Unlocking the mystery behind the activation phenomenon of T1 lipase: a molecular dynamics simulations approach

The activation of lipases has been postulated to proceed by interfacial activation, temperature switch activation, or aqueous activation. Recently, based on molecular dynamics (MD) simulation experiments, the T1 lipase activation mechanism was proposed to involve aqueous activation in addition to a double-flap mechanism. Because the open conformation structure is still unavailable, it is difficult to validate the proposed theory unambiguously to understand the behavior of the enzyme. In this study, we try to validate the previous reports and uncover the mystery behind the activation process using structural analysis and MD simulations. To investigate the effects of temperature and environmental conditions on the activation process, MD simulations in different solvent environments (water and water-octane interface) and temperatures (20, 50, 70, 80, and 100°C) were performed. Based on the structural analysis of the lipases in the same family of T1 lipase (I.5 lipase family), we proposed that the lid domain comprises α6 and α7 helices connected by a loop, thus forming a helix-loop-helix motif involved in interfacial activation. Throughout the MD simulations experiments, lid displacements were only observed in the water-octane interface, not in the aqueous environment with respect to the temperature effect, suggesting that the activation process is governed by interfacial activation coupled with temperature switch activation. Examining the activation process in detail revealed that the large structural rearrangement of the lid domain was caused by the interaction between the hydrophobic residues of the lid with octane, a nonpolar solvent, and this conformation was found to be thermodynamically favorable.     

定向进化-易错PCR方法提高华根霉Rhizopus chinensis CCTCC M201021脂肪酶的活力

运用定向进化-易错PCR的方法,提高了华根霉Rhizopus chinensis CCTCC M201021脂肪酶的活力。经过两轮易错PCR和pNPP顶层琼脂法筛选,从第一轮和第二轮突变库中分别筛选获得最佳突变株1-11和2-28,脂肪酶酶活与野生菌株相比分别提高2倍和4倍。基因比对结果表明,突变脂肪酶2-28有4个氨基酸发生了突变:A129S、K161R、A230T、K322R。蛋白质分子空间结构模拟显示,突变A129S、K161R、A230T位于脂肪酶分子表面。突变A230T增强了α-螺旋盖结构的稳定性。突变K322R处在loop上,靠近脂肪酶底物结合区域,与邻近的Asp(带负电)形成盐桥。静电引力将该loop向底物进入酶活性中心的通道口反方向牵引,使底物分子更易进入酶活性中心。酶学性质研究表明,突变株2-28脂肪酶的Km值比出发菌株下降了10%,Kcat值提高为原来的2.75倍。     

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.     

Conformational change in the activation of lipase: an analysis in terms of low-frequency normal modes.

The interfacial activation of Rhizomucor miehei lipase (RmL) involves the motion of an alpha-helical region (residues 82-96) which acts as a "lid" over the active site of the enzyme, undergoing a displacement from a "closed" to an "open" conformation upon binding of substrate. Normal mode analyses performed in both low and high dielectric media reveal that low-frequency vibrational modes contribute significantly to the conformational transition between the closed and open conformations. In these modes, the lid displacement is coupled to local motions of active site loops as well as global breathing motions. Atomic fluctuations of the first hinge of the lid (residues 83-84) are substantially larger in the low dielectric medium than in the high dielectric medium. Our results also suggest that electrostatic interactions of Arg86 play an important role in terms of both the intrinsic stability of the lid and its displacement, through enhancement of hinge mobility in a high dielectric medium. Additional calculations demonstrate that the observed patterns of atomic fluctuations are an intrinsic feature of the protein structure and not dependent on the nature of specific energy minima.     

In silico characterization of thermoactive, alkaline and detergent-stable lipase from a Staphylococcus aureus strain

Bacterial true lipases having thermo and alkaline stability are highly attractive for their industrial production of pharmaceuticals, agrochemicals, cosmetics, and flavour. Staphylococcus aureus lipase (SAL3) remains active at temperatures 40-60°C, with an optimum temperature of 55°C and an optimum pH of 9.5 stable over a range of 5-12. Detailed understanding of the structure and insight into the activity of such lipase would aid in engineering lipases that would function in the desired extreme industrial environments. In the present study, we carried out in silico characterization and structural modeling of SAL3 which is thermoactive, alkaline and detergent-stable. Comparison of SAL3 with other staphylococcal lipases indicates that SAL3 is a true lipase having the catalytic triad (residues Ser119, Asp310 & His352) and the calcium binding site (residues Asp351, Asp354, Asp359, Asp362 and Gly286). Conservation in sequence implies that interfacial activation mechanism is possible in SAL3 with the lid formed by helix (residues 180-196) and loop (residues 197-206). Three dimensional (3D) structure model of SAL3 has been predicted for the first time and aims at understanding its function and biochemical characteristics of possessing relatively high thermal and pH stability.     

Substrate specificity of lipoprotein lipase and endothelial lipase: studies of lid chimeras

The triglyceride (TG) lipase gene subfamily, consisting of LPL, HL, and endothelial lipase (EL), plays a central role in plasma lipoprotein metabolism. Compared with LPL and HL, EL is relatively more active as a phospholipase than as a TG lipase. The amino acid loop or "lid" covering the catalytic site has been implicated as the basis for the difference in substrate specificity between HL and LPL. To determine the role of the lid in the substrate specificity of EL, we studied EL in comparison with LPL by mutating specific residues of the EL lid and exchanging their lids. Mutation studies showed that amphipathic properties of the lid contribute to substrate specificity. Exchanging lids between LPL and EL only partially shifted the substrate specificity of the enzymes. Studies of a double chimera possessing both the lid and the C-terminal domain (C-domain) of EL in the LPL backbone showed that the role of the lid in determining substrate specificity does not depend on the nature of the C-domain of the lipase. Using a kinetic assay, we showed an additive effect of the EL lid on the apparent affinity for HDL(3) in the presence of the EL C-domain.     

Differential induction, purification and characterization of cold active lipase from Yarrowia lipolytica NCIM 3639

The production, purification and characterization of cold active lipases by Yarrowia lipolytica NCIM 3639 is described. The study presents a new finding of production of cell bound and extracellular lipase activities depending upon the substrate used for growth. The strain produced cell bound and extracellular lipase activity when grown on olive oil and Tween 80, respectively. The organism grew profusely at 20 °C and at initial pH of 5.5, producing maximum extracellular lipase. The purified lipase has a molecular mass of 400 kDa having 20 subunits forming a multimeric native protein. Further the enzyme displayed an optimum pH of 5.0 and optimum temperature of 25 °C. Peptide mass finger printing reveled that some peptides showed homologues sequence (42%) to Yarrowia lipolytica LIP8p. The studies on hydrolysis of racemic lavandulyl acetate revealed that extracellular and cell bound lipases show preference over the opposite antipodes of irregular monoterpene, lavandulyl acetate.     

Temperature and salt effects on proteolytic function of turnip mosaic potyvirus nuclear inclusion protein a exhibiting a low-temperature optimum activity

The nuclear inclusion protein a (NIa) of turnip mosaic potyvirus is a protease responsible for processing the viral polyprotein into functional proteins. The NIa protease exhibits an unusual optimum proteolytic activity at about 16 degrees C. In order to understand the origin of the low-temperature optimum activity, the effects of temperature and salt ions on the catalytic activity and the structure of the NIa protease have been investigated. The analysis of the temperature dependence of k(cat) and K(m) revealed that K(m) decreases more drastically than k(cat) as temperature decreases. The thermodynamic analysis showed that the decrease of K(m) is driven entropically, suggesting a possibility that the substrate binding might need a large entropy cost. The secondary structure of the NIa protease was significantly perturbed at temperatures between 20 and 40 degrees C and the protease was unfolded at very low concentrations of guanidine hydrochloride with a transition midpoint of 0.8 M. These results suggest that the NIa protease is highly flexible in structure. Interestingly, salt ions including NaCl, KCl, CaCl(2) and MgCl(2) stimulated the proteolytic activity by 2-6-fold and increased the optimum temperature to 20-25 degrees C. This stimulatory effect of the salt ions was due to the lowering of K(m). The salt ions promoted the structural rigidity as evidenced in the higher resistance to the heat-induced unfolding in the presence of the salt ions. The increase in rigidity may lead to the lowering of K(m) possibly by reducing the entropic cost for substrate binding. Taken together, these results suggest that the NIa protease is highly flexible in structure and the low-temperature optimum activity might possibly be attributed to lowered entropy cost for substrate binding at lower temperatures.     

Novel zinc-binding center and a temperature switch in the Bacillus stearothermophilus L1 lipase

The bacterial thermoalkalophilic lipases optimally hydrolyze saturated fatty acids at elevated temperatures. They also have significant sequence homology with staphylococcal lipases, and both the thermoalkalophilic and staphylococcal lipases are grouped as the lipase family I.5. We report here the first crystal structure of the lipase family I.5, the structure of a thermoalkalophilic lipase from Bacillus stearothermophilus L1 (L1 lipase) determined at 2.0-A resolution. The structure is in a closed conformation, and the active site is buried under a long lid helix. Unexpectedly, the structure exhibits a zinc-binding site in an extra domain that accounts for the larger molecular size of the family I.5 enzymes in comparison to other microbial lipases. The zinc-coordinated extra domain makes tight interactions with the loop extended from the C terminus of the lid helix, suggesting that the activation of the family I.5 lipases may be regulated by the strength of the interactions. The unusually long lid helix makes strong hydrophobic interactions with its neighbors. The structural information together with previous biochemical observations indicate that the temperature-mediated lid opening is triggered by the thermal dissociation of the hydrophobic interactions.     

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.     

Kinetics, mechanism, and time course analysis of lipase-catalyzed hydrolysis of high concentration olive oil in AOT-isooctane reversed micelles

Candida rugosa lipase has been used to investigate the hydrolysis of high concentration olive oil in the AOT-isooctane reversed micellar system at W(o) = 10, pH 7.1, and 37 degrees C. Results from this work show the hydrolytic reaction obeys Michaelis-Menten kinetics up to the initial substrate concentration of 1.37M, with turnover number k(cat) and Michaelis constant K(M) of 67.1 mumol/min mg enzyme and 0.717M, respectively. A competitive inhibition by the main product, oleic acid, has been found with a dissociation constant K(I) for the complex EP* of 0.089M. The rate equation was further analyzed in the time course reaction and was found in agreement with the experimental results for lower substrate concentrations, up to 0.341M. Large deviation occurred at high substrate concentrations, which may be due to the effects of large consumption of water on kinetics, on the formation of glycerol, and on the deactivation of lipase in the hydrolysis reaction as well.     

Different phyllosilicates as supports for lipase immobilisation

The aim of this work was to determine the enzymatic activities resulting from the adsorption of Rhizomucor miehei lipase (RML) and Candida cylindracea lipase (CCL) onto three different phyllosilicates (sepiolite, palygorskite and montmorillonite), comparing the resultant activities with those obtained following similar immobilisation technique on a widely used resin (Duolite A-568). Due to the different adsorption mechanisms produced, different derivatives with higher hydrolytic activities can be obtained. Comparing the clays tested, the results showed that, in comparison with the laminar silicate (montmorillonite sample) and Duolite A-568 (spherical particles), fibrous materials (palygorskite and sepiolite) resulted in derivatives with higher hydrolytic activities in the hydrolysis of different ethyl esters. Moreover, according to the data obtained with the electrophoresis, the selectivity of immobilisation for RML in the case of fibrous silicates was optimal. As a conclusion, and according to the activities and selectivities measured, at least two out of the four studied materials (sepiolite and palygorskite) would be useful as supports for immobilisation for proteins of relatively low molecular weight (such as RML) for further use in biotransformations, while for C. cylindracea the immobilisation onto duolite rendered a derivative specially active in the hydrolysis of ethyl formiate (esterasic activity).     

Modulating the activity of avian pancreatic lipases by an alkyl chain reacting with an accessible sulfhydryl group

Both turkey (TPL) and chicken (CPL) pancreatic lipases possess only one exposed sulfhydryl residue (Cystein114). After preincubation with the lipase, the sulfhydryl reagent C12 -TNB was found to be a powerful inhibitor of TPL whereas it had no effect on the CPL activity. Based on the 3D structure modelling and the molecular dynamics, the bulky dodecyl chain might hamper the lid movement of the TPL leading to the lipase inhibition upon reaction with C12 -TNB. Meanwhile, the predicted position of the C12 chain linked to Cystein114 of CPL could not block the lid opening mechanism which explains the absence of inhibition by C12 -TNB. Surprisingly, when added during the substrate hydrolysis, C12 -TNB activated the TPL but not the CPL that was slightly inhibited under these conditions. The 3D structure model generated for the open forms of C12 -TPL and C12 -CPL complexes showed that Cystein114 is still accessible and might react with C12 -TNB. Our models clearly explain the activation of TPL and the partial inhibition of CPL after the binding of the C12 chain to the enzyme.     

Ribonuclease Rs from Rhizopus stolonifer: lowering of optimum temperature in the presence of urea

RNase Rs showed an approx. 2-fold increase in its activity when incubated in the presence of 2 M urea at 37 degrees C. The increase in its activity, in the presence of urea, was comparable to the activity at its optimum temperature, i.e. 45 degrees C. Compared to the native enzyme at 37 degrees C, the K(m) and V(max) of RNase Rs at 45 degrees C and in the presence of 2 M urea at 37 degrees C showed an increase while k(cat)/K(m) decreased. Arrhenius plots in the presence and absence of urea showed a decrease in the activation energy in the presence of urea. Though there was no change in the secondary structure of the protein in the presence of urea, minor changes were observed in the tertiary structure. Hence, the increase in the activity of RNase Rs, in the presence of 2 M urea at 37 degrees C, is due to the lowering of the activation energy as a result of changes in the microenvironment of the active site.     

Structure prediction and functional analyses of a thermostable lipase obtained from Shewanella putrefaciens

Previous experimental studies on thermostable lipase from Shewanella putrefaciens suggested the maximum activity at higher temperatures, but with little information on its conformational profile. In this study, the three-dimensional structure of lipase was predicted and a 60 ns molecular dynamics (MD) simulation was carried out at temperatures ranging from 300 to 400 K to better understand its thermostable nature at the molecular level. MD simulations were performed in order to predict the optimal activity of thermostable lipase. The results suggested strong conformational temperature dependence. The thermostable lipase maintained its bio-active conformation at 350 K during the 60 ns MD simulations.     

The crystal structure of an EST2 mutant unveils structural insights on the H group of the carboxylesterase/lipase family

Esterase 2 (EST2) from the thermophilic eubacterium Alicyclobacillus acidocaldarius is a thermostable serine hydrolase belonging to the H group of the esterase/lipase family. This enzyme hydrolyzes monoacylesters of different acyl-chain length and various compounds with industrial interest. EST2 displays an optimal temperature at 70 degrees C and maximal activity with pNP-esters having acyl-chain bearing from six to eight carbon atoms. EST2 mutants with different substrate specificity were also designed, generated by site-directed mutagenesis, and biochemically characterized. To better define at structural level the enzyme reaction mechanism, a crystallographic analysis of one of these mutants, namely M211S/R215L, was undertaken. Here we report its three-dimensional structure at 2.10A resolution. Structural analysis of the enzyme revealed an unexpected dimer formation as a consequence of a domain-swapping event involving its N-terminal region. This phenomenon was absent in the case of the enzyme bound to an irreversible inhibitor having optimal substrate structural features. A detailed comparison of the enzyme structures before and following binding to this molecule showed a movement of the N-terminal helices resulting from a trans-cis isomerization of the F37-P38 peptide bond. These findings suggest that this carboxylesterase presents two distinct structural arrangements reminiscent of the open and closed forms already reported for lipases. Potential biological implications associated with the observed quaternary reorganization are here discussed in light of the biochemical properties of other lipolytic members of the H group.     

In silico and empirical approaches toward understanding the structural adaptation of the alkaline-stable lipase KV1 from Acinetobacter haemolyticus

Interests in Acinetobacter haemolyticus lipases are showing an increasing trend concomitant with growth of the enzyme industry and the widening search for novel enzymes and applications. Here, we present a structural model that reveals the key catalytic residues of lipase KV1 from A. haemolyticus. Homology modeling of the lipase structure was based on the structure of a carboxylesterase from the archaeon Archaeoglobus fulgidus as the template, which has a sequence that is 58% identical to that of lipase KV1. The lipase KV1 model is comprised of a single compact domain consisting of seven parallel and one anti-parallel β-strand surrounded by nine α-helices. Three structurally conserved active-site residues, Ser165, Asp259, and His289, and a tunnel through which substrates access the binding site were identified. Docking of the substrates tributyrin and palmitic acid into the pH 8 modeled lipase KV1 active sites revealed an aromatic platform responsible for the substrate recognition and preference toward tributyrin. The resulting binding modes from the docking simulation correlated well with the experimentally determined hydrolysis pattern, for which pH 8 and tributyrin being the optimum pH and preferred substrate. The results reported herein provide useful insights into future structure-based tailoring of lipase KV1 to modulate its catalytic activity.     

How gastric lipase, an interfacial enzyme with a Ser-His-Asp catalytic triad, acts optimally at acidic pH

Gastric lipase is active under acidic conditions and shows optimum activity on insoluble triglycerides at pH 4. The present results show that gastric lipase also acts in solution on vinyl butyrate, with an optimum activity above pH 7, which suggests that gastric lipase is able to hydrolyze ester bonds via the classical mechanism of serine hydrolases. These results support previous structural studies in which the catalytic triad of gastric lipase was reported to show no specific features. The optimum activity of gastric lipase shifted toward lower pH values, however, when the vinyl butyrate concentration was greater than the solubility limit. Experiments performed with long-chain triglycerides showed that gastric lipase binds optimally to the oil-water interface at low pH values. To study the effects of the pH on the adsorption step independently from substrate hydrolysis, gastric lipase adsorption on solid hydrophobic surfaces was monitored by total internal reflection fluorescence (TIRF), as well as using a quartz crystal microbalance. Both techniques showed a pH-dependent reversible gastric lipase adsorption process, which was optimum at pH 5 (Kd = 6.5 nM). Lipase adsorption and desorption constants (ka = 147,860 M(-1) s(-1) and kd = 139 x 10(-4) s(-1) at pH 6) were estimated from TIRF experiments. These results indicate that the optimum activity of gastric lipase at acidic pH is only "apparent" and results from the fact that lipase adsorption at lipid-water interfaces is the pH-dependent limiting step in the overall process of insoluble substrate hydrolysis. This specific kinetic feature of interfacial enzymology should be taken into account when studying any soluble enzyme acting on an insoluble substrate.     

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.     

Interfacial control of lid opening in Thermomyces lanuginosa lipase

Small unilamelar vesicles of anionic phospholipids (SUV), such as 1-palmitoyl-2-oleoylglycero-sn-3-phosphoglycerol (POPG), provide an interface where Thermomyces lanuginosa triglyceride lipase (TlL) binds and adopts a catalytically active conformation for the hydrolysis of substrate partitioned in the interface, such as tributyrin or p-nitrophenylbutyrate, with an increase in catalytic rate of more than 100-fold for the same concentration of substrate [Berg et al. (1998) Biochemistry 37, 6615-6627.]. This interfacial activation is not seen with large unilamelar vesicles (LUV) of the same composition, or with vesicles of zwitterionic phospholipids such as 1-palmitoyl-2-oleoylglycero-sn-3-phosphocholine (POPC), independently of the vesicle size. Tryptophan fluorescence experiments show that lipase binds to all those types of vesicles with similar affinity, but it adopts different forms that can be correlated with the enzyme catalytic activity. The spectral change on binding to anionic SUV corresponds to the catalytically active, or "open" form of the enzyme, and it is not modified in the presence of substrate partitioned in the vesicles, as demonstrated with inactive mutants. This indicates that the displacement of the lid characteristic of lipase interfacial activation is induced by the anionic phospholipid interface without blocking the accessibility of the active site to the substrate. Experiments with a mutant containing only Trp89 in the lid show that most of the spectral changes on binding to POPG-SUVs take place in the lid region that covers the active site; an increase in Trp anisotropy indicates that the lid becomes less flexible in the active form, and quenching experiments show that it is significantly buried from the aqueous phase. On the other hand, results with a mutant where Trp89 is changed to Leu show that the environment of the structural tryptophans in positions 117, 221, and 260 is somehow altered on binding, although their mobility and solvent accessibility remains the same as in the inactive form in solution. The form of TlL bound to POPC-SUV or -LUV vesicles as well as to LUV vesicles of POPG has the same spectral signatures and corresponds to an inactive or "closed" form of the enzyme. In these interfaces, the lid is highly flexible, and Trp89 remains accessible to solvent. Resonance energy transfer experiments show that the orientation of TlL in the interface is different in the active and inactive forms. A model of interaction consistent with these data and the available X-ray structures is proposed. This is a unique system where the composition and physical properties of the lipid interface control the enzyme activity.