Characteristics of nearly dry enzymes in organic solvents: implications for biocatalysis in the absence of water

We have examined enzymes in nearly anhydrous organic solvents spanning a wide range of dielectric constants using a combination of electron paramagnetic resonance (EPR) spectroscopy, molecular dynamics simulations, high-pressure kinetic studies and the electrostatic model of Kirkwood. This approach enabled us to investigate the relationship between catalytic activity, protein flexibility and solvent polarity for an enzymatic reaction proceeding through a highly polar transition state in the near absence of water. Further insights into water-protein interactions and the involvement of water in enzyme structure and function have been obtained by EPR and multinuclear nuclear magnetic resonance studies of enzymes suspended and immobilized in organic solvents with and without added water. In these systems, correlations were observed between the water content and enzyme activity, flexibility, and active-site polarity, although the structural properties of suspended and immobilized enzymes differed markedly. These results have helped to elucidate the role of water in molecular events at the enzymic active site leading to improved biocatalysis in low-water environments.     

Interaction of organic solvents with protein structures at protein-solvent interface

The effect of non-denaturing concentrations of three different organic solvents, formamide, acetone and isopropanol, on the structure of haloalkane dehalogenases DhaA, LinB, and DbjA at the protein-solvent interface was studied using molecular dynamics simulations. Analysis of B-factors revealed that the presence of a given organic solvent mainly affects the dynamical behavior of the specificity-determining cap domain, with the exception of DbjA in acetone. Orientation of organic solvent molecules on the protein surface during the simulations was clearly dependent on their interaction with hydrophobic or hydrophilic surface patches, and the simulations suggest that the behavior of studied organic solvents in the vicinity of hyrophobic patches on the surface is similar to the air/water interface. DbjA was the only dimeric enzyme among studied haloalkane dehalogenases and provided an opportunity to explore effects of organic solvents on the quaternary structure. Penetration and trapping of organic solvents in the network of interactions between both monomers depends on the physico-chemical properties of the organic solvents. Consequently, both monomers of this enzyme oscillate differently in different organic solvents. With the exception of LinB in acetone, the structures of studied enzymes were stabilized in water-miscible organic solvents.     

Effects of organic solvents on the enzyme activity of Trypanosoma cruzi glyceraldehyde-3-phosphate dehydrogenase in calorimetric assays

In drug discovery programs, dimethyl sulfoxide (DMSO) is a standard solvent widely used in biochemical assays. Despite the extensive use and study of enzymes in the presence of organic solvents, for some enzymes the effect of organic solvent is unknown. Macromolecular targets may be affected by the presence of different solvents in such a way that conformational changes perturb their active site structure accompanied by dramatic variations in activity when performing biochemical screenings. To address this issue, in this work we studied the effects of two organic solvents, DMSO and methanol (MeOH), in the isothermal titration calorimetry (ITC) kinetic assays for the catalyzed reaction of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Trypanosoma cruzi. The solvent effects on T. cruzi GAPDH had not yet been studied. This enzyme was shown here to be affected by the organic solvents content up to 5.0% for MeOH and up to 7.5% for DMSO. The results show that when GAPDH is assayed in the presence of DMSO (5%, v/v) using the ITC experiment, the enzyme exhibits approximately twofold higher activity than that of GAPDH with no cosolvent added. When MeOH (5%, v/v) is the cosolvent, the GAPDH activity is sixfold higher. The favorable effects of the organic solvents on the Michaelis-Menten enzyme-substrate complex formation ensure the consistency of the biological assays, structural integrity of the protein, and reproducibility over the measurement time. The reaction was also kinetically monitored by standard spectrophotometric assays to establish a behavioral performance of T. cruzi GAPDH when used for screening of potential inhibitors.     

Hydration of enzyme in nonaqueous media is consistent with solvent dependence of its activity

Water plays an important role in enzyme structure and function in aqueous media. That role becomes even more important when one focuses on enzymes in low water media. Here we present results from molecular dynamics simulations of surfactant-solubilized subtilisin BPN' in three organic solvents (octane, tetrahydrofuran, and acetonitrile) and in pure water. Trajectories from simulations are analyzed with a focus on enzyme structure, flexibility, and the details of enzyme hydration. The overall enzyme and backbone structures, as well as individual residue flexibility, do not show significant differences between water and the three organic solvents over a timescale of several nanoseconds currently accessible to large-scale molecular dynamics simulations. The key factor that distinguishes molecular-level details in different media is the partitioning of hydration water between the enzyme and the bulk solvent. The enzyme surface and the active site region are well hydrated in aqueous medium, whereas with increasing polarity of the organic solvent (octane --> tetrahydrofuran --> acetonitrile) the hydration water is stripped from the enzyme surface. Water stripping is accompanied by the penetration of tetrahydrofuran and acetonitrile molecules into crevices on the enzyme surface and especially into the active site. More polar organic solvents (tetrahydrofuran and acetonitrile) replace mobile and weakly bound water molecules in the active site and leave primarily the tightly bound water in that region. In contrast, the lack of water stripping in octane allows efficient hydration of the active site uniformly by mobile and weakly bound water and some structural water similar to that in aqueous solution. These differences in the active site hydration are consistent with the inverse dependence of enzymatic activity on organic solvent polarity and indicate that the behavior of hydration water on the enzyme surface and in the active site is an important determinant of biological function especially in low water media.     

Solvent-Dependent Gating Motions of an Extremophilic Lipase from Pseudomonas aeruginosa

Understanding how organic solvent-stable proteins can function in anhydrous and often complex solutions is essential for the study of the interaction of protein and molecular immiscible interfaces and the design of efficient industrial enzymes in nonaqueous solvents. Using an extremophilic lipase from Pseudomonas aeruginosa as an example, we investigated the conformational dynamics of an organic solvent-tolerant enzyme in complex solvent milieux. Four 100-ns molecular dynamics simulations of the lipase were performed in solvent systems: water, hexane, and two mixtures of hexane and water, 5% and 95% (w/w) hexane. Our results show a solvent-dependent structural change of the protein, especially in the region that regulates the admission of the substrate. We observed that the lipase is much less flexible in hexane than in aqueous solution or at the immiscible interface. Quantified by the size of the accessible channel, the lipase in water has a closed-gate conformation and no access to the active site, while in the hexane-containing systems, the lipase is at various degrees of open-gate state, with the immiscible interface setup being in the widely open conformation ensembles. The composition of explicit solvents in the access channel showed a significant influence on the conformational dynamics of the protein. Interestingly, the slowest step (bottleneck) of the hexane-induced conformational switch seems to be correlated with the slow dehydration dynamics of the channel.     

DMSO enhanced conformational switch of an interfacial enzyme

Interfacial proteins function in unique heterogeneous solvent environments, such as water–oil interfaces. One important example is microbial lipase, which is activated in an oil‐water emulsion phase and has many important enzymatic functions. A unique aprotic dipolar organic solvent, dimethyl sulfoxide (DMSO), has been shown to increase the activity of lipases, but the mechanism behind this enhancement is still unknown. Here, all‐atom molecular dynamics simulations of lipase in a binary solution were performed to examine the effects of DMSO on the dynamics of the gating mechanism. The amphiphilic α5 region of the lipase was a focal point for the analysis, since the structural ordering of α5 has been shown to be important for gating under other perturbations. Compared to the closed‐gorge ensemble in an aqueous environment, the conformational ensemble shifts towards open‐gorge structures in the presence of DMSO solvents. Increased width of the access channel is particularly prevalent in 45% and 60% DMSO concentrations (w/w). As the amount of DMSO increases, the α5 region of the lipase becomes more α‐helical, as we previously observed in studies that address water–oil interfacial and high pressure activation. We believe that the structural ordering of α5 plays an essential role on gating and lipase activity.     

Lipase in aqueous‐polar organic solvents: Activity,structure, and stability

Studying alterations in biophysical and biochemical behavior of enzymes in the presence of organic solvents and the underlying cause(s) has important implications in biotechnology. We investigated the effects of aqueous solutions of polar organic solvents on ester hydrolytic activity, structure and stability of a lipase. Relative activity of the lipase monotonically decreased with increasing concentration of acetone, acetonitrile, and DMF but increased at lower concentrations (upto ～20% v/v) of dimethylsulfoxide, isopropanol, and methanol. None of the organic solvents caused any appreciable structural change as evident from circular dichorism and NMR studies, thus do not support any significant role of enzyme denaturation in activity change. Change in 2D [15N, 1H]‐HSQC chemical shifts suggested that all the organic solvents preferentially localize to a hydrophobic patch in the active‐site vicinity and no chemical shift perturbation was observed for residues present in protein's core. This suggests that activity alteration might be directly linked to change in active site environment only. All organic solvents decreased the apparent binding of substrate to the enzyme (increased K_m); however significantly enhanced the k_cat. Melting temperature (T_m) of lipase, measured by circular dichroism and differential scanning calorimetry, altered in all solvents, albeit to a variable extent. Interestingly, although the effect of all organic solvents on various properties on lipase is qualitatively similar, our study suggest that magnitudes of effects do not appear to follow bulk solvent properties like polarity and the solvent effects are apparently dictated by specific and local interactions of solvent molecule(s) with the protein.     

Structural and dynamical studies of Humanin in water and TFE/water mixture: a molecular dynamics simulation

The structural and dynamical properties of Humanin, a small peptide with neuroprotective activity against the insults of the Alzheimer's disease-related genes and the neurotoxic amyloid peptide, are studied in two different environments by molecular dynamics simulation. In this study, we have performed comparative molecular dynamics simulations in the absence and in the presence of TFE. The resulting trajectories were analyzed in terms of structural and dynamical properties of peptide and compared to the available NMR data. In water humanin is observed to partly unfold. The peptide is readily stabilized in an ordered helical conformation in the TFE/water mixture. Our simulations show that the peptide is flexible with definite turn point in its structure in water environment. It is free to interact with receptors that mediate its action in polar environment. Humanin may also find an alpha helix structure necessary for passage through biomembranes and/or specific interactions.     

Toward understanding the inactivation mechanism of monooxygenase P450 BM-3 by organic cosolvents: a molecular dynamics simulation study

Cytochrome P450 BM-3 from Bacillus megaterium is an extensively studied enzyme for industrial applications. A major focus of current protein engineering research is directed to improving the catalytic performance of P450 BM-3 toward nonnatural substrates of industrial importance in the presence of organic solvents or cosolvents. For the latter reason, it is important to study the effect of organic cosolvent molecules on the structure and dynamics of the enzyme, in particular, the effect of cosolvent molecules on the active site's structure and dynamics. In this paper, we have studied, using molecular dynamics (MD) simulations, the F87A mutant of P450 BM-3 in the presence of DMSO as cosolvent, to understand the role of the F87A substitution for its catalytic activity. This mutant exhibits an altered regioselectivity and substrate specificity compared with wild-type; however, it has lower tolerance toward DMSO. The simulation results offer an explanation for the DMSO sensitivity of the F87A mutant. Our simulation results show that the F87 side chain prevents the disturbance of the water molecule bound to the heme iron by DMSO molecules. The absence of the phenyl ring in F87A mutant promotes interactions of the DMSO molecule with the heme iron resulting in water displacement by DMSO at the catalytic heme center.     

Polyethylene glycol-modified enzymes trap water on their surface and exert enzymic activity in organic solvents

Summary Polyethylene glycol-modified enzymes dissolved and had high enzymic activity in organic solvents. A trace amount of water was found to be necessary for the activity. It was reasoned that the amphipathic polymer covalently attached to enzymes kept water molecules around them. This was supported by findings that : (1) high enzymic activity was found in water- immiscible solvents, whereas activity was never observed in water-miscible solvents; (2) enzymic activity was inhibited by increasing the concentration of dimethyl sulfoxide in benzene; (3) activity of lipase was inhibited by a water-miscible alcohol substrate, but was steadily elevated by increasing the concentration of a water-immiscible alcohol substrate; (4) water was not absorbed from benzene solution containing a modified enzyme by molecular sieves, while it was easily absorbed in the presence of a water-miscible organic solvent, dimethyl sulfoxide.     

Why do water molecules around small hydrophobic solutes form stronger hydrogen bonds than in the bulk?

Molecular solutes are known to have a strong effect on the structural and dynamical properties of the surrounding water. In our recent study (PNAS, 114, 322 (2017)) we have identified the presence of strengthened water hydrogen bonds near hydrophobic solutes by using both IR spectroscopy and ab-initio molecular dynamics simulations. The water molecules involved in the enhanced hydrogen bonding have been shown to display extensive structural ordering and restricted mobility. We observed that an individual pair of water molecules can make stronger hydrogen bond to each other if it is not surrounded by intercalating water molecules. Here we present compelling simulation results which unravel a simple mechanistic picture of the emergence of the hydrogen bond (HB) strengthening around solvated methane. We show explicitly that actual absence of water molecules within the excluded volume due to the hydrophobic molecule assures smaller residual torque on neighboring water molecules enabling the formation of stronger HBs between them.     

Water in the active site of ketosteroid isomerase

Classical molecular dynamics simulations were utilized to investigate the structural and dynamical properties of water in the active site of ketosteroid isomerase (KSI) to provide insight into the role of these water molecules in the enzyme-catalyzed reaction. This reaction is thought to proceed via a dienolate intermediate that is stabilized by hydrogen bonding with residues Tyr16 and Asp103. A comparative study was performed for the wild-type (WT) KSI and the Y16F, Y16S, and Y16F/Y32F/Y57F (FFF) mutants. These systems were studied with three different bound ligands: equilenin, which is an intermediate analog, and the intermediate states of two steroid substrates. Several distinct water occupation sites were identified in the active site of KSI for the WT and mutant systems. Three additional sites were identified in the Y16S mutant that were not occupied in WT KSI or the other mutants studied. The number of water molecules directly hydrogen bonded to the ligand oxygen was approximately two in the Y16S mutant and one in the Y16F and FFF mutants, with intermittent hydrogen bonding of one water molecule in WT KSI. The molecular dynamics trajectories of the Y16F and FFF mutants reproduced the small conformational changes of residue 16 observed in the crystal structures of these two mutants. Quantum mechanical/molecular mechanical calculations of (1)H NMR chemical shifts of the protons in the active site hydrogen-bonding network suggest that the presence of water in the active site does not prevent the formation of short hydrogen bonds with far-downfield chemical shifts. The molecular dynamics simulations indicate that the active site water molecules exchange much more frequently for WT KSI and the FFF mutant than for the Y16F and Y16S mutants. This difference is most likely due to the hydrogen-bonding interaction between Tyr57 and an active site water molecule that is persistent in the Y16F and Y16S mutants but absent in the FFF mutant and significantly less probable in WT KSI.     

Molecular dynamics simulations of lignin peroxidase in solution

The dynamical and structural properties of lignin peroxidase and its Trp171Ala mutant have been investigated in aqueous solution using molecular dynamics (MD) simulations. In both cases, the enzyme retained its overall backbone structure and all its noncovalent interactions in the course of the MD simulations. Very interestingly, the analysis of the MD trajectories showed the presence of large fluctuations in correspondence of the residues forming the heme access channel; these movements enlarge the opening and facilitate the access of substrates to the enzyme active site. Moreover, steered molecular dynamics docking simulations have shown that lignin peroxidase natural substrate (veratryl alcohol) can easily approach the heme edge through the access channel.     

Molecular dynamics simulations of microstructure and mixing dynamics of cryoprotective solvents in water and in the presence of a lipid membrane

Molecular dynamics (MD) simulation is used to investigate the solubility behavior of cryoprotective (CP) solvents, such as DMSO, ethylene glycol (EG) and glycerol (GL), in pure water and in the presence of a lipid membrane. The MD study is focused on an equilibration timescale required for mixing large CP aggregates with aqueous and aqueous/lipid environments. The MD analysis demonstrates that DMSO mixes rapidly with water, so that all solute molecules are uniformly distributed in the equilibrium aqueous solution. Our investigation of the microstructure of binary EG/water and GL/water systems reveals that, despite the miscibility of both CP solvents with water, they are not ideally mixed in aqueous solutions at the molecular level. The MD simulations show that the mixing dynamics of the large CP cluster and surrounding water is found to be strongly dependent on nature of hydrophilic and hydrophobic interactions acting between cryoprotectant molecules. In particular, a spatial hydrogen-bond network formed between CP molecules plays an important role in the mixing dynamics between CP agents and water. A further analysis on the mixing behavior of the CP solvents with pure water and with aqueous solutions at a lipid membrane interface shows that, due to strong binding of the CP molecules to membrane surface, the equilibration process in the lipid environment becomes very slow, at least of the order of microseconds. The MD results are discussed in the context of the better understanding on the composition of the aqueous mixtures of the EG and GL solvents. Knowledge of the microstructure and the dynamics of these systems helps to develop better cryopreservation protocols and to propose more optimal cooling/warming regimes for cellular cryosolutions.     

Structural determinants of ligand imprinting: a molecular dynamics simulation study of subtilisin in aqueous and apolar solvents

The phenomenon known as "ligand imprinting" or "ligand-induced enzyme memory" was first reported in 1988, when Russell and Klibanov observed that lyophilizing subtilisin in the presence of competitive inhibitors (that were subsequently removed) could significantly enhance its activity in an apolar solvent. (Russell and Klibanov, J Biol Chem 1988;263:11624-11626). They further observed that this enhancement did not occur when similar assays were carried out in water. Herein, we shed light on the molecular determinants of ligand imprinting using a molecular dynamics (MD) approach. To simulate the effect of placing an enzyme in the presence of a ligand before its lyophilization, an inhibitor was docked in the active site of subtilisin and 20 ns MD simulations in water were performed. The ligand was then removed and the resulting structure was used for subsequent MD runs using hexane and water as solvents. As a control, the same simulation setup was applied using the structure of subtilisin in the absence of the inhibitor. We observed that the ligand maintains the active site in an open conformation and that this configuration is retained after the removal of the inhibitor, when the simulations are carried out in hexane. In agreement with experimental findings, the structural configuration induced by the ligand is lost when the simulations take place in water. Our analysis of fluctuations indicates that this behavior is a result of the decreased flexibility displayed by enzymes in an apolar solvent, relatively to the aqueous situation.     

Molecular dynamics simulations of a DMSO/water mixture using the AMBER force field

Due to its protective properties of biological samples at low temperatures and under desiccation, dimethyl sulfoxide (DMSO) in aqueous solutions has been studied widely by many experimental approaches and molecular dynamics (MD) simulations. In the case of the latter, AMBER is among the most commonly used force fields for simulations of biomolecular systems; however, the parameters for DMSO published by Fox and Kollman in 1998 have only been tested for pure liquid DMSO. We have conducted an MD simulation study of DMSO in a water mixture and computed several structural and dynamical properties such as of the mean density, self-diffusion coefficient, hydrogen bonding and DMSO and water ordering. The AMBER force field of DMSO is seen to reproduce well most of the experimental properties of DMSO in water, with the mixture displaying strong and specific water ordering, as observed in experiments and multiple other MD simulations with other non-polarizable force fields.

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Graphical abstract Hydration structure within hydrogen-bonding distance around a DMSOmolecule

     

Active-site hydration and water diffusion in cytochrome P450cam: a highly dynamic process

Long-timescale molecular dynamics simulations (300 ns) are performed on both the apo- (i.e., camphor-free) and camphor-bound cytochrome P450cam (CYP101). Water diffusion into and out of the protein active site is observed without biased sampling methods. During the course of the molecular dynamics simulation, an average of 6.4 water molecules is observed in the camphor-binding site of the apo form, compared to zero water molecules in the binding site of the substrate-bound form, in agreement with the number of water molecules observed in crystal structures of the same species. However, as many as 12 water molecules can be present at a given time in the camphor-binding region of the active site in the case of apo-P450cam, revealing a highly dynamic process for hydration of the protein active site, with water molecules exchanging rapidly with the bulk solvent. Water molecules are also found to exchange locations frequently inside the active site, preferentially clustering in regions surrounding the water molecules observed in the crystal structure. Potential-of-mean-force calculations identify thermodynamically favored trans-protein pathways for the diffusion of water molecules between the protein active site and the bulk solvent. Binding of camphor in the active site modifies the free-energy landscape of P450cam channels toward favoring the diffusion of water molecules out of the protein active site.     

Function and biotechnology of extremophilic enzymes in low water activity

ABSTRACT: Enzymes from extremophilic microorganisms usually catalyze chemical reactions in non-standard conditions. Such conditions promote aggregation, precipitation, and denaturation, reducing the activity of most non-extremophilic enzymes, frequently due to the absence of sufficient hydration. Some extremophilic enzymes maintain a tight hydration shell and remain active in solution even when liquid water is limiting, e.g. in the presence of high ionic concentrations, or at cold temperature when water is close to the freezing point. Extremophilic enzymes are able to compete for hydration via alterations especially to their surface through greater surface charges and increased molecular motion. These properties have enabled some extremophilic enzymes to function in the presence of non-aqueous organic solvents, with potential for design of useful catalysts. In this review, we summarize the current state of knowledge of extremophilic enzymes functioning in high salinity and cold temperatures, focusing on their strategy for function at low water activity. We discuss how the understanding of extremophilic enzyme function is leading to the design of a new generation of enzyme catalysts and their applications to biotechnology.     

Enhancement of the organic solvent‐stability of the LST‐03 lipase by directed evolution

LST‐03 lipase from an organic solvent‐tolerant Pseudomonas aeruginosa LST‐03 has high stability and activity in the presence of various organic solvents. In this research, enhancement of organic solvent‐stability of LST‐03 lipase was attempted by directed evolution. The structural gene of the LST‐03 lipase was amplified by the error prone‐PCR method. Organic solvent‐stability of the mutated lipases was assayed by formation of a clear zone of agar which contained dimethyl sulfoxide (DMSO) and tri‐n‐butyrin and which overlaid a plate medium. And the organic solvent‐stability was also confirmed by measuring the half‐life of activity in the presence of DMSO. Four mutated enzymes were selected on the basis of their high organic solvent‐stability in the presence of DMSO. The organic solvent‐stabilities of mutated LST‐03 lipase in the presence of various organic solvents were measured and their mutated amino acid residues were identified. The half‐lives of the LST‐03‐R65 lipase in the presence of cyclohexane and n‐decane were about 9 to 11‐fold longer than those of the wild‐type lipase, respectively. Some substituted amino acid residues of mutated LST‐03 lipases have been located at the surface of the enzyme molecules, while some other amino acid residues have been changed from neutral to basic residues. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Purification and characterization of a maltooligosaccharide-forming amylase that improves product selectivity in water-miscible organic solvents, from dimethylsulfoxide-tolerant Brachybacterium sp. strain LB25

A bacterium that secretes maltooligosaccharide-forming amylase in a medium containing 12.5% (vol/vol) dimethylsulfoxide (DMSO) was isolated and identified as Brachybacterium sp. strain LB25. The amylase of the strain was purified from the culture supernatant, and its molecular mass was 60 kDa. The enzyme was stable at pH 7.0–8.5 and active at pH 6.0–7.5. The optimum temperature at pH 7.0 was 35°C in the presence of 5 mM CaCl₂. The enzyme hydrolyzed starch to produce maltotriose primarily. The enzyme was active in the presence of various organic solvents. Its yield and product selectivity of maltooligosaccharides in the presence of DMSO or ethanol were compared with those of the industrial maltotriose-forming amylase from Microbacterium imperiale. Both enzymes improved the production selectivity of maltotriose by the addition of DMSO or ethanol. However, the total maltooligosaccharide yield in the presence of the solvents was higher for LB25 amylase than for M. imperiale amylase.