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.     

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.     

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.     

Effect of different parameters on the hydrolytic activity of cross-linked enzyme aggregates (CLEAs) of lipase from Thermomyces lanuginosa

Cross-linked enzyme aggregates (CLEAs) of lipase from Thermomyces lanuginosa (TLL) were synthesized using (NH₄)₂SO₄ as precipitant and glutaraldehyde as cross-linking agent. CLEAs were assayed for their hydrolytic activity in a reaction performed in an emulsioned medium. The effects of the amount of precipitant, cross-linker, and different additives such as protein cofeeder, oleic acid, n-heptane, sodium dodecyl sulfate (SDS), polyethylenglicol (PEG) and ethylendiamine were studied at selected ratios with respect to TLL mass. Traditional non-layered CLEAs of TLL showed recovered activities between 3 and 31% when compared with native lipase. Novel TLL layered CLEAs consisting of a protein cofeeder core and successive layers of target lipase showed an important increase in their retained activity. The highest recovered activity was found for the one-layered non-additivated CLEAs of TLL which showed a recovered activity of 75%.     

Dynamics of proteins in different solvent systems: analysis of essential motion in lipases.

We have investigated the effect of different solvents on the dynamics of Rhizomucor miehei lipase. Molecular dynamics simulations were performed in water, methyl hexanoate, and cyclohexane. Analysis of the 400-ps trajectories showed that the solvent has a pronounced effect on the geometrical properties of the protein. The radius of gyration and total accessibility surface decrease in organic solvents, whereas the number of hydrogen bonds increases. The essential motions of the protein in different solvents can be described in a low-dimensional "essential subspace," and the dynamic behavior in this subspace correlates with the polarity of the solvent. Methyl hexanoate, which is a substrate for R. miehei lipase, significantly increases the fluctuations in the active-site loop. During the simulation, a methyl hexanoate entered the active-site groove. This observation provides insight into the possible docking mechanism of the substrate.     

Characterization of cross-linked immobilized lipase from thermophilic mould Thermomyces lanuginosa using glutaraldehyde

Cross-linked enzyme aggregates (CLEAs) have emerged as an interesting biocatalyst design for immobilization. Using this approach, a 1,3 regiospecific, alkaline and thermostable lipase from Thermomyces lanuginosa was immobilized. Efficient cross-linking was observed when ammonium sulphate was used as precipitant along with a two fold increase in activity in presence of SDS. The TEM and SEM microphotographs of the CLEAs formed reveal that the enzyme aggregates are larger in size as compared to the free lipase due to the cross-linking of enzyme aggregates with glutaraldehyde. The stability and reusability of the CLEA with respect to olive oil hydrolysis was evaluated. The CLEA showed more than 90% residual activity even after 10 cycles of repeated use.     

Binding of Thermomyces (Humicola) lanuginosa lipase to the mixed micelles of cis-parinaric acid/NaTDC.

The binding of Thermomyces lanuginosa lipase and its mutants [TLL(S146A), TLL(W89L), TLL(W117F, W221H, W260H)] to the mixed micelles of cis-parinaric acid/sodium taurodeoxycholate at pH 5.0 led to the quenching of the intrinsic tryptophan fluorescence emission (300-380 nm) and to a simultaneous increase in the cis-parinaric acid fluorescence emission (380-500 nm). These findings were used to characterize the Thermomyces lanuginosa lipase/cis-parinaric acid interactions occurring in the presence of sodium taurodeoxycholate.The fluorescence resonance energy transfer and Stern-Volmer quenching constant values obtained were correlated with the accessibility of the tryptophan residues to the cis-parinaric acid and with the lid opening ability of Thermomyces lanuginosa lipase (and its mutants). TLL(S146A) was found to have the highest fluorescence resonance energy transfer. In addition, a TLL(S146A)/oleic acid complex was crystallised and its three-dimensional structure was solved. Surprisingly, two possible binding modes (sn-1 and antisn1) were found to exist between oleic acid and the catalytic cleft of the open conformation of TLL(S146A). Both binding modes involved an interaction with tryptophan 89 of the lipase lid, in agreement with fluorescence resonance energy transfer experiments.As a consequence, we concluded that TLL(S146A) mutant is not an appropriate substitute for the wild-type Thermomyces lanuginosa lipase for mimicking the interaction between the wild-type enzyme and lipids.     

Immobilization of lipase on cotton cloth using the layer-by-layer self-assembly technique

Lipase from Thermomyces lanuginosus was assembled into multiple layers on polyethylenimine treated cotton flannel cloth, utilising the enzymes property of forming bimolecular aggregates via layer-by-layer (LBL) immobilization technique. An increase in lipase activity with increasing enzyme layers confirmed lipase aggregation. A study to compare the activity of enzyme bound by classical LBL technique, containing alternate layers of polyethylenimine and lipase and the modified approach indicated above, showed that more enzyme was bound to cloth in the modified approach. A total of 13 U/cm² of enzyme were bound to cloth till the fifth layer whereas only 10.2 U/cm² were bound till the fifth bilayer in the classical approach. The successful assembly of lipase molecules has shown that this modified technique is a promising approach to immobilize enzymes that aggregate through hydrophobic interactions as nano-films on cloth.     

Substrate specificity and kinetic properties of enzymes belonging to the hormone-sensitive lipase family: comparison with non-lipolytic and lipolytic carboxylesterases

We have studied the kinetics of hydrolysis of triacylglycerols, vinyl esters and p-nitrophenyl butyrate by four carboxylesterases of the HSL family, namely recombinant human hormone-sensitive lipase (HSL), EST2 from Alicyclobacillus acidocaldarius, AFEST from Archeoglobus fulgidus, and protein RV1399C from Mycobacterium tuberculosis. The kinetic properties of enzymes of the HSL family have been compared to those of a series of lipolytic and non-lipolytic carboxylesterases including human pancreatic lipase, guinea pig pancreatic lipase related protein 2, lipases from Mucor miehei and Thermomyces lanuginosus, cutinase from Fusarium solani, LipA from Bacillus subtilis, porcine liver esterase and Esterase A from Aspergilus niger. Results indicate that human HSL, together with other lipolytic carboxylesterases, are active on short chain esters and hydrolyze water insoluble trioctanoin, vinyl laurate and olive oil, whereas the action of EST2, AFEST, protein RV1399C and non-lipolytic carboxylesterases is restricted to solutions of short chain substrates. Lipolytic and non-lipolytic carboxylesterases can be differentiated by their respective value of K(0.5) (apparent K(m)) for the hydrolysis of short chain esters. Among lipolytic enzymes, those possessing a lid domain display higher activity on tributyrin, trioctanoin and olive oil suggesting, then, that the lid structure contributes to enzyme binding to triacylglycerols. Progress reaction curves of the hydrolysis of p-nitrophenyl butyrate by lipolytic carboxylesterases with lid domain show a latency phase which is not observed with human HSL, non-lipolytic carboxylesterases, and lipolytic enzymes devoid of a lid structure as cutinase.     

Orientation and conformation of a lipase at an interface studied by molecular dynamics simulations

Electron density profiles calculated from molecular dynamics trajectories are used to deduce the orientation and conformation of Thermomyces lanuginosa lipase and a mutant adsorbed at an air-water interface. It is demonstrated that the profiles display distinct fine structures, which uniquely characterize enzyme orientation and conformation. The density profiles are, on the nanosecond timescale, determined by the average enzyme conformation. We outline a computational scheme that from a single molecular dynamics trajectory allows for extraction of electron density profiles referring to different orientations of the lipase relative to an implicit interface. Profiles calculated for the inactive and active conformations of the lipase are compared with experimental electron density profiles measured by x-ray reflectivity for the lipase adsorbed at an air-water interface. The experimental profiles contain less fine structural information than the calculated profiles because the resolution of the experiment is limited by the intrinsic surface roughness of water. Least squares fits of the calculated profiles to the experimental profiles provide areas per adsorbed enzyme and suggest that Thermomyces lanuginosa lipase adsorbs to the air-water interface in a semiopen conformation with the lid oriented away from the interface.     

Lipases fromRhizomucor miehei andHumicola lanuginosa: Modification of the lid covering the active site alters enantioselectivity

The homologous lipases fromRhizomucor miehei andHumicola lanuginosa showed approximately the same enantioselectivity when 2-methyldecanoic acid esters were used as substrates. Both lipases preferentially hydrolyzed theS-enantiomer of 1-heptyl 2-methyldecanoate (R. miehei:E _S =8.5;H. lanuginosa:E _S =10.5), but theR-enantiomer of phenyl 2-methyldecanoate (E _R =2.9). Chemical arginine specific modification of theR. miehei lipase with 1,2-cyclohexanedione resulted in a decreased enantioselectivity (E _R =2.0), only when the phenyl ester was used as a substrate. In contrast, treatment with phenylglyoxal showed a decreased enantioselectivity (E _S =2.5) only when the heptyl ester was used as a substrate. The presence of guanidine, an arginine side chain analog, decreased the enantioselectivity with the heptyl ester (E _S =1.9) and increased the enantioselectivity with the aromatic ester (E _R =4.4) as substrates. The mutation, Glu 87 Ala, in the lid of theH. lanuginosa lipase, which might decrease the electrostatic stabilization of the open-lid conformation of the lipase, resulted in 47% activity compared to the native lipase, in a tributyrin assay. The Glu 87 Ala mutant showed an increased enantioselectivity with the heptyl ester (E _S =17.4) and a decreased enantioselectivity with the phenyl ester (E _R =2.5) as substrates, compared to native lipase. The enantioselectivities of both lipases in the esterification of 2-methyldecanoic acid with 1-heptanol were unaffected by the lid modifications.     

Computational approach to solvent-free synthesis of ethyl oleate using Candida rugosa and Candida antarctica B Lipases. I. Interfacial activation and substrate (ethanol, oleic acid) adsorption

This paper presents the results of a MM2 study of the adsorption of oleic acid and ethanol/water in the tunnel and active-site models of lipases from Candida rugosa and Candida antarctica B. The role of an interface polar/no polar in the opening of C. rugosa lipase's lid is also addressed, discussed and analyzed at the level of the conformational changes needed to achieve the lipase open form. The adsorption of oleic acid and alcohols considering C. antarctica B, a lipase not interfacially activated, is also presented. In this case, the tunnel is shorter than in case of C. rugosa lipase. Two different pockets can be visualized at the active site-tunnel model of C. antarctica B lipase: one for the acyl group and another for the alcohol. Wrong location of alcohol and oleic acid severely hinders reaction because it hinders the H-transfer to histidine, a key step in the reaction mechanism. Right location of alcohol decreases the possibility of alcohol inhibition. In the case of C. rugosa, no restrictions for ethanol/water location are found. For that lipase, a second adsorption site for oleic acid (outside the tunnel) is presented. This site is the exit tunnel of the ester product when oleic acid is adsorbed in the tunnel. Experimental results of our own that correlate with this study are presented.     

Evolution of Stability in a Cold-Active Enzyme Elicits Specificity Relaxation and Highlights Substrate-Related Effects on Temperature Adaptation

Molecular aspects of thermal adaptation of proteins were studied by following the co-evolution of temperature dependence, conformational stability, and substrate specificity in a cold-active lipase modified via directed evolution. We found that the evolution of kinetic stability was accompanied by a relaxation in substrate specificity. Moreover, temperature dependence and selectivity turned out to be mutually dependent. While the wild-type protein was strictly specific for short-chain triglycerides (C4) in the temperature range 10-50 °C and displayed highest activity in the cold, its stabilized variant was able to accept C8 and C12 molecules and its selectivity was temperature dependent. We could not detect any improvement in the overall structural robustness of the mutant when the structure was challenged by temperature or chemical denaturants. There is, however, strong evidence for local stabilization effects in the active-site region provided by two independent approaches. Differential scanning fluorimetry revealed that the exposure of hydrophobic patches (as the active site is) precedes denaturation, and molecular dynamics simulations confirmed that stability was obtained by restriction of the mobility of the lid, a flexible structure that regulates the access to the enzyme active site and influences its stability. This reduction of lid movements is suggested to be accompanied by a concomitant increase in the mobility of other protein regions, thus accounting for the observed broadening of substrate specificity.     

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.     

Improving the catalytic activity of lipase LipK107 from Proteus sp. by site-directed mutagenesis in the lid domain based on computer simulation

The capacity of lipase LipK107 from Proteus sp. catalyzing the kinetic resolution of racemates was investigated. The resolution of racemic 1-phenylethanol in organic medium was selected as model reaction. The conversion was dramatically dependent on the water content and the LipK107 showed high activity in a wide range of water content without appreciable loss of enzyme enantiodiscrimination. Besides, the chain length of acyl donor also had a significant effect on the conversion, and the highest enantioselectivity was achieved when methyl palmitate was used. Based on the analysis of computer model structure of LipK107, different mutations were introduced into the lid region. Each derivative of LipK107 was expressed, purified, and assessed of the activity. According to the prediction, using mutants E130L + K131I and T138V as catalyst, respectively, the conversions of 1-phenylethanol improved greatly with a slight increase of enantiodiscrimination. In addition, the effects of hydrophobicity and electrostatic of the lid on lipase activity were determined. This work indicated that the modification of the lid might considerably enhance the activity and improve the yield of catalytic reactions, which could apply to other lipases. The computer simulations would make the process of identifying amino acids for substitution efficiently.     

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.     

Probing a functional role of Glu87 and Trp89 in the lid ofHumicola lanuginosa lipase through transesterification reactions in organic solvent

To reveal the functional role of Glu87 and Trp89 in the lid ofHumicola lanuginosa lipase, site-directed mutagenesis at Glu87 and Trp89 was carried out. The catalytic performance of wild-type and mutated lipases was studied in transesterification reactions in cyclohexane at a controlled water activity. Two different acyl donors were used in the investigation: tributyrin, a natural substrate for a lipase, and vinyl butyrate, an activated ester suitable for fast and efficient lipase-catalyzed transformations in preparative organic synthesis. As acyl acceptor 1-heptanol was used. The Glu87Ala mutation decreased theV _max,app value with tributyrin and vinyl butyrate by a factor of 1.5 and 2, respectively. TheK _m,app for tributyrin was not affected by the Glu87Ala mutation, but theK _m,app for vinyl butyrate increased twofold compared to the wild-type lipase. Changing Trp89 into a Phe residue afforded an enzyme with a 2.7- and 2-fold decreasedV _max,app with the substrates tributyrin and vinyl butyrate, respectively, compared to the wild-type lipase. No significant effects on theK _m,app values for tributyrin or vinyl butyrate were seen as a result of the Trp89Phe mutation. However, the introduction of a Glu residue at position 89 in the lid increased theK _m,app for tributyrin and vinyl butyrate by a factor of >5 and 2, respectively. The Trp89Glu mutated lipase could not be saturated with tributyrin within the experimental conditions (0–680 mM) studied here. With vinyl butyrate as a substrate theV _max,app was only 6% of that obtained with wild-type enzyme.     

The atypical lipase B from Candida antarctica is better adapted for organic media than the typical lipase from Thermomyces lanuginosa

Candida antarctica lipase B (CALB) and Thermomyces lanuginosa lipase (TLL) were evaluated as catalysts in different reaction media using hydrolysis of tributyrin as model reaction. In o/w emulsions, the enzymes were used in the free form and for use in monophasic organic media, the lipases were adsorbed on porous polypropylene (Accurel EP-100). In monophasic organic media, the highest specific activity of both lipases was obtained in pure tributyrin at a water activity of >0.5 and at an enzyme loading of 10 mg/g support. With tributyrin emulsified in water, the specific activities were 2780 micromol min(-1) mg(-1) for TLL and 535 micromol min(-1) mg(-1) for CALB. Under optimal conditions in pure tributyrin, CALB expressed 49% of the activity in emulsion (264 micromol min(-1) mg(-1)) while TLL expressed only 9.2% (256 micromol min(-1) mg(-1)) of its activity in emulsion. This large decrease is probably due to the structure of TLL, which is a typical lipase with a large lid domain. Conversion between open and closed conformers of TLL involves large internal movements and catalysis probably requires more protein mobility in TLL than in CALB, which does not have a typical lid region. Furthermore, TLL lost more activity than CALB when the water activity was reduced below 0.5, which could be due to further reduction in protein mobility.     

Structure and dynamics of Candida rugosa lipase: the role of organic solvent

The effect of organic solvent on the structure and dynamics of proteins was investigated by multiple molecular dynamics simulations (1 ns each) of Candida rugosa lipase in water and in carbon tetrachloride. The choice of solvent had only a minor structural effect. For both solvents the open and the closed conformation of the lipase were near to their experimental X-ray structures (C rms deviation 1–1.3 Å). However, the solvents had a highly specific effect on the flexibility of solvent-exposed side chains: polar side chains were more flexible in water, but less flexible in organic solvent. In contrast, hydrophobic residues were more flexible in organic solvent, but less flexible in water. As a major effect solvent changed the dynamics of the lid, a mobile element involved in activation of the lipase, which fluctuated as a rigid body about its average position. While in water the deviations were about 1.6 Å, organic solvent reduced flexibility to 0.9 Å. This increase rigidity was caused by two salt bridges (Lys85–Asp284, Lys75–Asp79) and a stable hydrogen bond (Lys75–Asn 292) in organic solvent. Thus, organic solvents stabilize the lid but render the side chains in the hydrophobic substrate-binding site more mobile. Figure Superimposition of open (black, PDB entry 1CRL) and closed (gray, PDB entry 1TRH) conformers of C. rugosa lipase. The mobile lid is indicatedThis revised version was published online in October 2004 with corrections to the Graphical Abstract.