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Amine Oxidation Mediated by N-Methyltryptophan Oxidase: Computational Insights into the Mechanism, Role of Active-Site Residues, and Covalent Flavin Binding

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    Research Article

    Amine Oxidation Mediated by N-Methyltryptophan Oxidase: Computational Insights into the Mechanism, Role of Active-Site Residues, and Covalent Flavin Binding
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    Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim, Germany
    *E-mail for W.T.: [email protected]
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    ACS Catalysis

    Cite this: ACS Catal. 2015, 5, 2, 1227–1239
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    https://doi.org/10.1021/cs501694q
    Published January 13, 2015
    Copyright © 2015 American Chemical Society
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    Abstract

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    Amine oxidation, a process widely utilized by flavoprotein oxidases, is the rate-determining step in the three-step demethylation of N-methyltryptophan (NMT) catalyzed by N-methyltryptophan oxidase (MTOX), which employs a covalently bound flavin adenine dinucleotide (FAD) as cofactor. For the required transfer of a hydride ion equivalent, three pathways (direct/concerted, radical, and adduct-forming/polar nucleophilic) have been proposed, without a consensus on which one is commonly used by amine oxidases. We combine theoretical pKa analysis, classical molecular dynamics (MD) simulations, and pure quantum mechanics (QM) and hybrid QM/molecular mechanics (QM/MM) calculations to provide molecular-level insights into the catalytic mechanism of NMT oxidation and to analyze the role of MTOX active-site residues and covalent FAD incorporation for NMT binding and oxidation. The QM(B3LYP-D2/6-31G(d))/CHARMM results clearly favor a direct concerted hydride transfer (HT) mechanism involving anionic NMT as the reactive species. On the basis of classical canonical MD simulations and QM/MM calculations of wild-type MTOX and two mutants (K341Q and H263N), we propose that the K341 residue acts as an active-site base and electrostatically, whereas H263 and Tyr249 only support substrate alignment. Covalent FAD binding leads to a more bent isoalloxazine moiety, which facilitates the binding of anionic NMT but increases the catalytic activity of FAD only slightly.

    Copyright © 2015 American Chemical Society

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    Supporting Information

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    The following file is available free of charge on the ACS Publications website at DOI: 10.1021/cs501694q.

    • Computational details, characteristic features from NVT ensembles, complete set of QM-only results (gas phase and water) of three NMT forms with four density functionals, effect of extending basis sets and QM part on QM/MM energies, key structural properties from QM-only and QM/MM geometries, effect of protonating the FAD-N1 position on the HT and SET pathways, average deviation from planarity of the isoalloxazine ring in noncovalently and covalently bound FAD, proton relay system via K341, Cs–N5 distances in selected MD trajectories, and visualization of QM-only and QM/MM geometries, of the extended QM region, and of spin densities (PDF)

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    Cited By

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    This article is cited by 17 publications.

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    ACS Catalysis

    Cite this: ACS Catal. 2015, 5, 2, 1227–1239
    Click to copy citationCitation copied!
    https://doi.org/10.1021/cs501694q
    Published January 13, 2015
    Copyright © 2015 American Chemical Society
    Request reuse permissions

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