Insights into generalization of the rate-limiting steps of the dehalogenation by LinB and DhaA: A computational approach.

LinB and DhaA are well-known haloalkane dehalogenases (HLDs) capable of converting a plethora of halogenated alkanes, also those considered persistent pollutants. The dehalogenation reaction that these two enzymes catalyze has been studied to determine its rate-limiting step (rls) for the last two decades now. As a result, it has been determined that HLDs can show different rate-limiting steps for individual substrates, and at this point we do not have a basis for any generalization in this matter. Therefore, in this work we aimed at gaining insights into the enzymatic dehalogenation of selected dibromo- and bromochloro-ethanes and propanes by LinB and DhaA using computational approach to determine whether defined structural similarities of the substrates result in a unified mechanism and the same rls. By predicting halogen binding isotope effects (BIEs) as well as computing interaction energy for each HLD-ligand complex the nature of the protein-ligand interactions has been characterized. Furthermore, C and Br kinetic isotope effects (KIEs) as well as the minimum free energy paths (MFEPs) were computed to investigate the chemical reaction for the selected systems. Accuracy of the approach and robustness of the computational predictions were validated by measuring KIEs on the selected reactions. Overall results strongly indicate that any generalization with respect to the enzymatic process involving various ligands in the case of DhaA is impossible, even if the considered ligands are structurally very similar as those analyzed in the present study. Moreover, even small structural differences such as changing of one of the (non-leaving) halogen substituents may lead to significant changes in the enzymatic process and result in a different rls in the case of LinB. It has also been demonstrated that KIEs themselves cannot be used as rls indicators in the reactions catalyzed by the studied HLDs.

Unravelling interactions between active site residues and DMAP in the initial steps of prenylated flavin mononucleotide biosynthesis catalyzed by PaUbiX.

BACKGROUND: Prenylated flavin mononucleotide (prFMN) is a recently discovered, heavily modified flavin compound. It is the only known cofactor that enables enzymatic 1,3-dipolar cycloaddition reactions. It is produced by enzymes from the UbiX family, from flavin mononucleotide and either dimethylallyl mono- or diphosphate. prFMN biosynthesis is currently reported to be initiated by protonation of the substrate by Glu140. METHODS: Computational chemistry methods are applied herein - Constant pH MD, classical MD simulations, and QM cluster optimizations. RESULTS: Glu140 competes for a single proton with Lys129 prior to prFMN biosynthesis, but it is the latter that adopted a protonated state. Once the prenyl-FMN adduct is formed, Glu140 occurs in a protonated state far more often, while the occupancy of protonated Lys129 does not change. Lys129, Glu140, and Arg122 seem to play a key role in either stabilizing or protonating DMAP phosphate group within the PaUbiX active site throughout initial steps of prFMN biosynthesis. CONCLUSIONS: The role of Lys129 in the functioning of PaUbiX is reported for the first time. Glu140 is unlikely to act as a proton donor in prFMN biosynthesis. Instead, Lys129 and Arg122 fulfil this role. Glu140 still plays a role in contributing to hydrogen-bond network. This behavior is most likely conserved throughout the UbiX family due to the structural similarity of the active sites of those proteins. SIGNIFICANCE: Mechanistic insights into a crucial biochemical process, the biosynthesis of prFMN, are provided. This study, although purely computational, extends and perfectly complements the knowledge obtained in classical laboratory experiments.

Isotope Effects on the Vaporization of Organic Compounds from an Aqueous Solution-Insight from Experiment and Computations.

An isotope fractionation analysis of organic groundwater pollutants can assess the remediation at contaminated sites yet needs to consider physical processes as potentially confounding factors. This study explores the predictability of water-air partitioning isotope effects from experiments and computational predictions for benzene and trimethylamine (both H-bond acceptors) as well as chloroform (H-bond donor). A small, but significant, isotope fractionation of different direction and magnitude was measured with ε = -0.12‰ ± 0.07‰ (benzene), εC = 0.49‰ ± 0.23‰ (triethylamine), and εH = 1.79‰ ± 0.54‰ (chloroform) demonstrating that effects do not correlate with expected hydrogen-bond functionalities. Computations revealed that the overall isotope effect arises from contributions of different nature and extent: a weakening of intramolecular vibrations in the condensed phase plus additional vibrational modes from a complexation with surrounding water molecules. Subtle changes in benzene contrast with a stronger coupling between intra- and intermolecular modes in the chloroform-water system and a very local vibrational response with few atoms involved in a specific mode of triethylamine. An energy decomposition analysis revealed that each system was affected differently by electrostatics and dispersion, where dispersion was dominant for benzene and electrostatics dominated for chloroform and triethylamine. Interestingly, overall stabilization patterns in all studied systems originated from contributions of dispersion rather than other energy terms.

Seeking the Source of Catalytic Efficiency of Lindane Dehydrochlorinase, LinA.

Herein we present the results of an in-depth simulation study of LinA and its two variants. In our analysis, we combined the exploration of protein conformational dynamics with and without bound substrates (hexachlorocyclohexane (HCH) isomers) performed using molecular dynamics simulation followed by the extraction of the most frequently visited conformations and their characteristics with a detailed description of the interactions taking place in the active site between the respective HCH molecule and the first shell residues by using symmetry-adapted perturbation theory (SAPT) calculations. A detailed investigation of the conformational space of LinA substates has been accompanied by description of enzymatic catalytic steps carried out using a hybrid quantum mechanics/molecular mechanics (QM/MM) potential along with the computation of the potential of mean force (PMF) to estimate the free energy barriers for the studied transformations: dehydrochlorination of γ-, (-)-α-, and (+)-α-HCH by LinA-type I and -type II variants. The applied combination of computational techniques allowed us not only to characterize two LinA types but also to point to the most important differences between them and link their features to catalytic efficiency each of them possesses toward the respective ligand. More importantly it has been demonstrated that type I protein is more mobile, its active site has a larger volume, and the dehydrochlorination products are stabilized more strongly than in the case of type II enzyme, due to differences in the residues present in the active sites. Additionally, interaction energy calculations revealed very interesting patterns not predicted before but having the potential to be utilized in any attempts of improving LinA catalytic efficiency. On the basis of all these observations, LinA-type I protein seems to be more preorganized for the dehydrochlorination reaction it catalyzes than the type II variant.

Computational Investigations of Position-Specific Vapor Pressure Isotope Effects in Ethanol-Toward More Powerful Isotope Models for Food Forensics.

With the advent of new experimental techniques, measurements of individual, per-position, vapor pressure isotope effects (VPIEs) became possible. Frequently, they are in opposite directions (larger and smaller than unity), leading to the cancellation when only bulk values are determined. This progress has not been yet paralleled by the theoretical description of phase change processes that would allow for computational prediction of the values of these isotope effects. Herein, we present the first computational protocol that allowed us to predict carbon VPIEs for ethanol-the molecule of great importance in authentication protocols that rely on the precise information about position-specific isotopic composition. Only the model comprising explicit treatment of the surrounding first-shell molecules provided good agreement with the measured values of isotope effects. Additionally, we find that the internal vibrations of molecules of the model to predict isotope effects work better than the entire set of normal modes of the system.

Compound-specific chlorine isotope fractionation in biodegradation of atrazine.

Atrazine is a frequently detected groundwater contaminant. It can be microbially degraded by oxidative dealkylation or by hydrolytic dechlorination. Compound-specific isotope analysis is a powerful tool to assess its transformation. In previous work, carbon and nitrogen isotope effects were found to reflect these different transformation pathways. However, chlorine isotope fractionation could be a particularly sensitive indicator of natural transformation since chlorine isotope effects are fully represented in the molecular average while carbon and nitrogen isotope effects are diluted by non-reacting atoms. Therefore, this study explored chlorine isotope effects during atrazine hydrolysis with Arthrobacter aurescens TC1 and oxidative dealkylation with Rhodococcus sp. NI86/21. Dual element isotope slopes of chlorine vs. carbon isotope fractionation (Λ = 1.7 ± 0.9 vs. Λ = 0.6 ± 0.1) and chlorine vs. nitrogen isotope fractionation (Λ = -1.2 ± 0.7 vs. Λ = 0.4 ± 0.2) provided reliable indicators of different pathways. Observed chlorine isotope effects in oxidative dealkylation (εCl = -4.3 ± 1.8‰) were surprisingly large, whereas in hydrolysis (εCl = -1.4 ± 0.6‰) they were small, indicating that C-Cl bond cleavage was not the rate-determining step. This demonstrates the importance of constraining expected isotope effects of new elements before using the approach in the field. Overall, the triple element isotope information brought forward here enables a more reliable identification of atrazine sources and degradation pathways.

Bromine isotope effects: Predictions and measurements.

Brominated organic compounds (BOCs), abundant in Nature, originate from its own sources or anthropogenic activity. Many of these compounds are harmful and constitute a serious threat, therefore it is important to study and understand their behavior and fate. In situ, BOCs undergo various chemical and biochemical reactions through distinctive mechanistic pathways. However, breaking C-Br specific bond is a crucial step in the transformation of brominated organic compounds. Understanding the mechanisms of debromination can be substantially enhanced by studying Br isotope effects. In this Mini-review we provide overlook of existing experimental techniques for Br isotope analysis, discuss Br kinetic isotope effects measured for selected chemical and biochemical reactions in the light of underlying reaction mechanisms, and review the outcome from computational study of performed to provide more insightful interpretation of observed findings.

Influence of short peptides with aromatic amino acid residues on aggregation properties of serum amyloid A and its fragments.

Serum amyloid A variant 1.1 (SAA1.1) is an acute phase protein. In response to injury, inflammation or infection its production increases highly, which may lead to aggregation of the protein and accumulation of its deposits in various organs. Due to the cellular toxicity of the aggregates, as well as the fact that accumulated deposits are a burden that obstructs proper functioning of the affected tissues, it is vital to find a way to suppress the process of pathological aggregates formation. To make this possible, it is necessary to investigate thoroughly the oligomerization process and recognize factors that may influence its course. Some previous studies showed that aromatic interactions are important to the potential of an inhibitor to suppress the aggregation process. In our research we had proved that a five-residue peptide RSFFS (saa1-5) is an efficient inhibitor of aggregation of the most amyloidogenic fragment of SAA1.1, SAA1-12. In the present work the oligomerization and aggregation propensity of SAA1-12 was compared to that of SAA1-27, in order to determine the contribution of the sequence which extends beyond the most amyloidogenic region but encompasses residues reportedly involved in the stabilization of the SAA native conformation. Thioflavin T fluorescence assay, quantitative chromatographic analysis of the insoluble fraction and transmission electron microscopy allowed for a deeper insight into the SAA aggregation process and the morphology of aggregates. Substitutions of Phe3 and/or Phe4 residues in saa1-5 sequence with tryptophan, tyrosine, homophenylalanine, naphthylalanine and ß,ß-diphenylalanine allowed to study the influence of different aromatic systems on the aggregation of SAA1-12 and SAA1-27, and evaluate these results in relation to hSAA1.1 protein. Our results indicate that compounds with aromatic moieties can affect the course of the aggregation process and change the ratio between the soluble and insoluble aggregates.

Role of Microsolvation and Quantum Effects in the Accurate Prediction of Kinetic Isotope Effects: The Case of Hydrogen Atom Abstraction in Ethanol by Atomic Hydrogen in Aqueous Solution.

Hydrogen abstraction from ethanol by atomic hydrogen in aqueous solution is studied using two theoretical approaches: the multipath variational transition state theory (MP-VTST) and a path-integral formalism in combination with free-energy perturbation and umbrella sampling (PI-FEP/UM). The performance of the models is compared to experimental values of H kinetic isotope effects (KIE). Solvation models used in this study ranged from purely implicit, via mixed-microsolvation treated quantum mechanically via the density functional theory (DFT) to fully explicit representation of the solvent, which was incorporated using a combined quantum mechanical-molecular mechanical (QM/MM) potential. The effects of the transition state conformation and the position of microsolvating water molecules interacting with the solute on the KIE are discussed. The KIEs are in good agreement with experiment when MP-VTST is used together with a model that includes microsolvation of the polar part of ethanol by five or six water molecules, emphasizing the importance of explicit solvation in KIE calculations. Both, MP-VTST and PI-FEP/UM enable detailed characterization of nuclear quantum effects accompanying the hydrogen atom transfer reaction in aqueous solution.

Can Alkaline Hydrolysis of γ-HCH Serve as a Model Reaction to Study Its Aerobic Enzymatic Dehydrochlorination by LinA?

Hexachlorocyclohexane (HCH) isomers constitute a group of persistent organic pollutants. Their mass production and treatment have led to a global environmental problem that continues to this day. The characterization of modes of degradation of HCH by isotope fractionation is a current challenge. Multi isotope fractionation analysis provides a concept to characterize the nature of enzymatic and chemical transformation reactions. The understanding of the kinetic isotope effects (KIE) on bond cleavage reaction contributes to analyses of the mechanism of chemical and enzymatic reactions. Herein, carbon, chlorine, and hydrogen kinetic isotope effects are measured and predicted for the dehydrochlorination reaction of γ-HCH promoted by the hydroxyl ion in aqueous solution. Quantum mechanical (QM) microsolvation with an implicit solvation model and path integral formalism in combination with free-energy perturbation and umbrella sampling (PI-FEP/UM) and quantum mechanical/molecular mechanical QM/MM potentials for including solvent effects as well as calculating isotope effects are used and analyzed with respect to their performance in reproducing measured values. Reaction characterization is discussed based on the magnitudes of obtained isotope effects. The comparative analysis between the chemical dehydrochlorination of γ-HCH in aqueous media and catalyzed reaction by dehydrochlorinase, LinA is presented and discussed. Based on the values of isotope effects, these two processes seem to occur via the same net mechanism.

Ligand-Driven Conformational Dynamics Influences Selectivity of UbiX.

Up until now, it has remained elusive as to why the flavin prenyltransferase UbiX requires dimethylallyl monophosphate (DMAP) as one of its cosubstrates instead of dimethylallyl pyrophosphate (DMAPP), even though the former is not used in metabolic pathways, while the latter is a common isoprenoid precursor. Herein, mainly on the basis of molecular dynamics (MD) simulations, we demonstrate that the selectivity of UbiX may be governed by its conformational dynamics. The hydrogen-bonding network of UbiX does not facilitate a proper encompassing of DMAPP. This induces significant conformational changes of the enzyme that result mostly in unreactive trajectories, whereas DMAP remains at a catalytically competent position throughout the performed simulations. Within the presented study, we provide a justification for the atypical selectivity of UbiX.

Can Path Integral Molecular Dynamics Make a Good Approximation for Vapor Pressure Isotope Effects Prediction for Organic Solvents? A Comparison to ONIOM QM/MM and QM Cluster Calculation.

Isotopic fractionation of volatile organic compounds (VOCs), which are under strict measures of control because of their potential harm to the environment and humans, has an important ecological aspect, as the isotopic composition of compounds may depend on the conditions in which such compounds are distributed in Nature. Therefore, detailed knowledge on isotopic fractionation, not only experimental but also based on theoretical models, is crucial to follow conditions and pathways within which these contaminants are spread throughout the ecosystems. In this work, we present carbon and, for the first time, bromine vapor pressure isotope effect (VPIE) on the evaporation process from pure-phase systems-dibromomethane and bromobenzene, the representatives of aliphatic and aromatic brominated VOCs. We combine isotope effects measurements with their theoretical prediction using three computational techniques, namely path integral molecular dynamics, QM cluster, and hybrid ONIOM models. While evaporation of both compounds resulted in normal bromine VPIEs, the difference in the direction of carbon isotopic fractionation is observed for the aliphatic and aromatic compounds, where VPIEs are inverse and normal, respectively. Even though theoretical models tested here turned out to be insufficient for quantitative agreement with the experimental values, cluster electronic structure calculations, as well as two-layer ONIOM computations, provided better reproduction of experimental trends.

Carbon-bromine bond cleavage - A perspective from bromine and carbon kinetic isotope effects on model debromination reactions.

In this work, we explore the effect of solvent on 13C and 81Br kinetic isotope effects (KIEs) during elimination of bromine substituent from brominated organic compounds promoted by hydroxyl anion. In the present study, we investigate HBr elimination from 2-bromoethylbenzene in three different polar media (water, ethanol, and acetonitrile) as well as bromide ion elimination from 1,2-dibromoethane upon nucleophilic substitution by the hydroxyl ion in aqueous solution using carbon and bromine isotope analysis as mechanistic tools. We reconsider the hypothesis that the magnitude of leaving group halide KIE should visibly depend on the solvent and bond-breaking in a protic solvent should be accompanied by hydrogen bonding which would result in less zero-point energy loss than in an aprotic solvent. Modeling the elimination reaction using the available popular theoretical methods along with different approaches for including environment effects we demonstrate in the presented study no interpretable effect of the solvent on the transition state structure and hence on the theoretically predicted KIEs. The comparison of the magnitudes of carbon and bromine kinetic isotope effects for two different mechanistic pathways (elimination vs substitution) is also discussed.

Measurement and Prediction of Chlorine Kinetic Isotope Effects in Enzymatic Systems.

Approaches to determine chlorine kinetic isotope effects (Cl-KIEs) on enzymatic dehalogenations are discussed and illustrated by representative examples. Three aspects are considered. First methodology for experimental measurement of Cl-KIEs, with stress being on FAB-IRMS technique developed in our laboratory, is described. Subsequently, we concentrate our discussion on the consequences of reaction complexity in the interpretation of experimental values, a problem especially important in cases of polychlorinated reactants. The most fruitful studies of enzymatic dehalogenations by Cl-KIEs require their theoretical evaluation, hence the computational focus of the second part of this chapter.

A Benchmark Study of Kinetic Isotope Effects and Barrier Heights for the Finkelstein Reaction.

Herein, we present a combined (experimental and computational) study of the Finkelstein reaction in condensed phase, where bromine is substituted by iodine in 2-bromoethylbenzene, in the presence of either acetone or acetonitrile as a solvent. Performance of various density functional theory and ab initio methods were tested for reaction barrier heights as well as for bromine and carbon kinetic isotope effects (KIEs). Two different implicit solvation models were examined (PCM and SMD). Theoretically predicted KIEs were compared with experimental values, while reaction barrier heights were assessed using the CCSD(T)-level and experimental energies as reference. In general, although the tested parameters (energies and KIEs) do not exhibit any substantial difference upon a change of the solvent, the different behavior of the theoretical methods was observed depending on the solvent. With respect to isotope effects, both PCM and SMD seem to perform very similarly, though results obtained with PCM are slightly closer to the experimental values. For predicting reaction barriers, utilization of either PCM or SMD solvation models yielded different results. Functionals from the ωB97 family: ωB97, ωB97X, and ωB97X-D provide the most accurate results for the studied system.

Characterization of Fluorescein-Based Monoboronate Probe and Its Application to the Detection of Peroxynitrite in Endothelial Cells Treated with Doxorubicin.

Boronate probes have emerged recently as a versatile tool for the detection of reactive oxygen and nitrogen species. Here, we present the characterization of a fluorescein-based monoboronate probe, a 4-(pinacol boronate)benzyl derivative of fluorescein methyl ester (FBBE), that proved to be useful to detect peroxynitrite in cell culture experiments. The reactivity of FBBE toward peroxynitrite as well hypochlorite, hydrogen peroxide, and tyrosyl hydroperoxide was determined. Second-order rate constants of the reactions of FBBE with peroxynitrite, HOCl, and H2O2 at pH 7.4 were equal to (2.8 ± 0.2) × 10(5) M(-1) s(-1), (8.6 ± 0.5) × 10(3) M(-1) s(-1), and (0.96 ± 0.03) M(-1) s(-1), respectively. The presence of glutathione completely blocked the oxidation of the probe by HOCl and significantly inhibited its oxidation by H2O2 and tyrosyl hydroperoxide but not by peroxynitrite. The oxidative conversion of the probe was also studied in the systems generating singlet oxygen, superoxide radical anion, and nitric oxide in the presence and absence of glutathione. Spectroscopic characterization of FBBE and its oxidation product has been also performed. The differences in the reactivity pattern were supported by DFT quantum mechanical calculations. Finally, the FBBE probe was used to study the oxidative stress in endothelial cells (Ea.hy926) incubated with doxorubicin, a quinone anthracycline antibiotic. In endothelial cells pretreated with doxorubicin, FBBE was oxidized, and this effect was reversed by PEG-SOD and L-NAME but not by catalase.

Kinetic Isotope Effects in Multipath VTST: Application to a Hydrogen Abstraction Reaction.

In this work we apply multipath canonical variational transition state theory with small-tunneling corrections (MP-CVT/SCT) to the hydrogen abstraction reaction from ethanol by atomic hydrogen in aqueous solution at room temperature. This reaction presents two transition states which can interconvert by internal rotations about single bonds and another two transition states that are non-interconvertible enantiomers to the former structures. The study also includes another three reactions with isotopically substituted species for which there are experimental values of thermal rate constants and kinetic isotope effects (KIEs). The agreement between the MP-CVT/SCT thermal rate constants and the experimental data is good. The KIEs obtained by the MP-CVT/SCT methodology are factorized in terms of individual transition state contributions to facilitate the analysis. It was found that the percentage contribution of each transition state to the total KIE is independent of the isotopic substitution.

Dehydrochlorination of Hexachlorocyclohexanes Catalyzed by the LinA Dehydrohalogenase. A QM/MM Study.

The elucidation of the catalytic role of LinA dehydrohalogenase in the degradation processes of hexachlorocyclohexane (HCH) isomers is extremely important to further studies on the bioremediation of HCH polluted areas. Herein, QM/MM free energy simulations are employed to provide the details of the dehydrochlorination reaction of two HCH isomers (γ and ß). In particular, the role of the protonation state of one of the catalytic residues-His73-is explored. Based on our calculations, two distinct minimum free energy pathways (concerted and stepwise) were found for γ-HCH and ß-HCH. The choice of the reaction channel for the dehydrochlorination reactions of γ- and ß-HCH was shown to depend on the initial mutual orientations of the reacting species in the active site and the protonation form of His73. The sequential pathway comprises the transfer of the proton (Hδ1) between His73 and Asp25 and subsequently the H1/Cl2 pair elimination from the substrate molecule. Within a concerted mechanism, the dehydrochlorination reaction of γ-/ß-HCH is initiated with neutral His73 and the Hδ1 proton is transferred upon final product formation. We found that the concerted pathway for ß-HCH results in significantly higher free energy of activation than the stepwise route and therefore can be disregarded as not a feasible mechanism. On the other hand, the reaction that occurs with much lower energetic barrier requires a stronger base (i.e., anionic His73) to abstract the proton (H1) from the substrate molecule. The presence of such transient form of His results in higher energy than the respective Michaelis complex and was observed only in the stepwise pathway for both isomers. Furthermore, we have concluded that both pathways (concerted and stepwise) are feasible for the dehydrochlorination reaction of γ-HCH. The activation free energies obtained from the M05-2X/6-31+G(d,p) corrected path coordinate PMF profiles for the dehydrochlorination reactions of the γ-/ß-HCH are in good agreement with the experimental values.

Characteristic isotope fractionation patterns in s-triazine degradation have their origin in multiple protonation options in the s-triazine hydrolase TrzN.

s-Triazine herbicides (atrazine, ametryn) are groundwater contaminants which may undergo microbial hydrolysis. Previously, inverse nitrogen isotope effects in atrazine degradation by Arthrobacter aurescens TC1 (i) delivered highly characteristic (13C/12C, 15N/14N) fractionation trends for pathway identification and (ii) suggested that the s-triazine ring nitrogen was protonated in the enzyme s-triazine hydrolase (TrzN) where (iii) TrzN crystal structure and mutagenesis indicated H+-transfer from the residue E241. This study tested the general validity of these conclusions for atrazine and ametryn with purified TrzN and a TrzN-E241Q site-directed mutant. TrzN-E241Q lacked activity with ametryn; otherwise, degradation consistently showed normal carbon isotope effects (εcarbon=-5.0‰±0.2‰ (atrazine/TrzN), εcarbon=-4.2‰±0.5‰ (atrazine/TrzN-E241Q), εcarbon=-2.4‰±0.3‰ (ametryn/TrzN)) and inverse nitrogen isotope effects (εnitrogen=2.5‰±0.1‰ (atrazine/TrzN), εnitrogen=2.1‰±0.3‰ (atrazine/TrzN-E241Q), εnitrogen=3.6‰±0.4‰ (ametryn/TrzN)). Surprisingly, TrzN-E241Q therefore still activated substrates through protonation implicating another proton donor besides E241. Sulfur isotope effects were larger in enzymatic (εsulfur=-14.7‰±1.0‰, ametryn/TrzN) than in acidic ametryn hydrolysis (εsulfur=-0.2‰±0.0‰, pH 1.75), indicating rate-determining C-S bond cleavage in TrzN. Our results highlight a robust inverse 15N/14N fractionation pattern for identifying microbial s-triazine hydrolysis in the environment caused by multiple protonation options in TrzN.

A computational study of the dechlorination of ß-hexachlorocyclohexane (ß-HCH) catalyzed by the haloalkane dehalogenase LinB.

LinB, a haloalkane dehalogenase from Sphingomonas paucimobilis UT26, is known to metabolize halohydrocarbons to halide ions and the respective alcohols. Its broad substrate specificity allowed its consideration for bioremediation. Herein, we have shown its catalytic action toward ß-hexachlorocyclohexane (ß-HCH) - an example of large-size substrates that can be accommodated in its active site. We have analyzed the capability of combined QM/MM schemes to describe in detail the SN2 dechlorination reaction between ß-HCH and Asp108 in the active site of LinB. Free energy surfaces have been calculated using one and two dimensional potentials of mean force (PMF) obtained at the PM3/MM (MM=amberff99SB, TIP3P) level of theory. The overestimated energetic barriers by the PM3 Hamiltonian were corrected using a DFT functional (M06-2X). The resulted activation energies (16 and 19 kcal mol(-1) from 1D and 2D-PMF profiles, respectively) for the dechlorination reaction of ß-HCH in the active site of LinB enzyme are in qualitative agreement with the experimentally determined value of 17 kcal mol(-1). The binding of ß-HCH to the active site of LinB has been compared to the binding of smaller 1-chlorobutane (1-CB) and larger δ-hexabromocyclododecane (δ-HBCD).