9 research outputs found

    MD results for the Y1124F mutant.

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    <p>(A) A representative active-site structure along with the average values of some structural parameters of the reactant complex for the first methyl transfer. (B) Left: the two-dimensional plot of <i>r</i>(C<sub>M</sub>…N<sub>ζ</sub>) and <i>θ</i> distributions of the reactant complex for the first methyl transfer; Middle: the free-energy change as a function of <i>r</i>(C<sub>M</sub>…N<sub>ξ</sub>) obtained from the distributions; Right: the free-energy change as a function of <i>θ</i> obtained from the distributions. (C) The structure of the reactant complex for the second methyl transfer. (D) Left: the two-dimensional plot of <i>r</i>(C<sub>M</sub>…N<sub>ζ</sub>) and <i>θ</i> distributions of the reactant complex for the second methyl transfer; Middle: the free-energy change as a function of <i>r</i>(C<sub>M</sub>…N<sub>ξ</sub>) obtained from the distributions; Right: the free-energy change as a function of <i>θ</i> obtained from the distributions. (E) The structure of the reactant complex for the third methyl transfer. (F) Left: the two-dimensional plot of <i>r</i>(C<sub>M</sub>…N<sub>ζ</sub>) and <i>θ</i> distributions of the reactant complex for the third methyl transfer; Middle: the free-energy change as a function of <i>r</i>(C<sub>M</sub>…N<sub>ξ</sub>) obtained from the distributions; Right: the free-energy change as a function of <i>θ</i> obtained from the distributions. (G) A representative active-site structure along with the average values of some structural parameters near the transition state for the first methyl transfer. (H) The structure near the transition state for the second methyl transfer. (I) The structure near the transition state for the third methyl transfer.</p

    MD results for the F1209Y mutant.

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    <p>(A) A representative active-site structure along with the average values of some structural parameters of the reactant complex for the first methyl transfer. (B) Left: the two-dimensional plot of <i>r</i>(C<sub>M</sub>…N<sub>ζ</sub>) and <i>θ</i> distributions of the reactant complex for the first methyl transfer; Middle: the free-energy change as a function of <i>r</i>(C<sub>M</sub>…N<sub>ξ</sub>) obtained from the distributions; Right: the free-energy change as a function of <i>θ</i> obtained from the distributions. (C) The structure of the reactant complex for the second methyl transfer. (D) Left: the two-dimensional plot of <i>r</i>(C<sub>M</sub>…N<sub>ζ</sub>) and <i>θ</i> distributions of the reactant complex for the second methyl transfer; Middle: the free-energy change as a function of <i>r</i>(C<sub>M</sub>…N<sub>ξ</sub>) obtained from the distributions; Right: the free-energy change as a function of <i>θ</i> obtained from the distributions. (E) The structure near the transition state for the first methyl transfer. (F) The structure near the transition state for the second methyl transfer.</p

    Free energy profiles of methyl transfer processes in the Y1124F mutant.

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    <p>The first methyl transfer: blue and solid line with a free energy barrier of 13.9 kcal/mol; the second methyl transfer: red and dashed line with a free energy barrier of 15.9 kcal/mol; the third methyl transfer: green and dashed line with a free energy barrier of 13.3 kcal/mol.</p

    MD results for the WT enzyme (GLP).

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    <p>(A) A representative active-site structure along with the average values of some structural parameters of the reactant complex for the first methyl transfer. GLP is shown in balls and sticks, and AdoMet and the H3K9 sidechain are in sticks. Hydrogen atoms are not shown for clarity except for those on N<sub>ζ</sub> and transferable methyl group. Hydrogen bonds are indicated by red dotted lines, and the distances related to the reactant coordinate are also shown. (B) Left: the two-dimensional plot of <i>r</i>(C<sub>M</sub>…N<sub>ζ</sub>) and <i>θ</i> distributions based on the 1.5-<i>ns</i> simulations of the reactant complex for the first methyl transfer; Middle: the free-energy change as a function of <i>r</i>(C<sub>M</sub>…N<sub>ξ</sub>) obtained from the distributions; Right: the free-energy change as a function of <i>θ</i> obtained from the distributions. (C) The active-site structure along with the average values of some structural parameters of the reactant complex for the second methyl transfer. (D) Left: the two-dimensional plot of <i>r</i>(C<sub>M</sub>…N<sub>ζ</sub>) and <i>θ</i> distributions of the reactant complex for the second methyl transfer; Middle: the free-energy change as a function of <i>r</i>(C<sub>M</sub>…N<sub>ξ</sub>) obtained from the distributions; Right: the free-energy change as a function of <i>θ</i> obtained from the distributions. (E) The structure of the reactant complex for the third methyl transfer. (F) Left: the two-dimensional plot of <i>r</i>(C<sub>M</sub>…N<sub>ζ</sub>) and <i>θ</i> distributions of the reactant complex for the third methyl transfer; Middle: the free-energy change as a function of <i>r</i>(C<sub>M</sub>…N<sub>ξ</sub>) obtained from the distributions; Right: the free-energy change as a function of <i>θ</i> obtained from the distributions. (G) A representative active-site structure along with the average values of some structural parameters near the transition state for the first methyl transfer obtained from the free energy (potential of mean force) simulations. (H) The structure along with the average values of some structural parameters near the transition state for the second methyl transfer. (I) The structure near the transition state for the third methyl transfer. All images were made by VMD <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037674#pone.0037674-HumphreyW1" target="_blank">[38]</a>. The distances shown on the structures are the calculated average distances from the trajectories over the 50-ps production runs in the corresponding window.</p

    QM/MM free energy simulations of the reaction catalysed by (<i>4S</i>)-limonene synthase involving linalyl diphosphate (LPP) substrate

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    <p>A large number of terpenoid natural products are known to exist in nature. Terpene synthases are pivotal enzymes for the biosynthesis of diverse terpenoid skelotons. Monoterpene synthases are one type of terpene synthases responsible for the production of several hundreds of natural monoterpenes based on a very limited pool of substrates. Therefore, understanding detailed catalytic mechanisms of those enzymes are important for understanding the product specificity of terpene synthases. In this study, we present a detailed mechanistic description of the biosynthesis of the (<i>4S</i>)-α-terpinyl carbocation from (<i>3S</i>)-linalyl diphosphate (LPP) catalysed by (<i>4S</i>)-limonene synthase (LS) using two-dimensional QM/MM free energy (2D-PMF) simulations. Our estimated free energy barrier is in a reasonable agreement with the corresponding experimental kinetic data. We also perform the one-dimensional QM/MM free energy (1D-PMF) simulations and show that His579 can act as a general base to deprotonate (<i>4S</i>)-α-terpinyl carbocation and to generate the limonene product.</p

    The definition of the structural parameters for monitoring the relative orientation of AdoMet and H3K9me1 [H3K9 and H3K9(me)<sub>2</sub>] in the reactant complex.

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    <p>The efficiency of the methyl transfer may be related to the distributions of <i>r</i>(C<sub>M</sub>…N<sub>ζ</sub>) and <i>θ</i> in the reactant complexes. <i>θ</i> is defined as the angle between the two vectors <i>r</i><sub>1</sub> and <i>r</i><sub>2</sub>. Here <i>r</i><sub>1</sub> is the direction of the lone pair of electrons on N<sub>ζ</sub> and <i>r</i><sub>2</sub> is the vector pointing from C<sub>M</sub> to S<sub>δ</sub>. The reaction coordinate for calculating the free energy profiles for the methyl transfers is <i>R</i> = <i>r</i>(C<sub>M</sub>…S<sub>δ</sub>)−<i>r</i>(C<sub>M</sub>…N<sub>ζ</sub>).</p

    Substrate-Assisted Catalysis in the Reaction Catalyzed by Salicylic Acid Binding Protein 2 (SABP2), a Potential Mechanism of Substrate Discrimination for Some Promiscuous Enzymes

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    Although one of an enzyme’s hallmarks is the high specificity for their natural substrates, substrate promiscuity has been reported more frequently. It is known that promiscuous enzymes generally show different catalytic efficiencies to different substrates, but our understanding of the origin of such differences is still lacking. Here we report the results of quantum mechanical/molecular mechanical simulations and an experimental study of salicylic acid binding protein 2 (SABP2). SABP2 has promiscuous esterase activity toward a series of substrates but shows a high activity toward its natural substrate, methyl salicylate (MeSA). Our results demonstrate that this enzyme may use substrate-assisted catalysis involving the hydroxyl group from MeSA to enhance the activity and achieve substrate discrimination

    Cloning, expression and biochemical characterization of a GH1 β-glucosidase from <i>Cellulosimicrobium cellulans</i>

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    <p>β-Glucosidase plays an important role in the degradation of cellulose. In this study, a novel β-glucosidase <i>ccbgl1b</i> gene for a glycosyl hydrolase (GH) family 1 enzyme was cloned from the genome of <i>Cellulosimicrobium cellulans</i> and expressed in <i>Escherichia coli</i> BL21 cells. The sequence contained an open reading frame of 1494 bp, encoded a polypeptide of 497 amino acid residues. The recombinant protein CcBgl1B was purified by Ni sepharose fastflow affinity chromatography and had a molecular weight of 57 kDa, as judged by SDS-PAGE. The optimum β-glucosidase activity was observed at 55 °C and pH 6.0. Recombinant CcBgl1B was found to be most active against aryl-glycosides <i>p</i>-nitrophenyl-β-D-glucopyranoside (<i>p</i>NPβGlc), followed by <i>p</i>-nitrophenyl-β-D-galactopyranoside (<i>p</i>NPβGal). Using disaccharides as substrates, the enzyme efficiently cleaved β-linked glucosyl-disaccharides, including sophorose (β-1,2-), laminaribiose (β-1,3-) and cellobiose (β-1,4-). In addition, a range of cello-oligosaccharides including cellotriose, cellotetraose and cellopentaose were hydrolysed by CcBgl1B to produce glucose. The interaction mode between the enzyme and the substrates driving the reaction was modelled using a molecular docking approach. Understanding how the GH1 enzyme CcBgl1B from <i>C. cellulans</i> works, particularly its activity against cello-oligosaccharides, would be potentially useful for biotechnological applications of cellulose degradation.</p
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