Seonah Kim

Diels–Alder Reactivities of Strained and Unstrained Cycloalkenes with Normal and Inverse-Electron-Demand Dienes: Activation Barriers and Distortion/Interaction Analysis

The Diels-Alder reactions of the cycloalkenes, cyclohexene through cyclopropene, with a series of dienes--1,3-dimethoxybutadiene, cyclopentadiene, 3,6-dimethyltetrazine, and 3,6-bis(trifluoromethyl)tetrazine--were studied with quantum mechanical calculations and compared with experimental values when available. The reactivities of cycloalkenes as dienophiles were found by a distortion/interaction analysis to be distortion controlled. The energies required for cycloalkenes to be distorted into the Diels-Alder transition states increase as the ring size of cycloalkenes increases from cyclopropene to cyclohexene, resulting in an increase in activation barriers. The reactivities of the dienes are controlled by both distortion and interaction energies. In normal Diels-Alder reactions with cycloalkenes, the electron-rich 1,3-dimethoxybutadiene exhibits stronger interaction energies than cyclopentadiene, but the high distortion energies required for 1,3-dimethoxybutadiene to achieve transition-state geometries overtake the favorable interaction, resulting in higher activation barriers. In inverse-electron-demand Diels-Alder reactions of 3,6-dimethyltetrazine and 3,6-bis(trifluoromethyl)tetrazine, the reactivities are mainly controlled by interaction energies.

Quantum mechanical calculations suggest that lytic polysaccharide monooxygenases use a copper-oxyl, oxygen-rebound mechanism

Significance Plant cell walls contain significant amounts of the polysaccharides cellulose and hemicellulose, which can be depolymerized by enzymes to sugars and upgraded to renewable fuels and chemicals. Traditionally, enzymes for biomass depolymerization were based on naturally occurring hydrolytic enzymes, until the recent discovery of another natural enzymatic paradigm for carbohydrate deconstruction. Namely, lytic polysaccharide monooxygenases (LPMOs), long thought to be hydrolases or carbohydrate-binding modules, were revealed to be oxidative, copper-containing enzymes. These enzymes are receiving significant attention as they could revolutionize biomass deconstruction to upgradeable intermediates for renewable energy applications. Here, we apply quantum mechanical calculations to elucidate the oxidative reaction mechanism to offer predictions into how LPMOs function. Lytic polysaccharide monooxygenases (LPMOs) exhibit a mononuclear copper-containing active site and use dioxygen and a reducing agent to oxidatively cleave glycosidic linkages in polysaccharides. LPMOs represent a unique paradigm in carbohydrate turnover and exhibit synergy with hydrolytic enzymes in biomass depolymerization. To date, several features of copper binding to LPMOs have been elucidated, but the identity of the reactive oxygen species and the key steps in the oxidative mechanism have not been elucidated. Here, density functional theory calculations are used with an enzyme active site model to identify the reactive oxygen species and compare two hypothesized reaction pathways in LPMOs for hydrogen abstraction and polysaccharide hydroxylation; namely, a mechanism that employs a η1-superoxo intermediate, which abstracts a substrate hydrogen and a hydroperoxo species is responsible for substrate hydroxylation, and a mechanism wherein a copper-oxyl radical abstracts a hydrogen and subsequently hydroxylates the substrate via an oxygen-rebound mechanism. The results predict that oxygen binds end-on (η1) to copper, and that a copper-oxyl–mediated, oxygen-rebound mechanism is energetically preferred. The N-terminal histidine methylation is also examined, which is thought to modify the structure and reactivity of the enzyme. Density functional theory calculations suggest that this posttranslational modification has only a minor effect on the LPMO active site structure or reactivity for the examined steps. Overall, this study suggests the steps in the LPMO mechanism for oxidative cleavage of glycosidic bonds.

Crystal Structure and Computational Characterization of the Lytic Polysaccharide Monooxygenase Gh61D from the Basidiomycota Fungus Phanerochaete Chrysosporium

Background: Lytic polysaccharide monooxygenases (LPMOs) represent a recently discovered enzymatic route to cleave carbohydrates. Results: We report the first basidiomycete LPMO structure and describe enzyme-cellulose interactions with simulation. Conclusion: We characterize the copper-containing active site and identify loops important for substrate recognition and binding. Significance: This structure is the first LPMO from a model basidiomycete fungus that contains many LPMO genes. Carbohydrate structures are modified and degraded in the biosphere by a myriad of mostly hydrolytic enzymes. Recently, lytic polysaccharide mono-oxygenases (LPMOs) were discovered as a new class of enzymes for cleavage of recalcitrant polysaccharides that instead employ an oxidative mechanism. LPMOs employ copper as the catalytic metal and are dependent on oxygen and reducing agents for activity. LPMOs are found in many fungi and bacteria, but to date no basidiomycete LPMO has been structurally characterized. Here we present the three-dimensional crystal structure of the basidiomycete Phanerochaete chrysosporium GH61D LPMO, and, for the first time, measure the product distribution of LPMO action on a lignocellulosic substrate. The structure reveals a copper-bound active site common to LPMOs, a collection of aromatic and polar residues near the binding surface that may be responsible for regio-selectivity, and substantial differences in loop structures near the binding face compared with other LPMO structures. The activity assays indicate that this LPMO primarily produces aldonic acids. Last, molecular simulations reveal conformational changes, including the binding of several regions to the cellulose surface, leading to alignment of three tyrosine residues on the binding face of the enzyme with individual cellulose chains, similar to what has been observed for family 1 carbohydrate-binding modules. A calculated potential energy surface for surface translation indicates that P. chrysosporium GH61D exhibits energy wells whose spacing seems adapted to the spacing of cellobiose units along a cellulose chain.

Experimental Diels–Alder Reactivities of Cycloalkenones and Cyclic Dienes Explained through Transition‐State Distortion Energies

Quantum chemical calculations are used to investigate the experimentally measured reactivities of cyclic dienes and cycloalkenones in the Diels-Alder reaction. The interaction energies (red) are nearly constant; differences arise in changes in distortion energies of both dienophile (blue) and diene (green; see picture, Ea=activation energy; values in kcal mol-1). Copyright

Bad Seeds Sprout Perilous Dynamics: Stochastic Thermostat Induced Trajectory Synchronization in Biomolecules

Molecular dynamics simulations starting from different initial conditions are commonly used to mimic the behavior of an experimental ensemble. We show in this article that when a Langevin thermostat is used to maintain constant temperature during such simulations, extreme care must be taken when choosing the random number seeds to prevent statistical correlation among the MD trajectories. While recent studies have shown that stochastically thermostatted trajectories evolving within a single potential basin with identical random number seeds tend to synchronize, we show that there is a synchronization effect even for complex, biologically relevant systems. We demonstrate this effect in simulations of alanine trimer and pentamer and in a simulation of a temperature-jump experiment for peptide folding of a 14-residue peptide. Even in replica-exchange simulations, in which the trajectories are at different temperatures, we find partial synchronization occurring when the same random number seed is employed. We explain this by extending the recent derivation of the synchronization effect for two trajectories in a harmonic well to the case in which the trajectories are at two different temperatures. Our results suggest several ways in which mishandling selection of a pseudorandom number generator initial seed can lead to corruption of simulation data. Simulators can fall into this trap in simple situations such as neglecting to specifically indicate different random seeds in either parallel or sequential restart simulations, utilizing a simulation package with a weak pseudorandom number generator, or using an advanced simulation algorithm that has not been programmed to distribute initial seeds.

Structural and Electronic Snapshots during the Transition from a Cu(II) to Cu(I) Metal Center of a Lytic Polysaccharide Monooxygenase by X-ray Photoreduction

Background: Lytic polysaccharide monooxygenases (LPMOs) exhibit a copper center that binds dioxygen for catalysis. Results: We present LPMO structures from Cu(II) to Cu(I) and analyze the transition with quantum mechanical calculations. Conclusion: Reduction changes the copper coordination state but requires only minor structural and electronic changes. Significance: These structures provide insight into LPMO catalytic activation for further mechanistic studies. Lytic polysaccharide monooxygenases (LPMOs) are a recently discovered class of enzymes that employ a copper-mediated, oxidative mechanism to cleave glycosidic bonds. The LPMO catalytic mechanism likely requires that molecular oxygen first binds to Cu(I), but the oxidation state in many reported LPMO structures is ambiguous, and the changes in the LPMO active site required to accommodate both oxidation states of copper have not been fully elucidated. Here, a diffraction data collection strategy minimizing the deposited x-ray dose was used to solve the crystal structure of a chitin-specific LPMO from Enterococcus faecalis (EfaCBM33A) in the Cu(II)-bound form. Subsequently, the crystalline protein was photoreduced in the x-ray beam, which revealed structural changes associated with the conversion from the initial Cu(II)-oxidized form with two coordinated water molecules, which adopts a trigonal bipyramidal geometry, to a reduced Cu(I) form in a T-shaped geometry with no coordinated water molecules. A comprehensive survey of Cu(II) and Cu(I) structures in the Cambridge Structural Database unambiguously shows that the geometries observed in the least and most reduced structures reflect binding of Cu(II) and Cu(I), respectively. Quantum mechanical calculations of the oxidized and reduced active sites reveal little change in the electronic structure of the active site measured by the active site partial charges. Together with a previous theoretical investigation of a fungal LPMO, this suggests significant functional plasticity in LPMO active sites. Overall, this study provides molecular snapshots along the reduction process to activate the LPMO catalytic machinery and provides a general method for solving LPMO structures in both copper oxidation states.

Ethanol dehydration in HZSM-5 studied by density functional theory: evidence for a concerted process.

Dehydration over acidic zeolites is an important reaction class for the upgrading of biomass pyrolysis vapors to hydrocarbon fuels or to precursors for myriad chemical products. Here, we examine the dehydration of ethanol at a Brønsted acid site, T12, found in HZSM-5 using density functional theory (DFT). The geometries of both cluster and mixed quantum mechanics/molecular mechanics (QM:MM) models are prepared from the ZSM-5 crystal structure. Comparisons between these models and different DFT methods are conducted to show similar results among the models and methods used. Inclusion of the full catalyst cavity through a QM:MM approach is found to be important, since activation barriers are computed on average as 7 kcal mol(-1) lower than those obtained with a smaller cluster model. Two different pathways, concerted and stepwise, have been considered when examining dehydration and deprotonation steps. The current study shows that a concerted dehydration process is possible with a lower (4-5 kcal mol(-1)) activation barrier while previous literature studies have focused on a stepwise mechanism. Overall, this work demonstrates that fairly high activation energies (∼50 kcal mol(-1)) are required for ethanol dehydration. A concerted mechanism is favored over a stepwise mechanism because charge separation in the transition state is minimized. QM:MM approaches appear to provide superior results to cluster calculations due to a more accurate representation of charges on framework oxygen atoms.

Through-Space Ultrafast Photoinduced Electron Transfer Dynamics of a C70-Encapsulated Bisporphyrin Covalent Organic Polyhedron in a Low-Dielectric Medium

Ultrafast photoinduced electron transfer (PIET) dynamics of a C70-encapsulated bisporphyrin covalent organic polyhedron hybrid (C70@COP-5) is studied in a nonpolar toluene medium with fluorescence and transient absorption spectroscopies. This structurally rigid donor (D)-acceptor (A) molecular hybrid offers a new platform featuring conformationally predetermined cofacial D-A orientation with a fixed edge-to-edge separation, REE (2.8 Å), without the aid of covalent bonds. Sub-picosecond PIET (τET ≤ 0.4 ps) and very slow charge recombination (τCR ≈ 600 ps) dynamics are observed. The origin of these dynamics is discussed in terms of enhanced D-A coupling (V = 675 cm-1) and extremely small reorganization energy (λ ≈ 0.18 eV), induced by the intrinsic structural rigidity of the C70@COP-5 complex.

Aromatic Claisen Rearrangements of O‐Prenylated Tyrosine and Model Prenyl Aryl Ethers: Computational Study of the Role of Water on Acceleration of Claisen Rearrangements

LynF, an enzyme from the TruF family, O-prenylates tyrosines in proteins; subsequent Claisen rearrangements give C-prenylated tyrosine products. These reactions in tyrosines and model phenolic systems have been explored with DFT and SCS-MP2 calculations. Various ab initio benchmarks have been computed (CBS-QB3, MP2, SCS-MP2) to examine the accuracy of commonly used density functionals, such as B3LYP and M06-2X. Solvent effects from water were considered using implicit and explicit models. Studies of the ortho-C-prenylation and Claisen rearrangement of tyrosine, and the Claisen rearrangement of α,α-dimethylallyl (prenyl) coumaryl ether establish the energetics of these reactions in the gas phase and in aqueous solution.

Crystal structure of glycoside hydrolase family 127 β-L-arabinofuranosidase from Bifidobacterium longum

Enzymes acting on β-linked arabinofuranosides have been unknown until recently, in spite of wide distribution of β-l-arabinofuranosyl oligosaccharides in plant cells. Recently, a β-l-arabinofuranosidase from the glycoside hydrolase family 127 (HypBA1) was discovered in the newly characterized degradation system of hydroxyproline-linked β-l-arabinooligosaccharides in the bacterium Bifidobacterium longum. Here, we report the crystal structure of HypBA1 in the ligand-free and β-l-arabinofuranose complex forms. The structure of HypBA1 consists of a catalytic barrel domain and two additional β-sandwich domains, with one β-sandwich domain involved in the formation of a dimer. Interestingly, there is an unprecedented metal-binding motif with Zn(2+) coordinated by glutamate and three cysteines in the active site. The glutamate residue is located far from the anomeric carbon of the β-l-arabinofuranose ligand, but one cysteine residue is appropriately located for nucleophilic attack for glycosidic bond cleavage. The residues around the active site are highly conserved among GH127 members. Based on biochemical experiments and quantum mechanical calculations, a possible reaction mechanism involving cysteine as the nucleophile is proposed.