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MD Simulations and QM/MM Calculations Show that Single-Site Mutations of Cytochrome P450 BM3 Alter the Active Site’s Complexity and the Chemoselectivity of Oxidation Without Having Changed the Active Species

Abstract

Abstract: It is a longstanding mechanistic consensus that the mutation of the proton-shuttle mediator Threonine (T) in Cytochrome P450 enzymes, severs also the water channel, and quench thereby the formation of the active species; the high-valent-iron(IV)–oxoporphyrin-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, in Ref. 6) on sulfoxidation and N-dealkylation of dimethyl-(4-methylsulfanyl-phenyl)-amine by the 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, originate in 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: in T268A, the shifted location of the new water channel 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 local surgery but one that may lead to a profound change in the active site, sufficiently so as to change chemoselectivity of catalyzed reactions.

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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, Accepted Manuscript
  • Open access: Creative Commons BY-NC license
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    MD Simulations and QM/MM Calculations Show that Single-Site Mutations of Cytochrome P450 BM3 Alter the Active Site’s Complexity and the Chemoselectivity of Oxidation Without Having Changed the Active Species

    K. D. Dubey, B. wang, M. Vajpai and S. Shaik, Chem. Sci., 2017, Accepted Manuscript , DOI: 10.1039/C7SC01932G

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