BM3 and its T268A vs. F87A mutants. We show that the observed variable ratio of S/Me oxidation for these enzymes, vis-à-vis the constant KIE, originates from Cpd I being the sole oxidant. Thus, while the conserved KIE probes the conserved nature of the transition state, the variable regiochemical S/Me ratio reflects the active-site reorganization in the mutants: the shifted location of the new water channel in T268A tightens the binding of the S-end by Cpd I and increases the S/Me ratio, whereas the absence of π-interaction with the S-end in F87A creates a looser binding that lowers the S/Me ratio. Our results match the experimental findings. As such, this study sheds light on puzzling experimental results, and may shift a central paradigm in P450 research. The broader implication on enzymatic research is that a single-site mutation is not a localised alteration but one that may lead to a profound change in the active site, sufficiently so as to change the chemoselectivity of catalyzed reactions.

Graphical abstract: MD simulations and QM/MM calculations show that single-site mutations of cytochrome P450BM3 alter the active site’s complexity and the chemoselectivity of oxidation without changing the active species

Back to tab navigation

Supplementary files

Publication details

The article was received on 30 Apr 2017, accepted on 11 Jun 2017 and first published on 13 Jun 2017


Article type: Edge Article
DOI: 10.1039/C7SC01932G
Citation: Chem. Sci., 2017,8, 5335-5344
  • Open access: Creative Commons BY-NC license
  •   Request permissions

    MD simulations and QM/MM calculations show that single-site mutations of cytochrome P450BM3 alter the active site’s complexity and the chemoselectivity of oxidation without changing the active species

    K. D. Dubey, B. Wang, M. Vajpai and S. Shaik, Chem. Sci., 2017, 8, 5335
    DOI: 10.1039/C7SC01932G

    This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Material from this article can be used in other publications provided that the correct acknowledgement is given with the reproduced material and it is not used for commercial purposes.

    Reproduced material should be attributed as follows:

    • For reproduction of material from NJC:
      [Original citation] - Published by The Royal Society of Chemistry (RSC) on behalf of the Centre National de la Recherche Scientifique (CNRS) and the RSC.
    • For reproduction of material from PCCP:
      [Original citation] - Published by the PCCP Owner Societies.
    • For reproduction of material from PPS:
      [Original citation] - Published by The Royal Society of Chemistry (RSC) on behalf of the European Society for Photobiology, the European Photochemistry Association, and RSC.
    • For reproduction of material from all other RSC journals:
      [Original citation] - Published by The Royal Society of Chemistry.

    Information about reproducing material from RSC articles with different licences is available on our Permission Requests page.

Search articles by author

Spotlight

Advertisements