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.     

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.     

Surface charge engineering of Thermomyces lanuginosus lipase improves enzymatic activity and biodiesel synthesis

Objectives

This study was aimed at engineering charged residues on the surface of Thermomyces lanuginosus lipase (TLL) to obtain TLL variant with elevated performance for industrial applications.

Results

Site-directed mutagenesis of eight charged amino acids on the TLL surface were conducted and substitutions on the negatively charged residues D111, D158, D165, and E239 were identified with elevated specific activities and biodiesel yields. Synergistic effect was not discovered in the double mutants, D111E/D165E and D165E/E239R, when compared with the corresponding single mutants. One TLL mutant, D165E, was identified with increased specific activity (456.60 U/mg), catalytic efficiency (k_cat/K_m: 44.14 s^?1 mM^?1), the highest biodiesel conversion yield (93.56%), and comparable thermostability with that of the TLL.

Conclusions

Our study highlighted the importance of surface charge engineering in improving TLL activity and biodiesel production, and the resulting TLL mutant, D165E, is a promising candidate for biodiesel industry.

     

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.     

Penicillium expansum lipase-catalyzed production of biodiesel in ionic liquids

Penicillium expansum lipase (PEL) was used to catalyze biodiesel production from corn oil in [BMIm][PF₆]¹ (an ionic liquid, IL) and tert-butanol. Both systems were optimized in terms of MeOH/oil molar ratio, reaction temperature, enzyme loading, solvent volume, and water content. The high conversion obtained in the IL (86%) as compared to that in tert-butanol (52%) demonstrates that the IL is a superior solvent for PEL-catalyzed biodiesel production. Poor yields were obtained in a series of hydrophilic ILs. Addition of salt hydrates affected biodiesel production predominantly through the specific ion (Hofmeister) effect. The impact of methanol on both activity and stability of PEL in the IL and in hexane was investigated, in comparison to the results obtained by two commonly used lipases, Novozym 435 and Lipozyme TLIM. The results substantiate that while different lipases show different resistance to methanol in different reaction systems, PEL is tolerant to methanol in both systems.     

Functional topology of a surface loop shielding the catalytic center in lipoprotein lipase.

Lipoprotein lipase (LPL), hepatic lipase, and pancreatic lipase show high sequence homology to one another. The crystal structure of pancreatic lipase suggests that it contains a trypsin-like Asp-His-Ser catalytic triad at the active center, which is shielded by a disulfide bridge-bounded surface loop that must be repositioned before the substrate can gain access to the catalytic residues. By sequence alignment, the homologous catalytic triad in LPL corresponds to Asp156-His241-Ser132, absolutely conserved residues, and the homologous surface loop to residues 217-238, a poorly conserved region. To verify these assignments, we expressed in vitro wild-type LPL and mutant LPLs having single amino acid mutations involving residue Asp156 (to His, Ser, Asn, Ala, Glu, or Gly), His241 (to Asn, Ala, Arg, Gln, or Trp), or Ser132 (to Gly, Ala, Thu, or Asp) individually. All 15 mutant LPLs were totally devoid of enzyme activity, while wild-type LPL and other mutant LPLs containing substitutions in other positions were fully active. We further replaced the 22-residue LPL loop which shields the catalytic center either partially (replacing 6 of 22 residues) or completely with the corresponding hepatic lipase loop. The partial loop-replacement chimeric LPL was found to be fully active, and the complete loop-replacement mutant had approximately 60% activity, although the primary sequence of the hepatic lipase loop is quite different. In contrast, replacement with the pancreatic lipase loop completely inactivated the enzyme. Our results are consistent with Asp156-His241-Ser132 being the catalytic triad in lipoprotein lipase.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Immobilization–stabilization of the lipase from Thermomyces lanuginosus: Critical role of chemical amination

This paper describes the immobilization and stabilization of the lipase from Thermomyces lanuginosus (TLL) on glyoxyl agarose. Enzymes attach to this support only by the reaction between several aldehyde groups of the support and several Lys residues on the external surface of the enzyme molecules at pH 10. However, this standard immobilization procedure is unsuitable for TLL lipase due to the low stability of TLL at pH 10 and its low content on Lys groups that makes that the immobilization process was quite slow. The chemical amination of TLL, after reversible immobilization on hydrophobic supports, has been shown to be a simple and efficient way to improve the multipoint covalent attachment of this enzyme. The modification enriches the enzyme surface in primary amino groups with low pKb, thus allowing the immobilization of the enzyme at lower pH values. The aminated enzyme was rapidly immobilized at pH 9 and 10, with activities recovery of approximately 70%. The immobilization of the chemically modified enzyme improved its stability by 5-fold when compared to the non-modified enzyme during thermal inactivation and by hundreds of times when the enzyme was inactivated in the presence of organic solvents, being both glyoxyl preparations more stable than the enzyme immobilized on bromocyanogen.     

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.     

Conformational plasticity of the calcium‐binding pocket in the Burkholderia glumae lipase: Remodeling induced by mutation of calcium coordinating residues

Most bacterial lipases bind one or more Ca²⁺ atoms at different locations and are a suitable case of study for investigating structural effects related to calcium binding, depletion, or mutation of calcium‐binding sites. Generally Ca²⁺ in microbial lipases can play a crucial role in the stabilization of the whole three‐dimensional structure by mediating long‐range effects. It has been recently demonstrated that calcium binding influences thermal stability of Burkholderia glumae lipase (BGL) through the restriction of conformational plasticity of specific regions. Moreover, calcium depletion results in a highly cooperative protein unfolding, eliciting protein aggregation. To further shed light on molecular mechanisms and structural features connected to calcium binding in microbial lipases, we present a molecular dynamics investigation, based on multiple‐replica approach at different temperatures, of BGL mutants targeting the calcium‐binding site. It turns out that additional acidic residues, which are conserved in other microbial lipases, help in overcoming effects induced by mutation of D241 Ca²⁺‐coordinating residue, upon rearrangements induced in the calcium binding site. © 2010 Wiley Periodicals, Inc. Biopolymers 95: 117–126, 2011.     

Generation of lipase-containing static emulsions in silicone spheres for synthesis in organic media

Because of the broad versatility of lipases as biocatalysts, interest has for some years been focused on the improvement of the economy of processes using these enzymes, especially by appropriate immobilisation. In this study, a method was developed to emulsify aqueous solutions of lipase A of Candida antarctica (CALA) and lipase of Thermomyces lanuginosa (TLL) in silicone elastomers yielding elastic beads. The persistent water-organic interface created by this static emulsion enabled an improved performance of the immobilised lipases due to the well known fact that from a kinetic point of view these enzymes show a higher efficiency in biphasic than in monophasic systems. The entrapped lipases catalysed the esterification of octanol and caprylic acid in hexane with an activity that, related to the free enzyme, was enhanced about 31-fold for CALA and 250-fold for TLL. Comparison to the activity of the same enzymes in sol–gels revealed that for CALA immobilisation in static emulsion was the only method yielding active biocatalysts, whereas activation of TLL was in the same range in static emulsion and sol–gels. However, apparent activity of TLL in static emulsion was considerably higher than in sol–gels due to the feasible high enzyme loading. The results indicate that immobilising lipases as static emulsion is a technique suitable for biotechnological application. Moreover, a transfer to enzymes of other classes seems possible.     

Towards a commercially potential process: Enzymatic recovery of phytosterols from plant oil deodoriser distillates mixture

In order to examine the industrial potential to indirectly isolate phytosterols from deodoriser distillates (DODs), enzymatic transesterification of an industrial rapeseed and soybean oil DOD mixture with bioethanol was investigated using commercial lipases and a few newly immobilised preparations of lipases. The lipases from different sources and differing preparation forms were evaluated, in terms of thermostability, enzyme efficiency, and toleration of ethanol. Lipozyme 435 and Lipozyme NS-40044 TLL were found to be most effective biocatalysts in catalysing ethanolysis of glycerides and steryl esters from DODs. The optimum conditions are 10% enzyme load (wt% of DODs), ethanol/DODs of 3.0:1.0 (mol/mol), water content 0.125% (based on the weight of total mixture), and reaction at 30 °C for 5 h. The results demonstrated that >95% sterols can be recovered as free form (>85% sterol esters were liberated as free sterols within 4 h). With this process, the system was simplified as fatty acid ethyl esters and free sterol as major components, where free sterols can be recovered via solvent extraction or molecular distillation. Furthermore, a reuse study of enzyme in consecutive batch reactions demonstrated an excellent operation stability and reusability of Lipozyme 435 and Lipozyme NS-40044 TLL with the developed process. This work indicated that the industrially refined waste DODs can be directly subjected to an enzymatic process for high efficacy recovery of phytosterol without any pre-process, driven by robust lipase preparations.     

Analysis of Comparative Sequence and Genomic Data to Verify Phylogenetic Relationship and Explore a New Subfamily of Bacterial Lipases

Thermostable and organic solvent-tolerant enzymes have significant potential in a wide range of synthetic reactions in industry due to their inherent stability at high temperatures and their ability to endure harsh organic solvents. In this study, a novel gene encoding a true lipase was isolated by construction of a genomic DNA library of thermophilic Aneurinibacillus thermoaerophilus strain HZ into Escherichia coli plasmid vector. Sequence analysis revealed that HZ lipase had 62% identity to putative lipase from Bacillus pseudomycoides. The closely characterized lipases to the HZ lipase gene are from thermostable Bacillus and Geobacillus lipases belonging to the subfamily I.5 with ≤ 57% identity. The amino acid sequence analysis of HZ lipase determined a conserved pentapeptide containing the active serine, GHSMG and a Ca²⁺-binding motif, GCYGSD in the enzyme. Protein structure modeling showed that HZ lipase consisted of an α/β hydrolase fold and a lid domain. Protein sequence alignment, conserved regions analysis, clustal distance matrix and amino acid composition illustrated differences between HZ lipase and other thermostable lipases. Phylogenetic analysis revealed that this lipase represented a new subfamily of family I of bacterial true lipases, classified as family I.9. The HZ lipase was expressed under promoter P_lac using IPTG and was characterized. The recombinant enzyme showed optimal activity at 65°C and retained ≥ 97% activity after incubation at 50°C for 1h. The HZ lipase was stable in various polar and non-polar organic solvents.     

A comparative study on two fungal lipases from Thermomyces lanuginosus and Yarrowia lipolytica shows the combined effects of detergents and pH on lipase adsorption and activity

The effects of various detergents and pH on the interfacial binding and activity of two fungal lipases from Yarrowia lipolytica (YLLIP2) and Thermomyces lanuginosus (TLL) were investigated using trioctanoin emulsions as well as monomolecular films spread at the air-water interface. Contrary to TLL, YLLIP2 was found to be more sensitive than TLL to interfacial denaturation but it was protected by detergent monomers and lowering the temperature. At pH 7.0, both the interfacial binding and the activities on trioctanoin of YLLIP2 and TLL were inhibited by sodium taurodeoxycholate (NaTDC). At pH 6.0, however, YLLIP2 remained active on trioctanoin in the presence of NaTDC, whereas TLL did not. YLLIP2 activity on trioctanoin was associated with strong interfacial binding of the enzyme to trioctanoin emulsion, whereas TLL was mostly detected in the water phase. The combined effects of bile salts and pH on lipase activity were therefore enzyme-dependent. YLLIP2 binds more strongly than TLL at oil-water interfaces at low pH when detergents are present. These findings are particularly important for lipase applications, in particular for enzyme replacement therapy in patients with pancreatic enzyme insufficiency since high detergent concentrations and highly variable pH values can be encountered in the GI tract.     

Biochemical and Structural Characterization of Two Site-Directed Mutants of <Emphasis Type="Italic">Staphylococcus xylosus</Emphasis> Lipase

Staphylococcus xylosus AF208229 lipase was expressed in E. coli containing an histidine-tag (WT-Val). In the present work, in order to check the importance of the residue 309 in the specific activity, the amino acid side chain residue valine 309 was substituted by aspartate or lysine through site-directed mutagenesis. Both mutant lipases (MUT-Lys and MUT-Asp) were expressed in E. coli and the recombinant histidine-tagged lipases were purified by immobilized metal ion affinity chromatography. The enzyme activity was determined using p-nitrophenyl butyrate as substrate and secondary structure content was evaluated by circular dichroism. MUT-Lys and MUT-Asp presented significant increase of lipase activity (P < 0.05) in comparison to WT-Val, although highest activities for the three enzymes were observed at the same pH and temperature (pH 9.0 and 42°C). The wild type and mutant lipases presented high thermal stability, after 30 min of incubation at 80°C all enzymes retained their initial activities.     

Lipase from Thermomyces lanuginosus: Uses and prospects as an industrial biocatalyst

The lipase from Thermomyces laguginosus (formerly Humicola laguginosa) (TLL) is a basophilic and noticeably thermostable enzyme, commercially available in both soluble and immobilized form. Although initially oriented toward the food industry, the enzyme has found applications in many different industrial areas, from biodiesel production to fine chemicals (mainly in enantio and regioselective or specific processes). This review intends to show some of the most relevant aspects of the use of this interesting enzyme. After checking the enzyme features, some of the most efficient methods of TLL immobilization will be commented. Finally, the main uses of the enzyme will be revised, with special emphasis in the modification of fats and oils, production of biodiesel, resolution of racemic mixtures, enantioselective hydrolysis of prochiral esters and regioselective process involving sugar preparations. In many instances, TLL has been compared to other lipases, the advantages or disadvantages of the enzyme will be discussed.     

Scanning electron microscopic studies of lipase-catalysed esterification catalysis for the synthesis of stearoyl lactate and p-cresyl laurate

The state of three lipases, two from Rhizomucor miehei and one from porcine pancreas, employed in the esterification reactions leading to the preparation of food additive esters were investigated by scanning electron microscopy (SEM). The lipases employed in the synthesis of stearoyl lactic acid and p-cresyl laurate in 10 ml solvent at 40–60 °C in shake-flask experiments and 150 ml in non-polar solvents at 50–60 °C in bench-scale level experiments were compared. All three lipases, which were subjected to high temperatures and non-polar solvents for a prolonged period of incubation of 72–120 h, showed decrease in the compactness when compared to unused lipase. The presence of buffer preserved the activity and compactness and the absence of the same reduced the amount of enzyme per unit area on the support. R. miehei lipase samples subjected to reaction in presence of 0.0004 ml of 0.1 M buffer/mg enzyme preparation at different pH values (4.0–9.0) showed a decrease in compactness of the enzyme on the surface which correlated to an increase in esterification activity. An increase in volume of buffer (0.0002–0.003 ml/mg enzyme preparation) in the reaction mixture at pH 7.0 showed a decrease in compactness and also a reduction in activity. The studies indicate that a compromise between pH and volume of buffer can lead to variation in the extent of adsorption, distribution and activity, enabling the achievement of maximum conversions in the esterification reactions.     

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

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

A novel thermostable lipase from Basidiomycete <Emphasis Type="Italic">Bjerkandera adusta </Emphasis>R59: characterisation and esterification studies

Microbial lipases are widely diversified in their enzymatic properties and substrate specificities, which make them very attractive for industrial application. Partially purified lipase from Bjerkandera adusta R59 was immobilized on controlled porous glass (CPG) and its properties were compared with those of the free enzyme. The free and immobilized lipases showed optimal activities at 45 and 50°C, respectively. Both enzyme forms were highly thermostable up to 60°C. The enzymes were stable at pH from 6.0 to 9.0 and their optimal pH for activity was 7.0. The free lipase was more thermostable in n-hexane than in aqueous environment. Both lipase preparations had good stabilities in non-polar solvents and were capable of hydrolysing a variety of synthetic and natural fats. Non-immobilized lipase activity was inhibited by disulphide bond reagents, serine and thiol inhibitors, while EDTA and eserine had no effect on enzyme activity. All anionic detergents tested in experiments inhibited lipase activity. The free lipase showed good stability in the presence of commercial detergents at laundry pH and temperatures. Applications of free and immobilized lipases for esterification were also presented.     

Combined docking and molecular dynamics simulations to enlighten the capacity of Pseudomonas cepacia and Candida antarctica lipases to catalyze quercetin acetylation

A combined docking and molecular dynamics protocol was applied to investigate quercetin binding modes within the catalytic cavity of Candida antarctica lipase B (CALB) and Pseudomonas cepacia lipase (PCL), aiming to explain the difference of specificity of these enzymes in acetylation reaction. For both lipases, docking of quercetin yielded two families of conformers with either the quercetin A or B-ring pointing towards the catalytic residues. Molecular dynamics (MD) calculations were subsequently performed on several complexes of each family. MD trajectories were analyzed focusing on the orientation of the acyl donor bound to the catalytic serine towards the oxyanion hole residues and the proximity of quercetin hydroxyl groups to the catalytic residues. Results showed that with CALB, the acetate was not correctly positioned within the oxyanion hole whatever the orientation of quercetin, suggesting that no product could be obtained. With PCL, the acetate remained within the oxyanion hole during all MD trajectories. Depending on quercetin orientation, either the 7-OH group or the 3, 5, 3′, 4′-OH groups came alternatively near the catalytic residues, suggesting that all of them could be acylated. The capacity of models to explain the regioselectivity of the reaction was discussed. Key residues and interactions involved in quercetin binding modes were identified and related to the reaction feasibility.