Insights into lid movements of Burkholderia cepacia lipase inferred from molecular dynamics simulations

The interfacial activation of many lipases at water/lipid interface is mediated by large conformational changes of a so‐called lid subdomain that covers up the enzyme active site. Here we investigated using molecular dynamic simulations in different explicit solvent environments (water, octane and water/octane interface) the molecular mechanism by which the lid motion of Burkholderia cepacia lipase might operate. Although B. cepacia lipase has so far only been crystallized in open conformation, this study reveals for the first time the major conformational rearrangements that the enzyme undergoes under the influence of the solvent, which either exposes or shields the active site from the substrate. In aqueous media, the lid switches from an open to a closed conformation while the reverse motion occurs in organic environment. In particular, the role of a subdomain facing the lid on B. cepacia lipase conformational rearrangements was investigated using position‐restrained MD simulations. Our conclusions indicate that the sole mobility of α9 helix side‐chains of B. cepacia lipase is required for the full completion of the lid conformational change which is essentially driven by α5 helix movement. The role of selected α5 hydrophobic residues on the lid movement was further examined. In silico mutations of two residues, V138 and F142, were shown to drastically modify the conformational behavior of B. cepacia lipase. Overall, our results provide valuable insight into the role played by the surrounding environment on the lid conformational rearrangement and the activation of B. cepacia lipase. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Solvent-induced lid opening in lipases: A molecular dynamics study

In most lipases, a mobile lid covers the substrate binding site. In this closed structure, the lipase is assumed to be inactive. Upon activation of the lipase by contact with a hydrophobic solvent or at a hydrophobic interface, the lid opens. In its open structure, the substrate binding site is accessible and the lipase is active. The molecular mechanism of this interfacial activation was studied for three lipases (from Candida rugosa, Rhizomucor miehei, and Thermomyces lanuginosa) by multiple molecular dynamics simulations for 25 ns without applying restraints or external forces. As initial structures of the simulations, the closed and open structures of the lipases were used. Both the closed and the open structure were simulated in water and in an organic solvent, toluene. In simulations of the closed lipases in water, no conformational transition was observed. However, in three independent simulations of the closed lipases in toluene the lid gradually opened. Thus, pathways of the conformational transitions were investigated and possible kinetic bottlenecks were suggested. The open structures in toluene were stable, but in water the lid of all three lipases moved towards the closed structure and partially unfolded. Thus, in all three lipases opening and closing was driven by the solvent and independent of a bound substrate molecule.     

Exploring the conformational states and rearrangements of Yarrowia lipolytica Lipase

We report the 1.7 Å resolution crystal structure of the Lip2 lipase from Yarrowia lipolytica in its closed conformation. The Lip2 structure is highly homologous to known structures of the fungal lipase family (Thermomyces lanuginosa, Rhizopus niveus, and Rhizomucor miehei lipases). However, it also presents some unique features that are described and discussed here in detail. Structural differences, in particular in the conformation adopted by the so-called lid subdomain, suggest that the opening mechanism of Lip2 may differ from that of other fungal lipases. Because the catalytic activity of lipases is strongly dependent on structural rearrangement of this mobile subdomain, we focused on elucidating the molecular mechanism of lid motion. Using the x-ray structure of Lip2, we carried out extensive molecular-dynamics simulations in explicit solvent environments (water and water/octane interface) to characterize the major structural rearrangements that the lid undergoes under the influence of solvent or upon substrate binding. Overall, our results suggest a two-step opening mechanism that gives rise first to a semi-open conformation upon adsorption of the protein at the water/organic solvent interface, followed by a further opening of the lid upon substrate binding.     

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.     

Computational studies of essential dynamics of Pseudomonas cepacia lipase

In order to investigate the interfacial activation of a lipase from Pseudomonas cepacia (PcL), molecular dynamics (MD) simulations and essential dynamics (ED) analysis were performed in different solvent environments: vacuum and explicit water solvents. Starting from the active (open) structure of PcL, the essential dynamics analysis of the simulations revealed large correlated motions, which may be responsible for the activation of the enzyme. Fluctuations in the U1 (active-site lid) and U2 domains are found to be important in the activation of PcL. In contrast, the catalytic triad exhibits very little displacement. These results are consistent with the previous X-ray structural determination. A combined analysis of the trajectories showed some differences for the simulations in different solvent environments. It was found that the region around the helix alpha5 showed larger displacements in the water simulations. It can be concluded that the open structure of PcL becomes unstable in water solvents, leading to the closing of the so-called 'lid' region. The simulations and ED analysis on the closed structure of PgL provided additional information concerning the structural changes involved in the activation of the lipases. It was found that structural changes for PcL and PgL, which are responsible for the essential motions of the protein, showed contrasting behavior in the different solvent environments.     

脂肪酶活性部位上的盖与界面活性

脂肪酶是一种非常重要的水解酶,在工业催化、医药和科学研究等领域中有广泛应用. 大部分脂肪酶的活性部位上方有一段被称为“盖”的α-螺旋,这种盖赋予脂肪酶在水/油界面上有特殊的催化活性,界面活性.而在单一水相或油相中却表现出低活性或无活性.界面活性与盖的组成、大小、构象及其存在环境等密切相关,探明盖与脂肪酶界面活性的关系对于脂肪酶的开发和利用是非常关键的.因此,长期以来人们对盖在脂肪酶催化作用中所扮演的角色进行了孜孜不倦的探索.本文从盖的构象、移动、组成和删除等方面综述了其对脂肪酶催化作用的影响,期望对人们认识脂肪酶盖与其催化作用之间的关系有一定的帮助.     

Aspergillus niger lipase: Heterologous expression in Pichia pastoris,molecular modeling prediction and the importance of the hinge domains at both sides of the lid domain to interfacial activation

Aspergillus niger lipase (ANL) is an important biocatalyst in the food processing industry. However, there is no report of its detailed three‐dimensional structure because of difficulties in crystallization. In this article, based on experimental data and bioinformational analysis results, the structural features of ANL were simulated. Firstly, two recombinant ANLs expressed in Pichia pastoris were purified to homogeneity and their corresponding secondary structure compositions were determined by circular dichroism spectra. Secondly, the primary structure, the secondary structure and the three‐dimensional structure of ANL were modeled by comparison with homologous lipases with known three‐dimensional structures using the BioEdit software, lipase engineering database ( http://www.led.uni‐stuttgart.de/ ), PSIPRED server and SwissModel server. The predicted molecular structure of ANL presented typical features of the α/β hydrolase fold including positioning of the putative catalytic triad residues and the GXSXG signature motif. Comparison of the predicted three‐dimensional structure of ANL with the X‐ray three‐dimensional structure of A. niger feruloyl esterase showed that the functional difference of interfacial activation between lipase and esterase was concerned with the difference in position of the lid. Our three‐dimensional model of ANL helps to modify lipase structure by protein engineering, which will further expand the scope of application of ANL. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Lipase-catalysed hydrolysis of short-chain substrates in solution and in emulsion: a kinetic study

We have studied the enzymatic hydrolysis of solutions and emulsions of vinyl propionate, vinyl butyrate and tripropionin by lipases of various origin and specificity. Kinetic studies of the hydrolysis of short-chain substrates by microbial triacylglycerol lipases from Rhizopus oryzae, Mucor miehei, Candida rugosa, Candida antarctica A and by (phospho)lipase from guinea-pig pancreas show that these lipolytic enzymes follow the Michaelis–Menten model. Surprisingly, the activity against solutions of tripropionin and vinyl esters ranges from 70% to 90% of that determined against emulsions. In contrast, a non-hyperbolic (sigmoidal) dependence of enzyme activity on ester concentration is found with human pancreatic lipase, triacylglycerol lipase from Humicola lanuginosa (Thermomyces lanuginosa) and partial acylglycerol lipase from Penicillium camembertii and the same substrates. In all cases, no abrupt jump in activity (interfacial activation) is observed at substrate concentration corresponding to the solubility limit of the esters. Maximal lipolytic activity is always obtained in the presence of emulsified ester. Despite progress in the understanding of structure–function of lipases, interpretation of the mode of action of lipases active against solutions of short-chain substrates remains difficult. Actually, it is not known whether these enzymes, which possess a lid structure, are in open or/and closed conformation in the bulk phase and whether the opening of the lid that gives access to the catalytic triad is triggered by interaction of the enzyme molecule with monomeric substrates or/and multimolecular aggregates (micelles) both present in the bulk phase. From the comparison of the behaviour of lipases used in this study which, in some cases, follow the Michaelis–Menten model and, in others, deviate from classical kinetics, it appears that the activity of classical lipases against soluble short-chain vinyl esters and tripropionin depends not only on specific interaction with single substrate molecules at the catalytic site of the enzyme but also on physico-chemical parameters related to the state of association of the substrate dispersed in the aqueous phase. It is assumed that the interaction of lipase with soluble multimolecular aggregates of tripropionin or short-chain vinyl esters or the formation of enzyme–substrate mixed micelles with ester bound to lipase, might represent a crucial step that triggers the structural transition to the open enzyme conformation by displacement of the lid.     

The open lid conformation of the lipase is explored in the compressed gas: New insights from molecular dynamic simulation

The advantages of enzymatic reactions in compressed gases such as supercritical CO₂ are limited due to the enzyme inactivation. But recent experimental observations reported the high activity of enzymes such as lipases in compressed propane. But there are no clear reasons at the molecular levels for such behavior. In this work using molecular dynamic simulation, we showed for the first time the possibility of interfacial activation of lipases in a compressed gas. The analysis showed that in compressed propane the lid of the lipase was opened and so the active conformation of the enzyme was resulted. Moreover it is found that in the compressed propane, similar to the aqueous solution, the enzyme has native conformation.     

Open and closed states of Candida antarctica lipase B: protonation and the mechanism of interfacial activation

Lipases (EC 3.1.1.3) are ubiquitous hydrolases for the carboxyl ester bond of water-insoluble substrates, such as triacylglycerols, phospholipids, and other insoluble substrates, acting in aqueous as well as in low-water media, thus being of considerable physiological significance with high interest also for their industrial applications. The hydrolysis reaction follows a two-step mechanism, or “interfacial activation,” with adsorption of the enzyme to a heterogeneous interface and subsequent enhancement of the lipolytic activity. Among lipases, Candida antarctica lipase B (CALB) has never shown any significant interfacial activation, and a closed conformation of CALB has never been reported, leading to the conclusion that its behavior was due to the absence of a lid regulating the access to the active site. The lid open and closed conformations and their protonation states are observed in the crystal structure of CALB at 0.91 Å resolution. Having the open and closed states at atomic resolution allows relating protonation to the conformation, indicating the role of Asp145 and Lys290 in the conformation alteration. The findings explain the lack of interfacial activation of CALB and offer new elements to elucidate this mechanism, with the consequent implications for the catalytic properties and classification of lipases.     

Influence of the organic solvents on the activity in water and the conformation of Candida rugosa lipase: Description of a lipase-activating pretreatment

The influence on lipase activity in water of a pretreatment on Candida rugosa lipase using water miscible and immiscible solvents was studied. The lipase activity in the hydrolysis of esteric substrates in aqueous media increases when the lipase was previously treated with various nearly anhydrous organic media. This activation, which was irreversible, was higher for longer pretreatment times. It was dependent on the pretreatment medium (water activity and solvent used). A relation between variations in the emission intensity and the activities of treated and untreated lipases was found. Activating pretreatment did not shift the peak of fluorescence emission but gave rise to variations in the secondary protein structure by increasing the helical nature. A similar increment in the hydrolysis rate in water can be obtained with the addition of an appropriate amount of solvent (acetonitrile or n-heptane) to the aqueous reaction medium.     

X-ray Crystallographic and MD Simulation Studies on the Mechanism of Interfacial Activation of a Family I.3 Lipase with Two Lids

The interfacial activation mechanism of family I.3 lipase from Pseudomonas sp. MIS38 (PML), which has two α-helical lids (lid1 and lid2), was investigated using a combination of X-ray crystallography and molecular dynamics (MD) simulation. The crystal structure of PML in an open conformation was determined at 2.1 Å resolution in the presence of Ca²⁺ and Triton X-100. Comparison of this structure with that in the closed conformation indicates that both lids greatly change their positions and lid1 is anchored by the calcium ion (Ca1) in the open conformation. This structure was not seriously changed even when the protein was dialyzed extensively against the Ca²⁺-free buffer containing Triton X-100 before crystallization, indicating that the open conformation is fairly stable unless a micellar substance is removed. The crystal structure of the PML derivative, in which the active site serine residue (Ser207) is diethylphosphorylated by soaking the crystal of PML in the open conformation in a solution containing diethyl p-nitrophenyl phosphate, was also determined. This structure greatly resembles that in the open conformation, indicating that PML structure in the open conformation represents that in the active form. MD simulation of PML in the open conformation in the absence of micelles showed that lid2 closes first, while lid1 maintains its open conformation. Likewise, MD simulation of PML in the closed conformation in the absence of Ca²⁺ and in the presence of octane or trilaurin micelles showed that lid1 opens, while lid2 remains closed. These results suggest that Ca1 functions as a hook for stabilization of a fully opened conformation of lid1 and for initiation of subsequent opening of lid2.     

3-D structure modelling of the Staphylococcus simulans lipase: conformational changes, substrate specificity and novel structural features

We have modelled, using the CHARMM27 energy force field, the structures of closed and open forms of Staphylococcus simulans lipase (SSL) on the basis of the crystal structures of Bacillus stearothermophilus and Staphylococcus hyicus lipases, respectively. The models suggested the presence of a main lid and a second lid that may act with the former as a double door to control the access to the active site. Superimposition of both closed and open forms of SSL allowed us to determine the hinge regions allowing the movements of the main and the second lid upon lipase activation. The flexibility of these hinge regions was checked by molecular dynamics simulations. The SSL models also allowed us to identify key residues involved in binding substrates, calcium or zinc ions.     

The lid is a structural and functional determinant of lipase activity and selectivity

In several lipases access to the enzyme active site is regulated by the position of a mobile structure named the lid. The role of this region in modulating lipase function is reviewed in this paper analysing the results obtained with three different recombinant lipases modified in the lid sequence: Candida rugosa lipase isoform 1 (CRL1), Pseudomonas fragi lipase (PFL) and Bacillus subtilis lipase A (BSLA). A CRL chimera enzyme obtained by replacing its lid with that of another C. rugosa lipase isoform (CRL1LID3) was found to be affected in both activity and enantioselectivity in organic solvent. Variants of the PFL protein in which three polar lid residues were replaced with amino acids strictly conserved in homologous lipases displayed altered chain length preference profile and increased thermostability. On the other hand, insertion of lid structures from structurally homologous enzymes into BSLA, a lipase that naturally does not possess such a lid structure, caused a reduction in the enzyme activity and an altered substrate specificity. These results strongly support the concept that the lid plays an important role in modulating not only activity but also specifity, enantioselectivity and stability of lipase enzymes.     

Pressure‐induced conformational switch of an interfacial protein

A special class of proteins adopts an inactive conformation in aqueous solution and activates at an interface (such as the surface of lipid droplet) by switching their conformations. Lipase, an essential enzyme for breaking down lipids, serves as a model system for studying such interfacial proteins. The underlying conformational switch of lipase induced by solvent condition is achieved through changing the status of the gated substrate‐access channel. Interestingly, a lipase was also reported to exhibit pressure activation, which indicates it is drastically active at high hydrostatic pressure. To unravel the molecular mechanism of this unusual phenomenon, we examined the structural changes induced by high hydrostatic pressures (up to 1500 MPa) using molecular dynamics simulations. By monitoring the width of the access channel, we found that the protein undergoes a conformational transition and opens the access channel at high pressures (>100 MPa). Particularly, a disordered amphiphilic α5 region of the protein becomes ordered at high pressure. This positive correlation between the channel opening and α5 ordering is consistent with the early findings of the gating motion in the presence of a water–oil interface. Statistical analysis of the ensemble of conformations also reveals the essential collective motions of the protein and how these motions contribute to gating. Arguments are presented as to why heightened sensitivity to high‐pressure perturbation can be a general feature of switchable interfacial proteins. Further mutations are also suggested to validate our observations. Proteins 2016; 84:820–827. © 2016 Wiley Periodicals, Inc.     

Structural basis for the cold adaptation of psychrophilic M37 lipase from Photobacterium lipolyticum

The M37 lipase from Photobacterium lipolyticum shows an extremely low activation energy and strong activity at low temperatures, with optimum activity seen at 298 K and more than 75% of the optimum activity retained down to 278 K. Though the M37 lipase is most closely related to the filamentous fungal lipase, Rhizomucor miehei lipase (RML) at the primary structure level, their activity characteristics are completely different. In an effort to identify structural components of cold adaptation in lipases, we determined the crystal structure of the M37 lipase at 2.2 A resolution and compared it to that of nonadapted RML. Structural analysis revealed that M37 lipase adopted a folding pattern similar to that observed for other lipase structures. However, comparison with RML revealed that the region beneath the lid of the M37 lipase included a significant and unique cavity that would be occupied by a lid helix upon substrate binding. In addition, the oxyanion hole was much wider in M37 lipase than RML. We propose that these distinct structural characteristics of M37 lipase may facilitate the lateral movement of the helical lid and subsequent substrate hydrolysis, which might explain its low activation energy and high activity at low temperatures.     

Requirement of lid2 for interfacial activation of a family I.3 lipase with unique two lid structures

A family I.3 lipase from Pseudomonas sp. MIS38 (PML) is characterized by the presence of two lids (lid1 and lid2) that greatly change conformation upon substrate binding. While lid1 represents the commonly known lid in lipases, lid2 is unique to PML and other family I.3 lipases. To clarify the role of lid2 in PML, a lid2 deletion mutant (ΔL2-PML) was constructed by deleting residues 35-64 of PML. ΔL2-PML requires calcium ions for both lipase and esterase activities as does PML, suggesting that it exhibits activity only when lid1 is fully open and anchored by the catalytically essential calcium ion, as does PML. However, when the enzymatic activity was determined using triacetin, the activity of PML exponentially increased as the substrate concentration reached and increased beyond the critical micellar concentration, while that of ΔL2-PML did not. These results indicate that PML undergoes interfacial activation, while ΔL2-PML does not. The activities of ΔL2-PML for long-chain triglycerides significantly decreased while its activity for fatty acid ethyl esters increased, compared with those of PML. Comparison of the tertiary models of ΔL2-PML in a closed and open conformation, which are optimized by molecular dynamics simulation, with the crystal structures of PML suggests that the hydrophobic surface area provided by lid1 and lid2 in an open conformation is considerably decreased by the deletion of lid2. We propose that the hydrophobic surface area provided by these lids is necessary to hold the micellar substrates firmly to the active site and therefore lid2 is required for interfacial activation of PML. DATABASE: Triacylglycerol lipase (EC 3.1.1.3).     

Crystal structure of a mono- and diacylglycerol lipase from Malassezia globosa reveals a novel lid conformation and insights into the substrate specificity

Most lipases contain a lid domain to shield the hydrophobic binding site from the water environment. The lid, mostly in helical form, can undergo a conformational change to expose the active cleft during the interfacial activation. Here we report the crystal structures of Malassezia globosa LIP1 (SMG1) at 1.45 and 2.60 ? resolution in two crystal forms. The structures present SMG1 in its closed form, with a novel lid in loop conformation. SMG1 is one of the few members in the fungal lipase family that has been found to be strictly specific for mono- and diacylglycerol. To date, the mechanism for this substrate specificity remains largely unknown. To investigate the substrate binding properties, we built a model of SMG1 in open conformation. Based on this model, we found that the two bulky hydrophobic residues adjacent to the catalytic site and the N-terminal hinge region of the lid both may act as steric hindrances for triacylglycerols binding. These unique structural features of SMG1 will provide a better understanding on the substrate specificity of mono- and diacylglycerol lipases and a platform for further functional study of this enzyme.     

Lipase-catalysed hydrolysis of short-chain substrates in solution and in emulsion: a kinetic study.

We have studied the enzymatic hydrolysis of solutions and emulsions of vinyl propionate, vinyl butyrate and tripropionin by lipases of various origin and specificity. Kinetic studies of the hydrolysis of short-chain substrates by microbial triacylglycerol lipases from Rhizopus oryzae, Mucor miehei, Candida rugosa, Candida antarctica A and by (phospho)lipase from guinea-pig pancreas show that these lipolytic enzymes follow the Michaelis-Menten model. Surprisingly, the activity against solutions of tripropionin and vinyl esters ranges from 70% to 90% of that determined against emulsions. In contrast, a non-hyperbolic (sigmoidal) dependence of enzyme activity on ester concentration is found with human pancreatic lipase, triacylglycerol lipase from Humicola lanuginosa (Thermomyces lanuginosa) and partial acylglycerol lipase from Penicillium camembertii and the same substrates. In all cases, no abrupt jump in activity (interfacial activation) is observed at substrate concentration corresponding to the solubility limit of the esters. Maximal lipolytic activity is always obtained in the presence of emulsified ester. Despite progress in the understanding of structure-function of lipases, interpretation of the mode of action of lipases active against solutions of short-chain substrates remains difficult. Actually, it is not known whether these enzymes, which possess a lid structure, are in open or/and closed conformation in the bulk phase and whether the opening of the lid that gives access to the catalytic triad is triggered by interaction of the enzyme molecule with monomeric substrates or/and multimolecular aggregates (micelles) both present in the bulk phase. From the comparison of the behaviour of lipases used in this study which, in some cases, follow the Michaelis-Menten model and, in others, deviate from classical kinetics, it appears that the activity of classical lipases against soluble short-chain vinyl esters and tripropionin depends not only on specific interaction with single substrate molecules at the catalytic site of the enzyme but also on physico-chemical parameters related to the state of association of the substrate dispersed in the aqueous phase. It is assumed that the interaction of lipase with soluble multimolecular aggregates of tripropionin or short-chain vinyl esters or the formation of enzyme-substrate mixed micelles with ester bound to lipase, might represent a crucial step that triggers the structural transition to the open enzyme conformation by displacement of the lid.     

不依赖油水界面激活的黑曲霉脂肪酶突变体的构建

为获得不依赖油水界面激活的黑曲霉脂肪酶 (ANL) 突变体,在生物信息学分析基础上,对黑曲霉脂肪酶盖子结构域两侧铰链区的氨基酸残基进行了置换突变,获得两个黑曲霉脂肪酶突变体 (ANL-Ser84Gly和ANL-Asp99Pro)。对不同浓度对硝基苯丁酸酯的水解活性检测结果表明：ANL-Ser84Gly的催化活性仍依赖油水界面,而ANL-Asp99Pro的催化活性不再依赖油水界面。底物特异性检测结果表明：较ANL而言,ANL-Ser84Gly的比活力显著降低,其水解对硝基苯棕榈酸酯、对硝基苯豆蔻酸酯、对硝基