ezAlign: A Tool for Converting Coarse-Grained Molecular Dynamics Structures to Atomistic Resolution for Multiscale Modeling.

Soft condensed matter is challenging to study due to the vast time and length scales that are necessary to accurately represent complex systems and capture their underlying physics. Multiscale simulations are necessary to study processes that have disparate time and/or length scales, which abound throughout biology and other complex systems. Herein we present ezAlign, an open-source software for converting coarse-grained molecular dynamics structures to atomistic representation, allowing multiscale modeling of biomolecular systems. The ezAlign v1.1 software package is publicly available for download at github.com/LLNL/ezAlign. Its underlying methodology is based on a simple alignment of an atomistic template molecule, followed by position-restraint energy minimization, which forces the atomistic molecule to adopt a conformation consistent with the coarse-grained molecule. The molecules are then combined, solvated, minimized, and equilibrated with position restraints. Validation of the process was conducted on a pure POPC membrane and compared with other popular methods to construct atomistic membranes. Additional examples, including surfactant self-assembly, membrane proteins, and more complex bacterial and human plasma membrane models, are also presented. By providing these examples, parameter files, code, and an easy-to-follow recipe to add new molecules, this work will aid future multiscale modeling efforts.

Bacterial Membranes Are More Perturbed by the Asymmetric Versus Symmetric Loading of Amphiphilic Molecules.

Characterizing the biophysical properties of bacterial membranes is critical for understanding the protective nature of the microbial envelope, interaction of biological membranes with exogenous materials, and designing new antibacterial agents. Presented here are molecular dynamics simulations for two cationic quaternary ammonium compounds, and the anionic and nonionic form of a fatty acid molecule interacting with a Staphylococcus aureus bacterial inner membrane. The effect of the tested materials on the properties of the model membranes are evaluated with respect to various structural properties such as the lateral pressure profile, lipid tail order parameter, and the bilayer's electrostatic potential. Conducting asymmetric loading of molecules in only one leaflet, it was observed that anionic and cationic amphiphiles have a large impact on the Staphylococcus aureus membrane's electrostatic potential and lateral pressure profile as compared to a symmetric distribution. Nonintuitively, we find that the cationic and anionic molecules induce a similar change in the electrostatic potential, which points to the complexity of membrane interfaces, and how asymmetry can induce biophysical consequences. Finally, we link changes in membrane structure to the rate of electroporation for the membranes, and again find a crucial impact of introducing asymmetry to the system. Understanding these physical mechanisms provides critical insights and viable pathways for the rational design of membrane-active molecules, where controlling the localization is key.

Solvation Dynamics of HEHEHP Ligand at the Liquid-Liquid Interface.

Actinide-lanthanide separation (ALSEP) has been a topic of interest in recent years as it has been shown to selectively extract problematic metals from spent nuclear fuel. However, the process suffers from slow kinetics, prohibiting it from being applied to nuclear facilities. In an effort to improve the process, many fundamental studies have been performed, but the majority have only focused on the thermodynamics of separation. Therefore, to understand the mechanism behind the ALSEP process, molecular dynamics (MD) simulations were utilized to obtain the dynamics and solvation characteristics for an organic extractant, 2-ethylhexylphosphonic acid mono-2-ethylhexyl ester (HEHEHP). Simulations were conducted with both pure and biphasic solvent systems to evaluate the complex solvent interactions within the ALSEP extraction method. The MD simulations revealed solvation and dynamical behaviors that are consistent with the experimentally observed chemical properties of HEHEHP for the pure solvent systems (e.g., hydrophobic/hydrophilic behaviors of the polar head group and alkyl chains and dimer formation between the ligands within an organic solvent). When present in a biphasic solvent system, interfacial behaviors of the ligand revealed that, at low concentrations, the alkyl side chains of HEHEHP were parallel to the interfacial plane. Upon increasing the concentration to 0.75 M, tendency for the parallel orientation decreased and a more perpendicular-like orientation was observed. Analysis of ligand solvation energies in different solvents through the thermodynamic integration method demonstrated favorability toward n-dodecane and biphasic solvents, which is in agreement with the previous experimental findings.

Correction: Unravelling the impact of hydrocarbon structure on the fumarate addition mechanism - a gas-phase ab initio study.

Correction for 'Unravelling the impact of hydrocarbon structure on the fumarate addition mechanism - a gas-phase ab initio study' by Vivek S. Bharadwaj et al., Phys. Chem. Chem. Phys., 2015, 17, 4054-4066.

Quantum-Mechanical Study of the Reaction Mechanism for 2π-2π Cycloaddition of Fluorinated Methylene Groups.

Perfluorocyclobutyl polymers are thermally and chemically stable, may be produced without a catalyst via thermal 2π-2π cycloaddition, and can form block structures, making them suitable for commercialization of specialty polymers. Thermal 2π-2π cycloaddition is a rare reaction that begins in the singlet state and proceeds through a triplet intermediate to form an energetically stable four-membered ring in the singlet state. This reaction involves two changes in spin state and, thus, two spin-crossover transitions. Presented here are density functional theory calculations that evaluate the energetics and reaction mechanisms for the dimerizations of two different polyfluorinated precursors, 1,1,2-trifluoro-2-(trifluoromethoxy)ethane and hexafluoropropylene. The spin-crossover transition states are thoroughly investigated, revealing important kinetics steps and an activation energy for the gas-phase cycloaddition of two hexafluoropropene molecules of 36.9 kcal/mol, which is in good agreement with the experimentally determined value of 34.3 kcal/mol. It is found that the first carbon-carbon bond formation is the rate-limiting step, followed by a rotation about the newly formed bond in the triplet state that results in the formation of the second carbon-carbon bond. Targeting the rotation of the C-C bond, a set of parameters were obtained that best produce high molecular weight polymers using this chemistry.

A Novel Optical Diagnostic for In Situ Measurements of Lithium Polysulfides in Battery Electrolytes.

An optical diagnostic technique to determine the order and concentration of lithium polysulfides in lithium-sulfur (Li-S) battery electrolytes has been developed. One of the major challenges of lithium-sulfur batteries is the problem of polysulfide shuttling between the electrodes, which leads to self-discharge and loss of active material. Here we present an optical diagnostic for quantitative in situ measurements of lithium polysulfides using attenuated total reflection Fourier transform infrared (FT-IR) spectroscopy. Simulated infrared spectra of lithium polysulfide molecules were generated using computational quantum chemistry routines implemented in Gaussian 09. The theoretical spectra served as a starting point for experimental characterization of lithium polysulfide solutions synthesized by the direct reaction of lithium sulfide and sulfur. Attenuated total reflection FT-IR spectroscopy was used to measure absorption spectra. The lower limit of detection with this technique is 0.05 M. Measured spectra revealed trends with respect to polysulfide order and concentration, consistent with theoretical predictions, which were used to develop a set of equations relating the order and concentration of lithium polysulfides in a sample to the position and area of a characteristic infrared absorption band. The diagnostic routine can measure the order and concentration to within 5% and 0.1 M, respectively.

Impact of Water-Dilution on the Solvation Properties of the Ionic Liquid 1-Methyltriethoxy-3-ethylimidazolium Acetate for Model Biomass Molecules.

Many studies have suggested that the processing of lignocellulosic biomass could provide a renewable feedstock to supplant much of the current demand on petroleum sources. Currently, alkyl imidazolium-based ionic liquids (ILs) have shown considerable promise in the pretreatment, solvation, and hydrolysis of lignocellulosic materials although their high cost and unfavorable viscosity has limited their widespread use. Functionalizing these ILs with an oligo(ethoxy) tail has previously been shown through experiment to decrease the IL's viscosity resulting in enhanced mass transport characteristics, in addition to other favorable traits including decreased inhibition of some enzymes. Additionally, the use of cosolvents to mitigate the cost and unfavorable traits of ILs is an area of growing interest with particular attention on water as the presence of water in biomass processes is inevitable. Through the use of biased and unbiased molecular dynamics (MD) simulations, this study provides a molecular-level perspective of the various solvent-solvent and solvent-solute interactions in binary mixtures of water and 1-methyltriethoxy-3-ethylimidazolium acetate ([Me-(OEt)3-Et-IM+] [OAc-]) in the presence of model cellulose compounds (i.e., glucose and cellobiose). It is observed that at ∼75% w/w IL and water a transition in the nanostructure of the solvent occurs between water-like and IL-like solvation characteristics. It is shown that H-bonding interactions between the anion and water are a major driving force that significantly impacts the solvent properties of the IL as well as conformational preferences of the cellulosic model compound. In addition, it is found that the oligo(ethoxy) cation tail is responsible for the reduction in the propensity for tail aggregation as compared to alkyl tails of similar length, which, combined with increased ionic shielding, results in increased diffusion and enhanced water-like solvation characteristics.

Coupling Protein Dynamics with Proton Transport in Human Carbonic Anhydrase II.

The role of protein dynamics in enzyme catalysis is one of the most highly debated topics in enzymology. The main controversy centers around what may be defined as functionally significant conformational fluctuations and how, if at all, these fluctuations couple to enzyme catalyzed events. To shed light on this debate, the conformational dynamics along the transition path surmounting the highest free energy barrier have been herein investigated for the rate limiting proton transport event in human carbonic anhydrase (HCA) II. Special attention has been placed on whether the motion of an excess proton is correlated with fluctuations in the surrounding protein and solvent matrix, which may be rare on the picosecond and subpicosecond time scales of molecular motions. It is found that several active site residues, which do not directly participate in the proton transport event, have a significant impact on the dynamics of the excess proton. These secondary participants are shown to strongly influence the active site environment, resulting in the creation of water clusters that are conducive to fast, moderately slow, or slow proton transport events. The identification and characterization of these secondary participants illuminates the role of protein dynamics in the catalytic efficiency of HCA II.

Overview: Nucleation of clathrate hydrates.

Molecular level knowledge of nucleation and growth of clathrate hydrates is of importance for advancing fundamental understanding on the nature of water and hydrophobic hydrate formers, and their interactions that result in the formation of ice-like solids at temperatures higher than the ice-point. The stochastic nature and the inability to probe the small length and time scales associated with the nucleation process make it very difficult to experimentally determine the molecular level changes that lead to the nucleation event. Conversely, for this reason, there have been increasing efforts to obtain this information using molecular simulations. Accurate knowledge of how and when hydrate structures nucleate will be tremendously beneficial for the development of sustainable hydrate management strategies in oil and gas flowlines, as well as for their application in energy storage and recovery, gas separation, carbon sequestration, seawater desalination, and refrigeration. This article reviews various aspects of hydrate nucleation. First, properties of supercooled water and ice nucleation are reviewed briefly due to their apparent similarity to hydrates. Hydrate nucleation is then reviewed starting from macroscopic observations as obtained from experiments in laboratories and operations in industries, followed by various hydrate nucleation hypotheses and hydrate nucleation driving force calculations based on the classical nucleation theory. Finally, molecular simulations on hydrate nucleation are discussed in detail followed by potential future research directions.

Molecular Simulations of Fatty-Acid Methyl Esters and Representative Biodiesel Mixtures.

Despite the importance of fatty-acid methyl esters (FAMEs) as key components of various green solvents, detergents, plasticizers, and biodiesels, our understanding of these systems at the molecular level is limited. An enhanced molecular-level perspective of FAMEs will enable a detailed analysis of the polymorph and crystallization phenomena that adversely impact flow properties at low temperatures. Presented here, is the parameterization and validation of a charge-modified generalized amber force field (GAFF) for eight common FAMEs and two representative biodiesel mixtures. Our simulations accurately reproduce available experimental data (e.g. densities and self-diffusivity coefficients) and their trends, with respect to temperature and degree of unsaturation. Structural analyses from our simulations provide a more detailed picture of liquid-phase molecular ordering in FAMEs and confirm recent experimental hypotheses. This study provides a firm foundation to initiate further studies into the mechanisms that drive crystallization phenomena at the molecular level.

The impact of active site protonation on substrate ring conformation in Melanocarpus albomyces cellobiohydrolase Cel7B.

The ability to utilize biomass as a feedstock for liquid fuel and value-added chemicals is dependent on the efficient and economic utilization of lignin, hemicellulose, and cellulose. In current bioreactors, cellulases are used to convert crystalline and amorphous cellulose to smaller oligomers and eventually glucose by means of cellulase enzymes. A critical component of the enzyme catalyzed hydrolysis reaction is the degree to which the enzyme can facilitate substrate ring deformation from the chair to a more catalytically active conformation (e.g. skewed boat) at the -1 subsite. Presented here is an evaluation of the impact of the protonation state for critical active site residues (i.e. Glu212, Asp214, Glu217, and His228) in Melanocarpus albomyces (Ma) Cellobiohydrolase Cel7B on the substrate's orientation and ring conformation. It is found that the protonation state of the active site can disrupt the intra-enzyme hydrogen bonding network and enhance the sampling of various ring puckering conformations for the substrate ring at the +1 and -1 subsites. In particular it is observed that the protonation state of Asp214 dictates the accessibility of the glycosidic bond to the catalytic acid/base Glu217 by influencing the φ/ψ dihedral angles and the puckering of the ring structure. The protonation-orientation-conformation analysis has revealed an active site that primarily utilizes two highly coupled protonation schemes; one protonation scheme to orient the substrate and generate catalytically favorable substrate geometries and ring puckering conformations and another protonation scheme to hydrolyze the glycosidic bond. In addition to identifying how enzymes utilize protonation state to manipulate substrate geometry, this study identifies possible directions for improving catalytic activity through protein engineering.

Elucidating the conformational energetics of glucose and cellobiose in ionic liquids.

A major challenge for the utilization of lignocellulosic feedstocks for liquid fuels and other value added chemicals has been the recalcitrant nature of crystalline cellulose to various hydrolysis techniques. Ionic liquids (ILs) are considered to be a promising solvent for the dissolution and conversion of cellulose to simple sugars, which has the potential to facilitate the unlocking of biomass as a supplement and/or replacement for petroleum as a feedstock. Recent studies have revealed that the orientation of the hydroxymethyl group, described via the ω dihedral, and the glycosidic bond, described via the φ-ψ dihedrals, are significantly modified in the presence of ILs. In this study, we explore the energetics driving the orientational preference of the ω dihedral and the φ-ψ dihedrals for glucose and cellobiose in water and three imidazolium based ILs. It is found that interactions between the cation and the ring oxygen in glucose directly impact the conformational preference of the ω dihedral shifting the distribution towards the gauche-trans (GT) conformation and creating an increasingly unfavorable gauche-gauche (GG) conformation with increasing tail length. This discovery modifies the current hypothesis that intramolecular hydrogen bonding is responsible for the shift in the ω dihedral distribution and illuminates the importance of the cation's character. In addition, it is found that the IL's interaction with the glycosidic bond results in the modification of the observed φ-ψ dihedrals, which may have implications for hydrolysis in the presence of ILs. The molecular level information gained from this study identifies the favorable IL-sugar interactions that need to be exploited in order to enhance the utilization of lignocellulosic biomass as a ubiquitous feedstock.

Unravelling the impact of hydrocarbon structure on the fumarate addition mechanism--a gas-phase ab initio study.

The fumarate addition reaction mechanism is central to the anaerobic biodegradation pathway of various hydrocarbons, both aromatic (e.g., toluene, ethyl benzene) and aliphatic (e.g., n-hexane, dodecane). Succinate synthase enzymes, which belong to the glycyl radical enzyme family, are the main facilitators of these biochemical reactions. The overall catalytic mechanism that converts hydrocarbons to a succinate molecule involves three steps: (1) initial H-abstraction from the hydrocarbon by the radical enzyme, (2) addition of the resulting hydrocarbon radical to fumarate, and (3) hydrogen abstraction by the addition product to regenerate the radical enzyme. Since the biodegradation of hydrocarbon fuels via the fumarate addition mechanism is linked to bio-corrosion, an improved understanding of this reaction is imperative to our efforts of predicting the susceptibility of proposed alternative fuels to biodegradation. An improved understanding of the fuel biodegradation process also has the potential to benefit bioremediation. In this study, we consider model aromatic (toluene) and aliphatic (butane) compounds to evaluate the impact of hydrocarbon structure on the energetics and kinetics of the fumarate addition mechanism by means of high level ab initio gas-phase calculations. We predict that the rate of toluene degradation is ∼100 times faster than butane at 298 K, and that the first abstraction step is kinetically significant for both hydrocarbons, which is consistent with deuterium isotope effect studies on toluene degradation. The detailed computations also show that the predicted stereo-chemical preference of the succinate products for both toluene and butane are due to the differences in the radical addition rate constants for the various isomers. The computational and kinetic modeling work presented here demonstrates the importance of considering pre-reaction and product complexes in order to accurately treat gas phase systems that involve intra and inter-molecular non-covalent interactions.

Electronic structure and spectroscopic analysis of 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ion pair.

Electronic and structural properties of the room temperature ionic liquid 1-ethyl-3-methylimidazolium bis(trifluoromethylsulonyl)imide are studied using density functional theory (DFT) methods in addition to infrared and UV-vis spectroscopy. The DFT methods were conducted for both gas phase and solution phase using the integral equation formalism polarizable continuum model, while optical absorption experiments were conducted using neat and dilute methanol solutions. Three energetically similar conformers were obtained for each of the gas phase and solution phase DFT calculations. These multiple configurations were considered when analyzing the molecular interactions between the ion pair and for a molecular-level interpretation of the experimental IR and UV-vis spectroscopy data. Excitation energies of low-lying singlet excited states of the conformers were calculated with time-dependent DFT and experimentally with UV-vis absorption spectra. Difference density plots and excited-state calculations in the gas phase are found to be in good agreement with the experimental findings, while the implicit solvation model calculations adversely impacted the accuracy of the predicted spectra.

Structural insight into activity enhancement and inhibition of H64A carbonic anhydrase II by imidazoles.

Human carbonic anhydrases (CAs) are zinc metalloenzymes that catalyze the hydration and dehydration of CO2 and HCO3 (-), respectively. The reaction follows a ping-pong mechanism, in which the rate-limiting step is the transfer of a proton from the zinc-bound solvent (OH(-)/H2O) in/out of the active site via His64, which is widely believed to be the proton-shuttling residue. The decreased catalytic activity (∼20-fold lower with respect to the wild type) of a variant of CA II in which His64 is replaced with Ala (H64A CA II) can be enhanced by exogenous proton donors/acceptors, usually derivatives of imidazoles and pyridines, to almost the wild-type level. X-ray crystal structures of H64A CA II in complex with four imidazole derivatives (imidazole, 1--methylimidazole, 2--methylimidazole and 4-methylimidazole) have been determined and reveal multiple binding sites. Two of these imidazole binding sites have been identified that mimic the positions of the 'in' and 'out' rotamers of His64 in wild-type CA II, while another directly inhibits catalysis by displacing the zinc-bound solvent. The data presented here not only corroborate the importance of the imidazole side chain of His64 in proton transfer during CA catalysis, but also provide a complete structural understanding of the mechanism by which imidazoles enhance (and inhibit when used at higher concentrations) the activity of H64A CA II.

Computational evaluation of the dynamic fluctuations of peripheral loops enclosing the catalytic tunnel of a family 7 cellobiohydrolase.

The size and character of the peripheral loops enclosing the active site for cellulase enzymes is believed to play a major role in dictating many critical enzymatic properties. For many cellulases it is observed that fully enclosed active sites forming a tunnel are more conducive to cellobiohydrolase activity and the ability to processively move along the substrate. Conversely, a more open active site groove is indicative of endoglucanase activity. For both cellobiohydrolases and endoglucanases, the loop regions have been implicated in the ability of the enzyme to bind substrate, influence the pKa of active site residues, modulate the catalytic activity, and influence thermal stability. Reported here are constant pH molecular dynamics (CpHMD) simulations that investigate the role of dynamic fluctuations, substrate interactions, and residue pKa values for the peripheral loops enclosing the active site of the cellobiohydrolase Melanocarpus albomyces Cel7B. Two highly flexible loop regions in the free enzyme have been identified, which impact the overall dynamical motions of the enzyme. Charge interactions between Asp198 and Asp367, which reside on two adjacent loops, were found to influence the overall loop conformations and dynamics. In the presence of a substrate the protonation of Asp367, Asp198, and Tyr370 were found to stabilize substrate binding and control the movement of two peripheral loops onto the active site containing the substrate (i.e., clamping down). The substrate-induced response of the loop regions secures the cellulose polymer in the catalytic tunnel and creates an environment that is conducive to hydrolysis of the glycosidic bond.

Acetylcholine promotes binding of α-conotoxin MII at α3 ß2 nicotinic acetylcholine receptors.

α-Conotoxin MII (α-CTxMII) is a 16-residue peptide with the sequence GCCSNPVCHLEHSNLC, containing Cys2-Cys8 and Cys3-Cys16 disulfide bonds. This peptide, isolated from the venom of the marine cone snail Conus magus, is a potent and selective antagonist of neuronal nicotinic acetylcholine receptors (nAChRs). To evaluate the impact of channel-ligand interactions on ligand-binding affinity, homology models of the heteropentameric α3ß2-nAChR were constructed. The models were created in MODELLER with the aid of experimentally characterized structures of the Torpedo marmorata-nAChR (Tm-nAChR, PDB ID: 2BG9) and the Aplysia californica-acetylcholine binding protein (Ac-AChBP, PDB ID: 2BR8) as templates for the α3- and ß2-subunit isoforms derived from rat neuronal nAChR primary amino acid sequences. Molecular docking calculations were performed with AutoDock to evaluate interactions of the heteropentameric nAChR homology models with the ligands acetylcholine (ACh) and α-CTxMII. The nAChR homology models described here bind ACh with binding energies commensurate with those of previously reported systems, and identify critical interactions that facilitate both ACh and α-CTxMII ligand binding. The docking calculations revealed an increased binding affinity of the α3ß2-nAChR for α-CTxMII with ACh bound to the receptor, and this was confirmed through two-electrode voltage clamp experiments on oocytes from Xenopus laevis. These findings provide insights into the inhibition and mechanism of electrostatically driven antagonist properties of the α-CTxMIIs on nAChRs.

Computational evaluations of charge coupling and hydrogen bonding in the active site of a family 7 cellobiohydrolase.

Solution pH and the pKa values of ionizable residues are critical factors known to influence enzyme catalysis, structural stability, and dynamical fluctuations. Presented here is an exhaustive computational study utilizing long time constant pH molecular dynamics, pH replica exchange simulations, and kinetic modeling to evaluate pH-dependent conformations, charge dynamics, residue pKa values, and the catalytic activity-pH profile for cellobiohydrolase Cel7B from Melanocarpus albomyces . The predicted pKa values support the role of Glu212 as the catalytic nucleophile and Glu217 as the acid-base residue. The presence of a charge-correlated active site and an extensive hydrogen bonding network is found to be critical in enabling favorable residue orientations for catalysis and shuttling excess protons around the active site. Clusters of amino acids are identified that act in concert to effectively modulate the optimal pH for catalysis while elevating the overall catalytic rate with respect to a noncoupled system. The work presented here demonstrates the complex and critical role of coupled ionizable residues to the proper functioning of cellobiohydrolase Cel7B, functionally related glycosyl hydrolases, and enzymes in general. The simulations also support the use of the CpHMD for the accurate prediction of residue pKa values and to evaluate the impact of pH on protein structure and charge dynamics.

Homology modeling and molecular docking for the science curriculum.

DockoMatic 2.0 is a powerful open source software program (downloadable from sourceforge.net) that allows users to utilize a readily accessible computational tool to explore biomolecules and their interactions. This manuscript describes a practical tutorial for use in the undergraduate curriculum that introduces students to macromolecular structure creation, ligand binding calculations, and visualization of docking results. A student procedure is provided that illustrates the use of DockoMatic to create a homology model for the amino propeptide region (223 amino acids with two disulfide bonds) of collagen α1 (XI), followed by molecular docking of the commercial drug Arixtra(®) to the homology model of α1 (XI), and finally, analysis of the results of the docking experiment. The activities and Supporting Information described are intended to educate students in the use of computational tools to create and investigate homology models for other systems of interest and to train students to perform and analyze molecular docking studies. The tutorial also serves as a foundation for investigators seeking to explore the viability of using computational biochemistry to study their receptor-ligand binding motifs. © 2013 by The International Union of Biochemistry and Molecular Biology, 42(2):179-182, 2014.

Insights into the glycyl radical enzyme active site of benzylsuccinate synthase: a computational study.

The fumarate addition reaction, catalyzed by the enzyme benzylsuccinate synthase (BSS), is considered to be one of the most intriguing and energetically challenging reactions in biology. BSS belongs to the glycyl radical enzyme family and catalyzes the fumarate addition reaction, which enables microorganisms to utilize hydrocarbons as an energy source under anaerobic conditions. Unfortunately, the extreme sensitivity of the glycyl radical to oxygen has hampered the structural and kinetic characterization of BSS, thereby limiting our knowledge on this enzyme. To enhance our molecular-level understanding of BSS, a computational approach involving homology modeling, docking studies, and molecular dynamics (MD) simulations has been used to deduce the structure of BSS's catalytic subunit (BSSα) and illuminate the molecular basis for the fumarate addition reaction. We have identified two conserved and distinct binding pockets at the BSSα active site: a hydrophobic pocket for toluene binding and a polar pocket for fumaric acid binding. Subsequent dynamical and energetic evaluations have identified Glu509, Ser827, Leu390, and Phe384 as active site residues critical for substrate binding. The orientation of substrates at the active site observed in MD simulations is consistent with experimental observations of the syn addition of toluene to fumaric acid. It is also found that substrate binding tightens the active site and restricts the conformational flexibility of the thiyl radical, leading to hydrogen transfer distances conducive to the proposed reaction mechanism. The stability of substrates at the active site and the occurrence of feasible radical transfer distances between the thiyl radical, substrates, and the active site glycine indicate a substrate-assisted radical transfer pathway governing fumarate addition.