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  • 1.
    Hopmann, Kathrin
    KTH, School of Biotechnology (BIO), Theoretical Chemistry.
    Nitrile Hydratases and Epoxide-Transforming Enzymes: Quantum Chemical Modeling of Reaction Mechanisms and Selectivities2008Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Quantum chemical studies of enzymatic reactions are able to provide detailed insight into mechanisms and catalytic strategies. The energetic feasibility of proposed mechanisms can be established, and new possible reaction pathways can be put forward. The role of the involved active site residues can be analyzed in detail and the origins for experimentally observed selectivities can be investigated. Density functional theory (DFT), in particular the hybrid functional B3LYP, is the method of choice in this kind of studies.

    In this thesis, the reaction mechanisms of several enzymes have been explored using the B3LYP functional. The studied enzymes include limonene epoxide hydrolase (LEH), soluble epoxide hydrolase (sEH), haloalcohol dehalogenase (HheC), and nitrile hydratase (NHase). Transition states and intermediates along various reaction pathways were optimized and evaluated.

    For the three epoxide-transforming enzymes, the role of the proposed catalytic residues could be confirmed. Analysis of in silico mutations helped to quantify the effect of various functional groups on the barriers and regioselectivities of epoxide opening. A detailed analysis of the factors governing the enzymatic regioselectivities is given.

    For nitrile hydratase, various putative first- and second-shell mechanisms have been studied. Active site models based on both the Co(III)-NHase and the Fe(III)-NHase were employed. The studied mechanisms include general base-catalyzed reaction pathways with water as nucleophile as well as two pathways involving cysteine-sulfenate as nucleophile. Several computed mechanisms exhibit similar barriers, making it difficult to pinpoint the true NHase mechanism.

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  • 2.
    Hopmann, Kathrin H.
    KTH, School of Biotechnology (BIO).
    Quantum chemical studies of epoxide-transforming enzymes2007Licentiate thesis, comprehensive summary (Other scientific)
    Abstract [en]

    Density functional theory is employed to study the reaction mechanisms of different epoxide-transforming enzymes. Calculations are based on quantum chemical active site models, which are build from X-ray crystal structures. The models are used to study conversion of various epoxides into their corresponding diols or substituted alcohols. Epoxide-transforming enzymes from three different families are studied. The human soluble epoxide hydrolase (sEH) belongs to the α/β-hydrolase fold family. sEH employs a covalent mechanism to hydrolyze various epoxides into vicinal diols. The Rhodococcus erythrobacter limonene epoxide hydrolase (LEH) constitutes a novel epoxide hydrolase, which is considered the founding member of a new family of enzymes. LEH mediates transformation of limone-1,2-epoxide into the corresponding vicinal diol by employing a general acid/general base-mediated mechanism. The Agrobacterium radiobacter AD1 haloalcohol dehalogenase HheC is related to the short-chain dehydrogenase/reductases. HheC is able to convert epoxides using various nucleophiles such as azide, cyanide, and nitrite. Reaction mechanisms of these three enzymes are analyzed in depth and the role of different active site residues is studied through in silico mutations. Steric and electronic factors influencing the regioselectivity of epoxide opening are identified. The computed energetics help to explain preferred reaction pathways and experimentally observed regioselectivities. Our results confirm the usefulness of the employed computational methodology for investigating enzymatic reactions.

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  • 3.
    Hopmann, Kathrin H.
    et al.
    KTH, School of Biotechnology (BIO), Theoretical Chemistry.
    Guo, Jing-Dong
    Department of Applied Chemistry, Jiangxi Science and Technology Normal University.
    Himo, Fahmi
    KTH, School of Biotechnology (BIO), Theoretical Chemistry.
    Theoretical Investigation of the First-Shell Mechanism of Nitrile Hydratase2007In: Inorganic Chemistry, ISSN 0020-1669, E-ISSN 1520-510X, Vol. 46, no 12, p. 4850-4856Article in journal (Refereed)
    Abstract [en]

    The first-shell mechanism of nitrile hydratase (NHase) is investigated theoretically using density functional theory. NHases catalyze the conversion of nitriles to amides and are classified into two groups, the non-heme Fe(III) NHases and the non-corrinoid Co(III) NHases. The active site of the non-heme iron NHase comprises a low-spin iron (S = (1)/(2)) with a remarkable set of ligands, including two deprotonated backbone nitrogens and both cysteine-sulfenic and cysteine-sulfinic acids. A widely proposed reaction mechanism of NHase is the first-shell mechanism in which the nitrile substrate binds directly to the low-spin iron in the sixth coordination site. We have used quantum chemical models of the NHase active site to investigate this mechanism. We present potential energy profiles for the reaction and provide characterization of the intermediates and transition-state structures for the NHase-mediated conversion of acetonitrile. The results indicate that the first-shell ligand Cys114-SO- could be a possible base in the nitrile hydration mechanism, abstracting a proton from the nucleophilic water molecule. The generally suggested role of the Fe(III) center as a Lewis acid, activating the substrate toward nucleophilic attack, is shown to be unlikely. Instead, the metal is suggested to provide electrostatic stabilization to the anionic imidate intermediate, thereby lowering the reaction barrier.

  • 4.
    Hopmann, Kathrin H.
    et al.
    KTH, School of Biotechnology (BIO), Theoretical Chemistry.
    Hallberg, B. Martin
    Biophysics, Department of Medical Biochemistry and Biophysics, Karolinska Institutet.
    Himo, Fahmi
    KTH, School of Biotechnology (BIO), Theoretical Chemistry.
    Catalytic mechanism of limonene epoxide hydrolase: a theoretical study2005In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 127, no 41, p. 14339-14347Article in journal (Refereed)
    Abstract [en]

    The catalytic mechanism of limonene epoxide hydrolase (LEH) was investigated theoretically using the density functional theory method B3LYP. LEH is part of a novel limonene degradation pathway found in Rhodococcus erythropolis DCL14, where it catalyzes the hydrolysis of limonene-1,2-epoxide to give limonene-1,2-diol. The recent crystal structure of LEH was used to build a model of the LEH active site composed of five amino acids and a crystallographically observed water molecule. With this model, hydrolysis of different substrates was investigated. It is concluded that LEH employs a concerted general acid/general base-catalyzed reaction mechanism involving protonation of the substrate by Asp101, nucleophilic attack by water on the epoxide, and abstraction of a proton from water by Asp132. Furthermore, we provide an explanation for the experimentally observed regioselective hydrolysis of the four stereoisomers of limonene-1,2-epoxide.

  • 5.
    Hopmann, Kathrin H.
    et al.
    KTH, School of Biotechnology (BIO), Theoretical Chemistry.
    Himo, Fahmi
    KTH, School of Biotechnology (BIO), Theoretical Chemistry.
    Cyanolysis and Azidolysis of Epoxides by Haloalcohol Dehalogenase: Theoretical Study of the Reaction Mechanism and Origins of Regioselectivity2008In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 47, no 17, p. 4973-4982Article in journal (Refereed)
    Abstract [en]

    Haloalcohol dehalogenase HheC catalyzes the reversible dehalogenation of vicinal haloalcohols to form epoxides and free halides. In addition, HheC is able to catalyze the irreversible and highly regioselective ring-opening of epoxides with nonhalide nucleophiles, such as CN- and N-3(-). For azidolysis of aromatic epoxides, the regioselectivity observed with HheC is opposite to the regioselectivity of the nonenzymatic epoxide-opening. This, together with a relatively broad substrate specificity, makes HheC a promising tool for biocatalytic applications. We have designed large quantum chemical models of the HheC active site and used density functional theory to study the reaction mechanism of the HheC-catalyzed ring-opening of (R)-styrene oxide with the nucleophiles CN- and N3-. Both the cyanolysis and the azidolysis reactions are shown to take place in a single concerted step. The results support the suggested role of the putative Ser132-Tyr145-Arg149 catalytic triad, where Tyr145 acts as a general acid, donating a proton to the substrate, and Arg149 interacts with Tyr145 and facilitates proton abstraction, while Ser132 positions the substrate and reduces the barrier for epoxide opening through interaction with the emerging oxyanion of the substrate. We have also studied the regioselectivity of (R)-styrene oxide opening for both the cyanolysis and the azidolysis reactions. The employed active site model was shown to be able to reproduce the experimentally observed beta-regioselectivity of HheC. In silico mutations of various groups in the HheC active site model were performed to elucidate the important factors governing the regioselectivity.

  • 6.
    Hopmann, Kathrin H.
    et al.
    KTH, School of Biotechnology (BIO), Theoretical Chemistry.
    Himo, Fahmi
    KTH, School of Biotechnology (BIO), Theoretical Chemistry.
    Insights into the Reaction Mechanism of Soluble Epoxide Hydrolase from Theoretical Active Site Mutants2006In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 110, no 42, p. 21299-21310Article in journal (Refereed)
    Abstract [en]

    Density functional theory calculations of active site mutants are used to gain insights into the reaction mechanism of the soluble epoxide hydrolases (sEHs). The quantum chemical model is based on the X-ray crystal structure of the human soluble epoxide hydrolase. The role of two conserved active site tyrosines is explored through in silico single and double mutations to phenylalanine. Full potential energy curves for hydrolysis of (1S,2S)-beta-methylstyrene oxide are presented. The results indicate that the two active site tyrosines act in concert to lower the activation barrier for the alkylation step. For the wild-type and three different tyrosine mutant models, the regioselectivity of epoxide opening is compared for the substrates (1S,2S)-beta-methylstyrene oxide and (S)-styrene oxide. An additional part of our study focuses on the importance of the catalytic histidine for the alkylation half-reaction. Different models are presented to explore the protonation state of the catalytic histidine in the alkylation step and to evaluate the possibility of an interaction between the nucleophilic aspartate and the catalytic histidine.

  • 7.
    Hopmann, Kathrin H.
    et al.
    KTH, School of Biotechnology (BIO), Theoretical Chemistry.
    Himo, Fahmi
    KTH, School of Biotechnology (BIO), Theoretical Chemistry.
    On the Role of Tyrosine as Catalytic Base in Nitrile Hydratase2008In: European Journal of Inorganic Chemistry, ISSN 1434-1948, E-ISSN 1099-1948, no 22, p. 3452-3459Article in journal (Refereed)
    Abstract [en]

    Nitrile Hydratases (NHases) catalyze the conversion of nitriles to their corresponding amides. Two NHase classes exist, the Fe-III-NHases and the Co-III-NHases. Both harbour an intriguing active site, with a low-spin metal ion coordinated to deprotonated back-bone amides and oxidized cysteine residues. So far it has not been possible to conclusively determine the reaction mechanism of NHase. Here we employ density functional theory to investigate the recent proposal that a fully conserved second-shell tyrosine residue is the catalytic base of nitrile hydratase (J. Biol. Chem. 2007, 282, 7397-7404). In the proposed mechanism, the tyrosine is suggested to be in the tyrosinate state and to mediate nitrile hydration through activation of a water molecule, which attacks the metal-bound substrate. We have explored this mechanism employing quantum chemical active site models on the basis of the Co-III-NHase from P. thermophila JCM 3095 and the Fe-III-NHase from R. erythropolis N-771. Potential energy curves and optimized transition states are presented. The computed barriers for the two models are a few kcal/mol above the experimental value, indicating that the conserved second-shell tyrosine could function as the catalytic base of NHase. To further evaluate the likelihood of this mechanism, we estimated the pK(a) value of the second-shell tyrosine in each model. We also provide estimates of the energy involved in the exchange of a metal-bound water molecule with a nitrile substrate.

  • 8.
    Hopmann, Kathrin H.
    et al.
    KTH, School of Biotechnology (BIO), Theoretical Chemistry.
    Himo, Fahmi
    KTH, School of Biotechnology (BIO), Theoretical Chemistry.
    Quantum Chemical Modeling of the Dehalogenation Reaction of Haloalcohol Dehalogenase2008In: Journal of Chemical Theory and Computation, ISSN 1549-9618, E-ISSN 1549-9626, Vol. 4, no 7, p. 1129-1137Article in journal (Refereed)
    Abstract [en]

    The dehalogenation reaction of haloalcohol dehalogenase HheC from Agrobacterium radiobacter AD1 was investigated theoretically using hybrid density functional theory methods. HheC catalyzes the enantioselective conversion of halohydrins into their corresponding epoxides. The reaction is proposed to be mediated by a catalytic Ser132-Tyr145-Arg149 triad, and a distinct halide binding site is suggested to facilitate halide displacement by stabilizing the free ion. We investigated the HheC-mediated dehalogenation of (R)-2-chloro-1-phenylethanol using three quantum chemical models of various sizes. The calculated barriers and reaction energies give support to the suggested reaction mechanism. The dehalogenation occurs in a single concerted step, in which Tyr145 abstracts a proton from the halohydrin substrate and the substrate oxyanion displaces the chloride ion, forming the epoxide. Characterization of the involved stationary points is provided. Furthermore, by using three different models of the halide binding site, we are able to assess the adopted modeling methodology.

  • 9.
    Hopmann, Kathrin H.
    et al.
    KTH, School of Biotechnology (BIO), Theoretical Chemistry.
    Himo, Fahmi
    KTH, School of Biotechnology (BIO), Theoretical Chemistry.
    Theoretical Investigation of the Second-Shell Mechanism of Nitrile Hydratase2008In: European Journal of Inorganic Chemistry, ISSN 1434-1948, E-ISSN 1099-1948, no 9, p. 1406-1412Article in journal (Refereed)
    Abstract [en]

    Nitrile hydratases (NHases) are biocatalytically important enzymes that are utilized in the industrial production of acrylamide and nicotinamide. There are two different classes of NHases, harbouring either a low-spin Fe-III or a low-spin Co-III ion in the active site, in each case with the same peculiar set of ligands, involving deprotonated backbone amides and oxidized cysteine residues. The detailed reaction mechanism of NHase has not been established yet, but different proposals have been put forward. Depending on the binding site of the substrate, these can be divided into first-shell and second-shell mechanisms, respectively, Recently, we have investigated different first-shell mechanisms using quantum-chemical active-site models based on the iron-dependent NHase (Inorg. Chem. 2007, 46, 4850). Here we continue our investigation of the NHase reaction by exploring two different variations of the second-shell mechanism of the iron-dependent NHase. In the first, a metal-bound hydroxide ion performs a nucleophilic attack on the nitrile substrate, while in the second investigated mechanism, the oxidized cysteine, Cys114-SO-, acts as the nucleophile. We report energies, optimized transition state, and intermediate geometries for both investigated mechanisms. The calculated barriers are similar to the previously reported first-shell mechanism involving Cys114-SO- as catalytic base.

  • 10.
    Hopmann, Kathrin H.
    et al.
    KTH, School of Biotechnology (BIO), Theoretical Chemistry.
    Himo, Fahmi
    KTH, School of Biotechnology (BIO), Theoretical Chemistry.
    Theoretical study of the full reaction mechanism of human soluble epoxide hydrolase2006In: Chemistry - A European Journal, ISSN 0947-6539, E-ISSN 1521-3765, Vol. 12, no 26, p. 6898-6909Article in journal (Refereed)
    Abstract [en]

    The complete reaction mechanism of soluble epoxide hydrolase (sEH) has been investigated by using the B3LYP density functional theory method. Epoxide hydrolases catalyze the conversion of epoxides to their corresponding vicinal diols. In our theoretical study, the sEH active site is represented by quantum-chemical models that are based on the X-ray crystal structure of human soluble epoxide hydrolase. The trans-substituted epoxide (1S,2S)-beta-methyl styrene oxide has been used as a substrate in the theoretical investigation of the sEH reaction mechanism. Both the alkylation and the hydrolytic half-reactions have been studied in detail. We present the energetics of the reaction mechanism as well as the optimized intermediates and transition-state structures. Full potential energy curves for the reactions involving nucleophilic attack at either the benzylic or the homo-benzylic carbon atom of (1S,2S)-beta-methylstyrene oxide have been computed. The regioselectivity of epoxide opening has been addressed for the two substrates (1S,2S)-beta-methylstyrene oxide and (S)-styrene oxide.

  • 11.
    Hopmann, Kathrin Helen
    et al.
    KTH, School of Biotechnology (BIO), Theoretical Chemistry.
    Himo, Fahmi
    KTH, School of Biotechnology (BIO), Theoretical Chemistry.
    Quantum chemical modeling of enzymatic reactions - applications to epoxide-transforming enzymes2010In: Comprehensive Natural Products II: Chemistry and Biology, Elsevier, 2010, Vol. 8, p. 719-747Chapter in book (Refereed)
  • 12.
    Hopmann, Kathrin
    et al.
    KTH, School of Biotechnology (BIO), Theoretical Chemistry.
    Himo, Fahmi
    KTH, School of Biotechnology (BIO), Theoretical Chemistry.
    A theoretical study of th azidolysis and cyanolysis of epozides by haloalcohol dehalogenaseManuscript (Other academic)
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