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.

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.

2-OMe-lysophosphatidylcholine analogues are GPR119 ligands and activate insulin secretion from ßTC-3 pancreatic cells: Evaluation of structure-dependent biological activity.

GPR119 receptor has been proposed as a metabolic regulator playing a pivotal role in the modulation of glucose homeostasis in type 2 diabetes. GPR119 was identified on pancreatic ß cells and its ligands have the ability to enhance glucose-stimulated insulin secretion (GSIS). Lysophosphatidylcholine (LPC) was shown to potentiate GSIS and our present studies indicate that 2-methoxy-lysophosphatidylcholine (2-OMe-LPC) analogues, unable to undergo 1→2 acyl migration, stimulate GSIS from murine ßTC-3 pancreatic cells even more efficiently. Moreover, biological assays in engineered Tango™ GPR119-bla U2OS cells were carried out to ascertain the agonist activity of 2-OMe-LPC at GPR119. 2-OMe-LPC possessing in sn-1 position the residues of myristic, palmitic, stearic and oleic acid were also evaluated as factors regulating [Ca2+]i mobilization and cAMP levels. Extension of these studies to R- and S-enantiomers of 14:0 2-OMe-LPC revealed that the overall impact on GSIS does not depend on chirality, however, the intracellular calcium mobilization data show that the R enantiomer is significantly more active than S one. Taking into account differences in chemical structure between various native LPCs and their 2-methoxy counterparts the possible binding mode of 2-OMe-LPC to the GPR119 receptor was determined using molecular modeling approach.

Lysophosphatidylcholine elicits intracellular calcium signaling in a GPR55-dependent manner.

The GPR55 signaling is fertile ground for drug discovery, however despite considerable research progress during the past 10 years, many open questions remain. The GPR55 pharmacology remains controversial, as many ligands have been reported with inconsistent results. Here, we show that various molecular species of lysophosphatidylcholine (LPC) elicit intracellular Ca2+ mobilization in GPR55-expressing PC-3 human prostate carcinoma cells. The response was even stronger than [Ca2+]i flux evoked by endogenous (OEA) and synthetic (Abn-CBD) agonists. Treatment with GPR55 antagonists CID16020046 and ML193 as well as the lipid raft disrupter methyl-ß-cyclodextrin strongly blunted LPC-induced calcium signal. Additionally, molecular modeling analysis revealed that LPC 16:0 and LPC 18:1 interact stronger with the receptor than to OEA. Identified electrostatic interactions between GPR55 residues and the ligands overlap with the binding site identified previously for lysophosphatidylinositol. Therefore, we prove that LPC is another GPR55-sensitive ligand. This finding is relevant in understanding lysophospolipids-mediated signaling and opens new avenues to develop therapeutic approach based on GPR55 targeting.

The chemical synthesis and preliminary biological studies of phosphodiester and phosphorothioate analogues of 2-methoxy-lysophosphatidylethanolamine.

The chemical synthesis of phosphorothioate/phosphodiester analogues of 2-methoxy-lysophosphatidylethanolamine has been described. For the preparation of phosphorothioate derivatives oxathiaphospholane approach has been employed. The phosphodiester compounds were prepared by OXONE® oxidation of corresponding phosphorothioates. Each lysophospholipid analogue was synthesized as a series of four compounds, bearing different fatty acid residues both saturated (14:0, 16:0, 18:0) and unsaturated (18:1). The methylation of glycerol 2-hydroxyl function was applied in order to increase the stability of prepared analogues by preventing 1→2 acyl migration. The cytotoxicity of newly synthesized 2-methoxy-lysophosphatidylethanolamine derivatives was evaluated with resazurin-based method in prostate cancer PC3 cell line. The highest reduction of cell viability was noted for LPE analogues containing myristoyl acyl chain.

Lysophosphatidylcholine and lysophosphatidylinosiol--novel promissing signaling molecules and their possible therapeutic activity.

For many years the role of lysophospholipids (LPLs) was associated only with structural and storage components of the cell without any informational function. Today, based on many research projects performed during the last decades, it is clear that some of the LPLs act as hormone-like signaling molecules and thus are very important inter- and intracellular lipid mediators. They can activate specific membrane receptors and/or nuclear receptors regulating many crucial physiological and pathophysiological processes. The LPLs were iden- tified as involved in a majority of cellular processes, including modulation of disease-related mechanisms observed, for instance, in case of diabetes, obesity, atherosclerosis and cancer. Among LPLs, lysophosphatidylcholine (LPC) and lysophosphatidylinositol (LPI) are becoming attractive research topics. Their recently revealed activities as novel ligands of orphan G protein-coupled receptors (i.e., GPR55 and GPR119) involved in modulation of tumor physiology and insulin secretion seem to be one of the most interesting aspects of these compounds. Moreover, the most recent scientific reports emphasize the significance of the acyl chain structure bound to the glycerol basis of LPL, as it entails different biological properties and activities of the compounds.