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.     

Structure,mechanism, and enantioselectivity shifting of lipase LipK107 with a simple way

Because of the complex mechanisms of enzymatic reactions, no precise and simple method of understanding and controlling the chiral selectivity of enzymes has been developed. However, structure-based rational design is a powerful approach to engineering enzymes with desired catalytic activities. In this work, a simple, structure-based, large-scale in silico design and virtual screening strategy was developed and successfully applied to enzyme engineering. We first performed protein crystallization and X-ray diffraction to determine the structure of lipase LipK107, which is a novel family I.1 lipase displaying activity for both R and S isomers in chiral resolution reactions. The catalytic mechanism of family I.1, which includes LipK107, was ascertained first through comparisons of the sequences and structures of lipases from other families. The binding states of LipK107, including the energy and the conformation of complexes with the R and S enantiomers, have been evaluated by careful biocomputation to figure out the reason for the higher S selectivity. Based on this study, a simple strategy for manipulating the chiral selectivity by modulating a crucial distance in the enzyme–substrate complex and judging virtual mutations in silico is recommended. Then, a novel electrostatic interaction analysis protocol was used to design LipK107 mutants to validate our strategy. Both positive and negative mutations determined using this theoretical protocol have been implemented in wet experiments and were proved to produce the desired enantioselectivity, showing a 176% increase or 50% decrease in enantioselectivity as desired. Because of its accuracy and versatility, the strategy is promising for practical applications.     

Membrane phospholipid bilayer as a determinant of monoacylglycerol lipase kinetic profile and conformational repertoire

The membrane‐associated serine hydrolase, monoacylglycerol lipase (MGL), is a well‐recognized therapeutic target that regulates endocannabinoid signaling. Crystallographic studies, while providing structural information about static MGL states, offer no direct experimental insight into the impact of MGL's membrane association upon its structure–function landscape. We report application of phospholipid bilayer nanodiscs as biomembrane models with which to evaluate the effect of a membrane system on the catalytic properties and conformational dynamics of human MGL (hMGL). Anionic and charge‐neutral phospholipid bilayer nanodiscs enhanced hMGL's kinetic properties [apparent maximum velocity (V_max) and substrate affinity (K_m)]. Hydrogen exchange mass spectrometry (HX MS) was used as a conformational analysis method to profile experimentally the extent of hMGL–nanodisc interaction and its impact upon hMGL structure. We provide evidence that significant regions of hMGL lid‐domain helix α4 and neighboring helix α6 interact with the nanodisc phospholipid bilayer, anchoring hMGL in a more open conformation to facilitate ligand access to the enzyme's substrate‐binding channel. Covalent modification of membrane‐associated hMGL by the irreversible carbamate inhibitor, AM6580, shielded the active site region, but did not increase solvent exposure of the lid domain, suggesting that the inactive, carbamylated enzyme remains intact and membrane associated. Molecular dynamics simulations generated conformational models congruent with the open, membrane‐associated topology of active and inhibited, covalently‐modified hMGL. Our data indicate that hMGL interaction with a phospholipid membrane bilayer induces regional changes in the enzyme's conformation that favor its recruiting lipophilic substrate/inhibitor from membrane stores to the active site via the lid, resulting in enhanced hMGL catalytic activity and substrate affinity.     

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.     

A method to rationally increase protein stability based on the charge–charge interaction,with application to lipase LipK107

We report a suite of enzyme redesign protocol based on the surface charge–charge interaction calculation, which is potentially applied to improve the stability of an enzyme without compromising its catalytic activity. Together with the experimental validation, we have released a suite of enzyme redesign algorithm Enzyme Thermal Stability System, written based on our model, for open access to meet the needs in wet labs. Lipk107, a lipase of a versatile industrial use, was chosen to test our software. Our calculation determined that four residues, D113, D149, D213, and D253, located on the surface of LipK107 were critical to the stability of the enzyme. The model was validated with mutagenesis at these four residues followed by stability and activity tests. LipK107 mutants D113A and D149K were more resistant to thermal inactivation with ～10°C higher half‐inactivation temperature than wild‐type LipK107. Moreover, mutant D149K exhibited significant retention in residual activity under constant heat, showing a 14‐fold increase in the half‐inactivation time at 50°C. Activity tests showed that these mutants retained the equal or higher specific activity, among which noteworthy was the mutant D253A with as much as 20% higher activity. We suggest that our protocol could be used as a general guideline to redesign protein enzymes with increased stabilities and enhanced activities.     

Engineering of Yarrowia lipolytica lipase Lip8p by circular permutation to alter substrate and temperature characteristics

Applications of lipases are mainly based on their catalytic efficiency and substrate specificity. In this study, circular permutation (CP), an unconventional protein engineering technique, was employed to acquire active mutants of Yarrowia lipolytica lipase Lip8p. A total of 21 mutant lipases exhibited significant shifts in substrate specificity. Cp128, the most active enzyme mutant, showed higher catalytic activity (14.5-fold) and higher affinity (4.6-fold) (decreased K _m) to p-nitrophenyl-myristate (pNP-C₁₄) than wild type (WT). Based on the three-dimensional (3D) structure model of the Lip8p, we found that most of the functional mutation occurred in the surface-exposed loop region in close proximity to the lid domain (S112–F122), which implies the steric effect of the lid on lipase activity and substrate specificity. The temperature properties of Cp128 were also investigated. In contrast to the optimal temperature of 45 °C for the WT enzyme, Cp128 exhibited the maximal activity at 37 °C. But it is noteworthy that there is no change in thermostability.     

Open and closed states of Mrlip1 DAG lipase revealed by molecular dynamics simulation

ABSTRACT

The lid and flap domains control the catalytic activity of lipase through the opening and closing motion. However, this gating mechanism of diacylglycerol (DAG) lipase is poorly understood due to the lack of 3D structures in open conformations. In this study, the opening and closing states of Mrlip1 DAG lipase are revealed by the homology modelling and molecular dynamic simulations. It was found that the active residues (Ser₁₇₁, His₂₈₁ and Asp₂₂₈) in the catalytic pocket of Mrlip1 DAG lipase are covered by the lid domain in the closed conformation, and exposed to the solvent in the open conformation. The role of residues Phe₂₇₈ and Gln₂₈₂ in the flap domain, as well as that of Thr₁₀₁ and Thr₁₀₇ in the lid domains are also identified in gating mechanism. The site-directed mutagenesis have been carried out to illustrate the putative alterations of enzyme specificity. Our results suggest that the substrate specificity is achieved by these two key residues Phe₂₇₈ and Gln₂₈₂, and the irreversible conversion from DAG to TAG (Triacylglycerol) lipase are enabled by the two-point mutations.     

Open–closed conformational change revealed by the crystal structures of 3-keto-l-gulonate 6-phosphate decarboxylase from Streptococcus mutans

The 3-keto-l-gulonate 6-phosphate decarboxylase (KGPDC) catalyses the decarboxylation of 3-keto-l-gulonate 6-phosphate to l-xylulose in the presence of magnesium ions. The enzyme is involved in l-ascorbate metabolism and plays an essential role in the pathway of glucuronate interconversion. Crystal structures of Streptococcus mutans KGPDC were determined in the absence and presence of the product analog d-ribulose 5-phosphate. We have observed an 8 Å αB-helix movement and other structural rearrangements around the active site between the apo-structures and product analog bound structure. These drastic conformational changes upon ligand binding are the first observation of this kind for the KGPDC family. The flexibilities of both the α-helix lid and the side chains of Arg144 and Arg197 are associated with substrate binding and product releasing. The open–closed conformational changes of the active site, through the movements of the α-helix lid and the arginine residues are important for substrate binding and catalysis.     

Purification and Characterization of a Novel (<Emphasis Type="Italic">R</Emphasis>)-1-Phenylethanol Dehydrogenase from <Emphasis Type="Italic">Lysinibacillus</Emphasis> sp. NUST506

A novel (R)-1-phenylethanol dehydrogenase was successfully purified from Lysinibacillus sp. NUST506 by preparative polyacrylamide gel electrophoresis. The enzyme is a NAD+-dependent oxidoreductase. The molecular weight of the (R)-1-phenylethanol dehydrogenase measured by SDS-PAGE was about 28 kDa. Furthermore, the optimal reaction conditions for the oxidative reaction were 70°C and pH 9.5 and for the reductive reaction were 65°C and pH 6.5. Under the optimal conditions, the K_M and k_cat values with (R)-1-phenylethanol as a substrate were found to be 0.78 mM and 123 s^–1 and with acetophenone they were 0.56 mM and 125 s^–1, respectively. The (R)-1-phenylethanol dehydrogenase became more stable at pH 9.5 in comparison with pH 5.0 and high stability was noticed at 4 and 37°C. Properties of the enzyme place it as a promising candidate for industrial applications.     

Impact of the configuration of a chiral,activating carrier on the enantioselectivity of entrapped lipase from Candida rugosa in cyclohexane

Lipase from Candida rugosa was loaded into an amphiphilic polymer co-network (APCN) composed of the chiral poly[(R)-N-(1-hydroxybutan-2-yl) acrylamide] [P-(R)-HBA] and P-(S)-HBA, respectively, linked by poly(dimethylsiloxane). The nanophase-separated amphiphilic morphology affords a 38,000-fold activation of the enzyme in the esterification of 1-phenylethanol with vinyl acetate. Further, the enantioselectivity of the entrapped lipase was influenced by the configuration of the chiral, hydrophilic polymer matrix. While the APCN with the (S)-configuration of the APCN affords 5.4 faster conversion of the (R)-phenylethanol compared to the respective (S)-enantiomer, the (R)-APCN allows an only a 2.8 faster conversion of the (R)-enantiomer of the alcohol. Permeation-experiments reveal that the enantioselectivity of the reaction is at least partially caused by specific interactions between the substrates and the APCN.     

Lid closure dynamics of porcine pancreatic lipase in aqueous solution

BackgroundPancreatic lipases hydrolyze fatty acids in dietary pathway. The activity of porcine pancreatic lipase (PPL) is controlled by lid domain along with a coenzyme, colipase. The active open-state conformation of the protein could be induced by detergents or bile salts which would be further stabilized by binding of colipase. In the absence of these interactions, the lid preferably attains a closed conformation in water.MethodsMolecular dynamic simulation was used to monitor the lid movement of PPL in open and closed conformations in water. Free energy surface was constructed from the simulation. Energy barriers and major structural changes during lid opening were evaluated.ResultsThe lid closure of PPL in water from its open conformation might be initiated by columbic interactions which initially move the lid away from domain 1. This is followed by major dihedral changes on the lid residues which alter the trajectory of motion. The lid then swirls back towards domain 1 to attain closed conformation. This is accompanied with conformational changes around β5- and β9-loops as well. However, PPL in closed conformation shows only the domain movements and the lid remains in its closed conformation.ConclusionsPPL in closed conformation is stable in water and the open conformation is driven towards closed state. The lid follows a swirling trajectory during the closure.General significanceConformational state of the lid regulates the activity and substrate specificity of PPL. Hence, it is essential to understand the lid dynamics and the role of specific amino acid residues involved.     

Immobilization of Candida rugosa lipase on electrospun cellulose nanofiber membrane

A biocatalyst with high activity retention of lipase was fabricated by the covalent immobilization of Candida rugosa lipase on a cellulose nanofiber membrane. This nanofiber membrane was composed of nonwoven fibers with 200 nm nominal fiber diameter. It was prepared by electrospinning of cellulose acetate (CA) and then modified with alkaline hydrolysis to convert the nanofiber surface into regenerated cellulose (RC). The nanofiber membrane was further oxidized by NaIO₄. Aldehyde groups were simultaneously generated on the nanofiber surface for coupling with lipase. Response surface methodology (RSM) was applied to model and optimize the modification conditions, namely NaIO₄ content (2–10 mg/mL), reaction time (2–10 h), reaction temperature (25–35 °C) and reaction pH (5.5–6.5). Well-correlating models were established for the residual activity of the immobilized enzyme (R² = 0.9228 and 0.8950). We found an enzymatic activity of 29.6 U/g of the biocatalyst was obtained with optimum operational conditions. The immobilized lipase exhibited significantly higher thermal stability and durability than equivalent free enzyme.     

Conformation studies on Burkholderia cenocepacia lipase via resolution of racemic 1-phenylethanol in non-aqueous medium and its process optimization

In this study, the effect of various organic solvents on enzyme activity and substrate enantiomeric excess (ee_s) of the lipase from Burkholderia cenocepacia (BCL) was investigated in the enantioselective transesterification of 1-phenylethanol. Secondary structure analysis by Fourier transform-infrared spectroscopy (FT-IR) showed that the variations in secondary structure element content (α-helix, β-sheet, β-turn and random coil) were probably responsible for the changes in enzyme activity and ee_s. Furthermore, the change in fluorescence intensity indicated, to some extent, the alteration in tertiary structure, which may also explain why organic solvents affect enzyme activity and ee_s. Moreover, response surface methodology (RSM) was employed to optimize the reaction parameters. The optimized reaction conditions were: substrate molar ratio 4.7:1; reaction time 18.6 h, and reaction temperature 53.4 °C. Under the optimal reaction conditions, the ee_s and ee_p were respectively 99.22% and 98.74%, and the corresponding enzyme activity was 1392.2 U/min/g protein. Compared with other lipases, BCL exhibited better catalytic efficiency and has significant potential in industrial applications.     

Production of chiral alcohols by enantioselective reduction with NADH-dependent phenylacetaldehyde reductase from Corynebacterium strain,ST-10

Phenylacetaldehyde reductase (PAR) (systematic name, 2-phenylethanol: NAD⁺ oxidoreductase) isolated from styrene-assimilating Corynebacterium strain ST-10 was used to produce chiral alcohols. This enzyme with a broad substrate range reduced various prochiral 2-alkanones and aromatic ketones to yield optically active secondary alcohols with an enantiomeric purity of 87–100% enantiomeric excess (e.e.). The stereochemistry of PAR revealed that the pro-R hydrogen of NADH was transferred to carbonyl moiety of acetophenone derivatives or alkanones through its re face. The combination with a NADH-regenerating system using formate dehydrogenase and formate was able to practically produce optically pure alcohols.     

Studies of the mechanism of action and regulation of cAMP-dependent protein kinase

Magnetic resonance and kinetic studies of the catalytic subunit of a Type II cAMP-dependent protein kinase from bovine heart have established the active complex to be an enzyme-ATP-metal bridge. The metal ion is β,γ coordinated with Δ chirality at the β-phosphorous atom. The binding of a second metal ion at the active site which bridges the enzyme to the three phosphoryl groups of ATP, partially inhibits the reaction. Binding of the metal-ATP substrate to the enzyme occurs in a diffusion-controlled reaction followed by a 40 ° change in the glycosidic torsional angle. This conformational change results from strong interaction of the nucleotide base with the enzyme. NMR studies of four ATP-utilizing enzymes show a correlation between such conformational changes and high nucleotide base specificity. Heptapeptide substrates and substrate analogs bind to the active site of the catalytic subunit at a rate significantly lower than collision frequency indicating conformational selection by the enzyme or a subsequent slow conformational change. NMR studies of the conformation of the enzyme-bound peptide substrates have ruled out α-helical and β-pleated sheet structures. The results of kinetic studies of peptide substrates in which the amino acid sequence was systematically varied were used to rule out the obligatory requirement for all possible β-turn conformations within the heptapeptide although an enzymatic preference for a β_2–5 or β_3–6 turn could not be excluded. Hence if protein kinase has an absolute requirement for a specific secondary structure, then this structure must be a coil. In the enzyme-substrate complex the distance along the reaction coordinate between the γ-P of ATP and the serine oxygen of the peptide substrate (5.3 ± 0.7 Å) allows room for a metaphosphate intermediate. This finding together with kinetic observations as well as the location of the inhibitory metal suggest a dissociative mechanism for protein kinase, although a mechanism with some associative character remains possible. Regulation of protein kinase is accomplished by competition between the regulatory subunit and peptide or protein substrates at the active site of the catalytic subunit. Thus, the regulatory subunit is found by NMR to block the binding of the peptide substrate to the active site of protein kinase but allows the binding of the nucleotide substrate and divalent cations. The dissociation constant of the regulatory subunit from the active site (10^?10m) is increased ~10-fold by phosphorylation and ~10⁴-fold by the binding of cAMP, to a value (10^?5m) which exceeds the intracellular concentration of the R₂C₂ holoenzyme complex (10^?6m). The resulting dissociation of the holoenzyme releases the catalytic subunit, permitting the active site binding of peptide or protein substrates.     

The crystal structure analysis of group B Streptococcus sortase C1: a model for the "lid" movement upon substrate binding

A unique feature of the class-C-type sortases, enzymes essential for Gram-positive pilus biogenesis, is the presence of a flexible “lid” anchored in the active site. However, the mechanistic details of the “lid” displacement, suggested to be a critical prelude for enzyme catalysis, are not yet known. This is partly due to the absence of enzyme-substrate and enzyme-inhibitor complex crystal structures. We have recently described the crystal structures of the Streptococcus agalactiae SAG2603 V/R sortase SrtC1 in two space groups (type II and type III) and that of its “lid” mutant and proposed a role of the “lid” as a protector of the active-site hydrophobic environment. Here, we report the crystal structures of SAG2603 V/R sortase C1 in a different space group (type I) and that of its complex with a small-molecule cysteine protease inhibitor. We observe that the catalytic Cys residue is covalently linked to the small-molecule inhibitor without lid displacement. However, the type I structure provides a view of the sortase SrtC1 lid displacement while having structural elements similar to a substrate sorting motif suitably positioned in the active site. We propose that these major conformational changes seen in the presence of a substrate mimic in the active site may represent universal features of class C sortase substrate recognition and enzyme activation.     

Lipase and its modulator from Pseudomonas sp. strain KFCC 10818: proline-to-glutamine substitution at position 112 induces formation of enzymatically active lipase in the absence of the modulator

A lipase gene, lipK, and a lipase modulator gene, limK, of Pseudomonas sp. strain KFCC 10818 have been cloned, sequenced, and expressed in Escherichia coli. The limK gene is located immediately downstream of the lipK gene. Enzymatically active lipase was produced only in the presence of the limK gene. The effect of the lipase modulator LimK on the expression of active lipase was similar to those of the Pseudomonas subfamily I.1 and I.2 lipase-specific foldases (Lifs). The deduced amino acid sequence of LimK shares low homology (17 to 19%) with the known Pseudomonas Lifs, suggesting that Pseudomonas sp. strain KFCC 10818 is only distantly related to the subfamily I.1 and I.2 Pseudomonas species. Surprisingly, a lipase variant that does not require LimK for its correct folding was isolated in the study to investigate the functional interaction between LipK and LimK. When expressed in the absence of LimK, the P112Q variant of LipK formed an active enzyme and displayed 63% of the activity of wild-type LipK expressed in the presence of LimK. These results suggest that the Pro(112) residue of LipK is involved in a key step of lipase folding. We expect that the novel finding of this study may contribute to future research on efficient expression or refolding of industrially important lipases and on the mechanism of lipase folding.     

Closed conformation of the active site loop of rabbit muscle triosephosphate isomerase in the absence of substrate: evidence of conformational heterogeneity

The active site loop of triosephosphate isomerase (TIM) exhibits a hinged-lid motion, alternating between the two well defined "open" and "closed" conformations. Until now the closed conformation had only been observed in protein complexes with substrate analogues. Here, we present the first rabbit muscle apo TIM structure, refined to 1.5A resolution, in which the active site loop is either in the open or in the closed conformation in different subunits of the enzyme. In the closed conformation described here, the lid loop residues participate in stabilizing hydrogen bonds characteristic of holo TIM structures, whereas chemical interactions observed in the open loop conformation are similar to those found in the apo structures of TIM. In the closed conformation, a number of water molecules are observed at the projected ligand atom positions that are hydrogen bonded to the active site residues. Additives used during crystallization (DMSO and Tris molecules and magnesium atoms) were modeled in the electron density maps. However, no specific binding of these molecules is observed at, or close to, the active site and the lid loop. To further investigate this unusual closed conformation of the apo enzyme, two more rabbit muscle TIM structures, one in the same and another in a different crystal form, were determined. These structures present the open lid conformation only, indicating that the closed conformation cannot be explained by crystal contact effects. To rationalize why the active site loop is closed in the absence of ligand in one of the subunits, extensive comparison with previously solved TIM structures was carried out, supported by the bulk of available experimental information about enzyme kinetics and reaction mechanism of TIM. The observation of both open and closed lid conformations in TIM crystals might be related to a persistent conformational heterogeneity of this protein in solution.     

Lid dynamics of porcine pancreatic lipase in non-aqueous solvents

BackgroundUnderstanding the dynamics of enzymes in organic solvents has wider implications on their industrial applications. Pancreatic lipases, which show activity in their lid open-state, demonstrate enhanced activity in organic solvents at higher temperatures. However, the lid dynamics of pancreatic lipases in non-aqueous environment is yet to be clearly understood.MethodsDynamics of porcine pancreatic lipase (PPL) in open and closed conformations was followed in ethanol, toluene, and octanol using molecular simulation methods. In silico double mutant D250V and E254L of PPL (PPL_mut-Cl) was created and its lid opening dynamics in water and in octanol was analyzed.ResultsPPL showed increase in solvent accessible surface area and decrease in packing density as the polarity of the surrounded solvent decreased. Breaking the interactions between D250-Y115, and D250-E254 in PPL_mut-Cl directed the lid to attain open-state conformation. Major energy barriers during the lid movement in water and in octanol were identified. Also, the trajectories of lid movement were found to be different in these solvents.ConclusionsOnly the double mutant at higher temperature showed lid opening movement suggesting the essential role of the three residues in holding the lid in closed conformation. The lid opening dynamics was faster in octanol than water suggesting that non-polar solvents favor open conformation of the lid.General significanceThis study identifies important interactions between the lid and the residues in domain 1 which possibly keeps the lid in closed conformation. Also, it explains the rearrangements of residue–residue interactions during lid opening movement in water and in octanol.