Lipase A from Bacillus subtilis: Substrate Binding, Conformational Dynamics, and Signatures of a Lid.

Protein-ligand binding studies are crucial for understanding the molecular basis of biological processes and for further advancing industrial biocatalysis and drug discovery. Using computational modeling and molecular dynamics simulations, we investigated the binding of a butyrate ester substrate to the lipase A (LipA) enzyme of Bacillus subtilis. Besides obtaining a close agreement of the binding free energy with the experimental value, the study reveals a remarkable reorganization of the catalytic triad upon substrate binding, leading to increased essential hydrogen bond populations. The investigation shows the distortion of the oxyanion hole in both the substrate-bound and unbound states of LipA and highlights the strengthening of the same in the tetrahedral intermediate complex. Principal component analysis of the unbound ensemble reveals the dominant motion in LipA to be the movement of Loop-1 (Tyr129-Arg142) between two states that cover and uncover the active site, mirroring that of a lid prevalent in several lipases. This lid-like motion of Loop-1 is also supported by its tendency to spontaneously open up at an oil-water interface. Overall, this study provides valuable insights into the impact of substrate binding on the structure, flexibility, and conformational dynamics of the LipA enzyme.

Revisiting the Burden Borne by Fumarase: Enzymatic Hydration of an Olefin.

Fumarate hydratase (FH) is a remarkable catalyst that decreases the free energy of the catalyzed reaction by 30 kcal mol-1, much larger than most exceptional enzymes with extraordinary catalytic rates. Two classes of FH are observed in nature: class-I and class-II, which have different folds, yet catalyze the same reversible hydration/dehydration reaction of the dicarboxylic acids fumarate/malate, with equal efficiencies. Using class-I FH from the hyperthermophilic archaeon Methanocaldococcus jannaschii (Mj) as a model along with comparative analysis with the only other available class-I FH structure from Leishmania major (Lm), we provide insights into the molecular mechanism of catalysis in this class of enzymes. The structure of MjFH apo-protein has been determined, revealing that large intersubunit rearrangements occur across apo- and holo-protein forms, with a largely preorganized active site for substrate binding. Site-directed mutagenesis of active site residues, kinetic analysis, and computational studies, including density functional theory (DFT) and natural population analysis, together show that residues interacting with the carboxylate group of the substrate play a pivotal role in catalysis. Our study establishes that an electrostatic network at the active site of class-I FH polarizes the substrate fumarate through interactions with its carboxylate groups, thereby permitting an easier addition of a water molecule across the olefinic bond. We propose a mechanism of catalysis in FH that occurs through transition-state stabilization involving the distortion of the electronic structure of the substrate olefinic bond mediated by the charge polarization of the bound substrate at the enzyme active site.

Molecular simulations explain the exceptional thermal stability, solvent tolerance and solubility of protein-polymer surfactant bioconjugates in ionic liquids.

Proteins complexed electrostatically with polymer surfactants constitute a viscous liquid by themselves, called the solvent-free protein liquid (SFPL). A solution of SFPL in a room temperature ionic liquid (PS-IL) offers the protein hyperthermal stability, higher solubility and greater IL tolerance. A generic understanding of these protein-polymer systems is obtained herein through extensive atomistic molecular dynamics simulations of three different enzymes (lipase A, lysozyme and myoglobin) under various conditions. Along with increased intra-protein hydrogen bonding, the surfactant coating around the proteins imparts greater thermal stability, and also aids in screening protein-IL interactions, endowing them IL tolerance. The reduced surface polarity of the protein-polymer bioconjugate and hydrogen bonding between the ethylene glycol groups of the surfactant and the IL cation contribute to the facile solvation of the protein in its PS-IL form. The results presented here rationalize several experimental observations and will aid in the improved design of such hybrid materials for sustainable catalysis.

Insights into substrate behavior in a solvent-free protein liquid to rationalize its reduced catalytic rate.

When proteins are engineered with a polymer surfactant coating on their surface, they can form a liquid phase by themselves, without the need for a solvent, such as, say, water. However, such solvent-free protein liquids (SFPL), despite their capability to function at temperatures above those in aqueous solutions, exhibit much reduced catalytic rates. A comprehensive understanding of the nature of substrates in such liquids is crucial to reason out the reduced catalytic activity of enzymes as SFPL media, and thus identify the means to improve the same. Employing atomistic molecular dynamics simulations of lipase A from Bacillus subtilis in its SFPL form, we demonstrate that at low concentrations, the substrate molecules are located mostly in the hydrophilic layer of the surfactant shell that ensheaths the enzyme; substrates in this SFPL are present in various conformations with similar propensities as in the aqueous solution. Slower translational diffusion and reorientational dynamics, as well as the reduced tendency of a substrate molecule to closely interact with the enzymes in the SFPL medium have been identified herein as the contributing factors for the reduced activity of enzymes in this hybrid liquid. At high concentrations of substrates corresponding to those used in in vitro experiments, the formation of an enzyme-substrate complex is observed. Microscopic insights reported here can aid in the choice of surfactants to improve the catalytic rate of enzymes in SFPL.

An atomistic view of solvent-free protein liquids: the case of Lipase A.

Solvent-free enzymes hold the promise of being able to deliver higher activity at elevated temperatures by virtue of them being not limited by the boiling point of the solvent. They have been realized in the liquid phase through a polymer surfactant coating on the protein surface. However, a clear understanding of intermolecular interactions, structure, dynamics, and the behaviour of the minuscule amount of water present in the solvent-free protein liquid is essential to enhance the activity of these biofluids. Using atomistic molecular dynamics simulations, we demonstrate that the scaled spatial correlations between proteins in the hybrid liquid phase of Lipase A enzymes are comparable to the inter-particle correlations in a noble gas fluid. The hydrophilic region of the surfactants forms a coronal layer around each enzyme which percolates throughout the liquid, while the hydrophobic parts are present as disjointed clusters. Inter-surfactant interactions, determined to be attractive and in the range of -200 to -300 kcal mol-1, stabilize the liquid state. While the protein retains its native state conformational dynamics in the solvent-free form, the fluxionality of its side chains is much reduced; at 333 K, the latter is found to be equivalent to that of the enzyme in an aqueous solution at 249 K. Despite the sluggishness of the solvent-free enzyme, some water molecules exhibit high mobility and transit between enzymes primarily via the interspersed hydrophilic regions. These microscopic insights offer ideas to improve substrate diffusion in the liquid to enable the enhancement of catalytic activity.

Clinicoepidemiological profile of trauma patients admitting to the emergency department of a tertiary care hospital in eastern India.

CONTEXT: Trauma is an immediate cause of patients flowing to the emergency department of any hospital. Besides epidemiology, clinical profile and treatment strategy forms an important aspect to reflect the gap in the existing public sector health-care system and the requirement. AIMS: To evaluate the clinicoepidemiological profile of trauma patients admitting to an apex trauma hospital in east India. SETTINGS AND DESIGN: A prospective observational study was performed during the time period of December 2018 to July 2019 on trauma patients admitted to the Trauma and Emergency department. METHODS AND MATERIAL: Patient's demographic profile, injury type, mechanism, the vehicle involved in the accident, and transportation were recorded. Various trauma scores (clinical) and outcome measures were recorded. STATISTICAL ANALYSIS USED: Statistical analysis was done by R version 3.6.1. RESULTS: Male: female ratio was 407:93 with the 21-30 age group predominantly. 2-6 PM was the most common time of injury and ambulance was the predominant mode of transport (58%). Road traffic injury (RTI) accounted for 75% victims; two-wheelers (68%) dominated over others. Thirty percent (drivers 18%, pillion riders 12%) were wearing helmet; 41% were wearing seat belts (drivers 34%, passenger 12%). Twenty-five percent of drivers consumed alcohol. The median ± Interquartile range of injury severity score (ISS), revised trauma and trauma score and injury severity score were 17 (11-26), 7.8 (4.1-7.8), and 98.41 (95.95-99.30), respectively. Extremity injury (54% fractures) and head injury (50%) were the frontrunners in the pattern of injury, with half of the victims were polytrauma (ISS > 15). CONCLUSIONS: The injury was prominently RTI and the trauma victims/patients were young male drivers on two wheelers. The focus should be directed to make use of safety measures among the youth.

Orientational Switch of the Lipase A Enzyme at the Oil-Water Interface: An Order of Magnitude Increase in Turnover Rate with a Single Surfactant Tag Explained.

Interfacially active lipases can be immobilized at a biphasic interface to enhance turnover recyclability and to facilitate product separation. Extensive coarse-grained molecular dynamics simulations of lipase A (LipA) from Bacillus subtilis show a bimodal orientational distribution of the enzyme at an oil-water interface, arising from its ellipsoidal Janus particle-like character. The relative orientational preference can be tuned by pH. The simulations rationalize a rare experimental observation of an order of magnitude increase in the turnover rate of this lipase upon its noncovalent tagging by a single surfactant molecule at the interface, compared to its rate in bulk water. The adsorption free energy, the interfacial activation, a decrease in the number of orientational fluctuations, and an increased rate of translational diffusion, to all of which the Janus character of LipA contributes, are the factors responsible for this enhancement. This study can spur further investigations of the Janus behavior of enzymes to enhance their activity as well as to stabilize the biphasic emulsion needed for interfacial catalysis.