Stereoselectivity of Pseudomonas cepacia lipase toward secondary alcohols: a quantitative model

The lipase from Pseudomonas cepacia represents a widely applied catalyst for highly enantioselective resolution of chiral secondary alcohols. While its stereopreference is determined predominantly by the substrate structure, stereoselectivity depends on atomic details of interactions between substrate and lipase. Thirty secondary alcohols with published E values using P. cepacia lipase in hydrolysis or esterification reactions were selected, and models of their octanoic acid esters were docked to the open conformation of P. cepacia lipase. The two enantiomers of 27 substrates bound preferentially in either of two binding modes: the fast-reacting enantiomer in a productive mode and the slow-reacting enantiomer in a nonproductive mode. Nonproductive mode of fast-reacting enantiomers was prohibited by repulsive interactions. For the slow-reacting enantiomers in the productive binding mode, the substrate pushes the active site histidine away from its proper orientation, and the distance d(H(N epsilon) - O(alc)) between the histidine side chain and the alcohol oxygen increases, d(H(N epsilon) - O(alc)) was correlated to experimentally observed enantioselectivity: in substrates for which P. cepacia lipase has high enantioselectivity (E > 100), d(H(N epsilon) - O(alc)) is >2.2 A for slow-reacting enantiomers, thus preventing efficient catalysis of this enantiomer. In substrates of low enantioselectivity (E < 20), the distance d(H(N epsilon) - O(alc)) is less than 2.0 A, and slow- and fast-reacting enantiomers are catalyzed at similar rates. For substrates of medium enantioselectivity (20 < E < 100), d(H(N epsilon) - O(alc)) is around 2.1 A. This simple model can be applied to predict enantioselectivity of P. cepacia lipase toward a broad range of secondary alcohols.     

The Mechanisms of Action and Inhibition of Pancreatic Lipase and Acetylcholinesterase: A Comparative Modeling Study

Abstract

Pancreatic lipase and acetylcholinesterase are both serine esterases. Their X-ray structures reveal a similar overall fold, but no sequence homology can be detected. A catalytic triad like in the trypsin family of serine proteases consisting of serine, histidine and aspartate (glutamate in acetylcholinesterase) suggests mechanistic similarities. Models of the transition states of the substrate cleavage have been built and possible catalytic pathways were examined. The model that could produce a consistent pathway throughout the reactions had a transition state of the opposite handedness compared to trypsin. These models could be used to rationalise binding modes of inhibitors of both enzymes. The lipase inhibitor tetrahydrolipstatin (THL) contains a gamma-lactone which is opened by the catalytic serine; the alcohol leaving group prohibits deacylation by locking the pathway for incoming water and thus inactivates the enzyme. Carbamate inhibitors of acetylcholinesterase transfer a carbamoyl group to the serine-OH which deacylates slowly. These observations can be used as a starting point for the discovery of new classes of inhibitors.     

Burkholderia cepacia lipase is a promising biocatalyst for biofuel production

Lipases resistant to inhibition and denaturation by methanol are valuable tools for biotechnological applications, in particular for biofuel production. Microbial lipases have attracted a great deal of interest because of their stability at high concentrations of organic solvents. Burkholderia cepacia lipase (BCL) is tested here for robustness towards methanol in terms of conformational stability and catalytic activity in transesterification assays. This lipase turns out to be even more tolerant than the homologous and better characterized enzyme from Burkholderia glumae. BCL unfolding transition, as monitored by far‐UV circular dichroism (CD) and intrinsic fluorescence, displays a T_m above 60°C in the presence of 50% methanol. The protein unfolds at low pH, and the organic solvent affects the nature of the denatured state under acidic conditions. The protein performs well in transesterification assays upon prolonged incubations at high methanol concentrations. BCL is highly tolerant to methanol and displays particularly high conformational stability under conditions employed for transesterification reactions. These features depict BCL as a promising enzyme for biofuel industry.     

A comparison of lipase-catalysed ester and lactone synthesis in low-water systems: analysis of optimum water activity.

We investigated the effects of the lyophilisation medium (enzyme plus buffer salt and additives) and of water activity (a(w)) on the catalytic properties of lipase from Chromobacterium viscosum (lipase CV) in organic solvents; catalysis of ester and lactone synthesis were compared and, despite the similarities of the reactive groups involved in these reactions, some interesting differences were observed. Including 2-[N-morpholino]ethanesulfonic acid (MES) buffer in the lyophilisation medium of lipase CV increased its catalytic activity in transesterification and lactonisation, although the buffer salt requirement for maximal activity differed between the two reactions. Sorbitol, glucose, lactose, 18-crown-6 (crown ether 18-C-6), beta-cyclodextrin and bovine serum albumin were employed as alternative additives in the transesterification reaction, but were not as effective as MES buffer. Salt hydrates were used to investigate the effect of a(w) on esterification and lactonisation reactions catalysed by lipase CV. The maximum rate of hexadecanolide synthesis in toluene occurred at a(w) = 0.48. The optimum a(w) for the transesterification reaction in heptane/alcohol mixtures depended on the alcohol substrate employed (1-heptanol, 2-heptanol, or 3-methyl-3-hexanol) but not on the acyl donor (p-NP acetate or caprylate). The optimum a(w) values for both reactions were unchanged when a common solvent system (toluene/1-heptanol) was employed, indicating that the dependence of enzyme activity on a(w) is an intrinsic property of the enzyme-catalysed reaction and not a function of the solvent or other additives.     

Molecular basis for substrate selectivity of a mono- and diacylglycerol lipase from Malassezia globosa

The lipase from Malassezia globosa (SMG1) was identified to be strictly specific for mono- and diacylglycerol but not triacylglycerol. The crystal structures of SMG1 were solved in the closed conformation, but they failed to provide direct evidence of factors responsible for this unique selectivity. To address this problem, we constructed a structure in the open, active conformation and modeled a diacylglycerol analogue into the active site. Molecular dynamics simulations were performed on this enzyme-analogue complex to relax steric clashes. This bound diacylglycerol analogue unambiguously identified the position of two pockets which accommodated two alkyl chains of substrate. The structure of SMG1-analogue complex revealed that Leu103 and Phe278 divided the catalytic pocket into two separated moieties, an exposed groove and a narrow tunnel. Analysis of the binding model suggested that the unique selectivity of this lipase mainly resulted from the shape and size of this narrow tunnel, in which there was no space for the settlement of the third chain of triacylglycerol. These results expand our understanding on the mechanism underlying substrate selectivity of enzyme, and could pave the way for site-directed mutagenesis experiments to improve the enzyme for application.     

Gas-phase enzyme catalysis using immobilized lipase for ester production

Bench-scale reactors were operated in continuous recycle and single-pass modes using immobilized porcine lipase to catalyze gas-phase esterification of ethyl alcohol with two carboxylic acids (acetic acid and propionic acid). Approximately one order of magnitude increases (over uncatalyzed reactions) in conversion were achieved; produc-tion concentrations ranged from 0.1 to 0.5 mM in air, and were affected strongly by substrate concentration and acid-induced enzyme inactivation.     

Human lipoprotein lipase: the loop covering the catalytic site is essential for interaction with lipid substrates.

Lipoprotein lipase (LPL), a key enzyme which initiates the hydrolysis of triglycerides present in chylomicrons and very low density lipoproteins, consists of multiple functional domains which are necessary for normal activity. The catalytic domain of LPL mediates the esterase function of the enzyme but separate lipid binding sites have been proposed to be involved in the interaction of LPL with emulsified lipid substrates at the water-lipid interface. Like pancreatic lipase (PL), LPL contains a surface loop covering the catalytic pocket that may modulate access of the substrate to the active site of the enzyme. Secondary structural analysis of this loop reveals a helix-turn-helix motif with two short amphipathic helices that have hydrophobic moments of 0.64 and 0.68. In order to investigate the role of the loop in the initial interaction of LPL with its substrate, we utilized site-directed mutagenesis to generate eight constructs in which the amphipathic properties of the loop were altered and expressed them in human embryonal kidney-293 cells. Reducing the amphiphilicity without changing the predicted secondary structure of the loop abolished the ability of the lipase to hydrolyze emulsified, long chain fatty acid triglycerides (triolein) but not the water soluble substrate tributyrin. Replacing the loop of LPL with the loop of hepatic lipase, which differs in 15 of 22 amino acids but is also amphiphilic, led to the expression of an enzyme that retained both triolein and tributyrin hydrolyzing activity. Substitution of the LPL loop by a short four amino acid peptide, which may allow more direct access to the active site than the 22 amino acid loop, enhanced hydrolysis of short chain fatty acid triglycerides by more than 2-fold, while the ability to hydrolyze emulsified substrates was abolished. Thus, disruption of the amphipathic structure of the LPL loop selectively decreases the hydrolysis of emulsified lipid substrate without affecting the esterase or catalytic function of the enzyme. These studies establish that the loop with its two amphipathic helices is essential for hydrolysis of long chain fatty acid substrate by LPL providing new insight into the role of the LPL loop in lipid-substrate interactions. We propose that the interaction between the lipoprotein substrates and the amphipathic helices within this loop may in part determine lipase substrate specificity.     

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.     

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.     

无溶剂体系脂肪酶动力学拆分2-辛醇及产物的非均相共沸蒸馏提取

为了提高脂肪酶在非水相中对手性仲醇的拆分效率,以2-辛醇为模式底物,建立了以辛酸为酰基供体的无溶剂脂肪酶动力学拆分手性仲醇的反应体系,采用1.5∶1的酸醇摩尔比,45℃条件下反应12 h,2-辛醇的转化率达到49.9%,并且S-2-辛醇的对映体过量率e.e.s=98.2%,反应的对映体比率E600。利用脂肪族仲醇与水形成共沸物的特性,通过非均相共沸蒸馏的方法提取拆分得到的手性仲醇,S-2-辛醇的光学纯度并未降低,并且产率大于90%,产品纯度大于98%。     

Mapping Conformational Dynamics to Individual Steps in the TEM-1 β-Lactamase Catalytic Mechanism

Conformational dynamics are increasingly recognized as being essential for enzyme function. However, there is virtually no direct experimental evidence to support the notion that individual dynamic modes are required for specific catalytic processes, apart from the initial step of substrate binding. In this work, we use a unique approach based on millisecond hydrogen–deuterium exchange mass spectrometry to identify dynamic modes linked to individual catalytic processes in the antibiotic resistance enzyme TEM-1 β-lactamase. Using a “good” substrate (ampicillin), a poorly hydrolyzed substrate (cephalexin) and a covalent inhibitor (clavulanate), we are able to isolate dynamic modes that are specifically linked to substrate binding, productive lactam ring hydrolysis and deacylation. These discoveries are ultimately translated into specific targets for allosteric TEM-1 inhibitor development.     

Unusual commonality in active site structural features of substrate promiscuous and specialist enzymes

Enzyme promiscuity is the ability of (some) enzymes to perform alternate reactions or catalyze non-cognate substrate(s). The latter is referred to as substrate promiscuity, widely studied for its biotechnological applications and understanding enzyme evolution. Insights into the structural basis of substrate promiscuity would greatly benefit the design and engineering of enzymes. Previous studies on some enzymes have suggested that flexibility, hydrophobicity, and active site protonation state could play an important role in enzyme promiscuity. However, it is not known yet whether substrate promiscuous enzymes have distinctive structural characteristics compared to specialist enzymes, which are specific for a substrate. In pursuit to address this, we have systematically compared substrate/catalytic binding site structural features of substrate promiscuous with those of specialist enzymes. For this, we have carefully constructed dataset of substrate promiscuous and specialist enzymes. On careful analysis, surprisingly, we found that substrate promiscuous and specialist enzymes are similar in various binding/catalytic site structural features such as flexibility, surface area, hydrophobicity, depth, and secondary structures. Recent studies have also alluded that promiscuity is widespread among enzymes. Based on these observations, we propose that substrate promiscuity could be defined as a continuum feature that varies from narrow (specialist) to broad range of substrate preferences. Moreover, diversity of conformational states of an enzyme accessible for ligand binding may possibly regulate its substrate preferences.     

Coenzyme binding in alcohol dehydrogenase

Crystallographic investigations of horse liver alcohol dehydrogenase have demonstrated that NAD is not a passive participant in the redox reactions catalysed by the enzyme. On the molecular level NAD acts as an activator which induces an active form of the enzyme. This is mediated by a large conformational change, making the active site dehydrated and by providing one part of the substrate-binding cleft. The catalytic events, substrate binding, inhibitor binding and the role of the catalytic zinc ion are discussed in relation to the role of NAD. Human alcohol dehydrogenase isoenzymes which have very different substrate specificities are discussed in relation to sequence differences.     

Lipase-catalyzed transesterification in organic media: solvent effects on equilibrium and individual rate constants

The kinetics of the immobilized lipase B from Candida antarctica have been studied in organic solvents. This enzyme has been shown to be slightly affected by the water content of the organic media, and it does not seem to be subject to mass transfer limitations. On the other hand, some evidence indicates that the catalytic mechanism of reactions catalyzed by this lipase proceeds through the acyl-enzyme intermediate. Moreover, despite the fact that the immobilization support dramatically enhances the catalytic power of the enzyme, it does not interfere with the intrinsic solvent effect. Consequently, this enzyme preparation becomes optimum for studying the role played by the organic solvent in catalysis. To this end, we have measured the acylation and deacylation individual rate constants, and the binding equilibrium constant for the ester, in several organic environments. Data obtained show that the major effect of the organic solvent is on substrate binding, and that the catalytic steps are almost unaffected by the solvent, indicating the desolvation of the transition state. However, the strong decrease in binding for hydrophilic solvents such as THF and dioxane, compared to the rest of solvents, cannot be easily explained by means of thermodynamic arguments (desolvation of the ester substrate). For this reason, data have been considered as an indication of the existence of an unknown step in the catalytic pathway occurring prior to formation of the acyl-enzyme intermediate.     

Kinetic and structural characterization of triacylglycerol lipases possessing phospholipase A1 activity

The pancreatic lipase gene family displays various substrate selectivities for triglycerides and phospholipids. The structural basis for this difference in substrate specificity has not been definitively established. Based on a kinetic comparative study between various pancreatic lipase family members, we showed here that porcine pancreatic lipase (PPL), which was so far classified as “classical lipase”, was able to hydrolyze phosphatidylcholine (PC). Amino acid sequence alignments revealed that Val260 residue in PPL lid could be critical for the interaction with lipid substrate. Molecular dynamics was applied to investigate PC binding modes within the catalytic cavity of PPL and human pancreatic lipase (HPL), aiming to explain the difference of specificity of these enzymes towards phospholipids. Results showed that with HPL, the oxyanion hole was not able to accommodate the PC molecule, suggesting that no activity could be obtained. With PPL, the formation of a large pocket involving Val260 allowed the PC molecule to come near the catalytic residues, suggesting that it could be hydrolyzed. One more interesting finding is that human pancreatic lipase related protein 2 could hydrolyze phospholipids through its PLA₁ and PLA₂ activities. Overall, our study shed the light on new structural features of the phospholipase activity of pancreatic lipase family members.     

The effect of metal ions on the binding of ethanol to human alcohol dehydrogenase β<Subscript>2</Subscript>β<Subscript>2</Subscript>

Molecular docking simulations were performed in this study to investigate the importance of both structural and catalytic zinc ions in the human alcohol dehydrogenase beta(2)beta(2) on substrate binding. The structural zinc ion is not only important in maintaining the structural integrity of the enzyme, but also plays an important role in determining substrate binding. The replacement of the catalytic zinc ion or both catalytic and structural zinc ions with Cu(2+) results in better substrate binding affinity than with the wild-type enzyme. The width of the bottleneck formed by L116 and V294 in the substrate binding pocket plays an important role for substrate entrance. In addition, unfavorable contacts between the substrate and T48 and F93 prevent the substrate from moving too close to the metal ion. The optimal binding position occurs between 1.9 and 2.4 A from the catalytic metal ion.     

Molecular characterization and identification of surrogate substrates for diacylglycerol lipase α

Diacylglycerol lipase α is the key enzyme in the formation of the most prevalent endocannabinoid, 2-arachidonoylglycerol in the brain. In this study we identified the catalytic triad of diacylglycerol lipase α, consisting of serine 472, aspartate 524 and histidine 650. A truncated version of diacylglycerol lipase α, spanning residues 1-687 retains complete catalytic activity suggesting that the C-terminal domain is not required for catalysis. We also report the discovery and the characterization of fluorogenic and chromogenic substrates for diacylglycerol lipase α. Assays performed with these substrates demonstrate equipotent inhibition of diacylglycerol lipase α by tetrahydrolipastatin and RHC-20867 as compared to reactions performed with the native diacylglycerol substrate. Thus, confirming the utility of assays using these substrates for identification and kinetic characterization of inhibitors from pharmaceutical collections.     

基于分子对接的洋葱假单胞菌脂肪酶催化制备R-N-（2-甲基-6-乙基苯基）丙氨酸的分子机制

采用分子对接方法,研究了洋葱假单胞菌脂肪酶（BCL）不对称酯水解制备光学纯的N-（2-甲基-6-乙基苯基）丙氨酸（NEMPA）的分子机制。通过将立体电子效应的构象约束条件引入计算机分子对接中,在Autodock 4.2软件中筛选到不同烷基链长的（R,S）-NEMPA与BCL活性口袋合适的反应型构象,对实验数据进行了合理解释;基于能量最优、空间互补原则,预测了Val266和Leu287为调控BCL对映体选择性的关键氨基酸位点,为定点突变提高BCL的对映体选择性提供了理论指导。     

De novo design and utilization of photolabile caged substrates as probes of hydrogen tunneling with horse liver alcohol dehydrogenase at sub-zero temperatures: a cautionary note

In order to understand the influence of protein dynamics on enzyme catalysis and hydrogen tunneling, the horse liver alcohol dehydrogenase (HLADH) catalyzed oxidation of benzyl alcohol was studied at sub-zero temperatures. Previous work showed that wild type HLADH has significant kinetic complexity down to -50 degrees C due to slow binding and loss of substrate [S.-C. Tsai, J.P. Klinman, Biochemistry, 40 (2001) 2303]. A strategy was therefore undertaken to reduce kinetic complexity at sub-zero temperatures, using a photolabile (caged) benzyl alcohol that prebinds to the enzyme and yields the active substrate upon photolysis. By computer modeling, a series of caged alcohols were designed de novo, synthesized, and characterized with regard to photolysis and binding properties. The o-nitrobenzyl ether 15, with a unique long tail, was found to be most ideal. At sub-zero temperatures in 50% MeOH, a two-phase kinetic trace and a rate enhancement by the use of 15 were observed. Despite the elimination of substrate binding as a rate-limiting step, the use of caged benzyl alcohol does not produce a measurable H/D kinetic isotope effect. Unexpectedly, the observed fast phase corresponds to multiple enzyme turnovers, based on the stoichiometry of the substrate to enzyme. Possible side reactions and their effects, such as the re-oxidation of bound NADH and the dissipation of photo-excitation energy, may offer an explanation for the observed multiple-turnovers. The lack of observable deuterium isotope effects offers a cautionary note for the application of caged substrates to isolate and study chemical steps of enzyme reactions, particularly when NADH is involved in the reaction pathway.     

Production and solid-phase refolding of human glucagon-like peptide-1 using recombinant Escherichia coli

A cDNA encoding cinnamyl alcohol dehydrogenase (CAD), catalyzing conversion of cinnamyl aldehydes to corresponding cinnamyl alcohols, was cloned from secondary xylem of Leucaena leucocephala. The cloned cDNA was expressed in Escherichia coli BL21 (DE3) pLysS cells. Temperature and Zn(2+) ion played crucial role in expression and activity of enzyme, such that, at 18°C and at 2 mM Zn(2+) the CAD was maximally expressed as active enzyme in soluble fraction. The expressed protein was purified 14.78-folds to homogeneity on Ni-NTA agarose column with specific activity of 346 nkat/mg protein. The purified enzyme exhibited lowest Km with cinnamyl alcohol (12.2 μM) followed by coniferyl (18.1 μM) and sinapyl alcohol (23.8 μM). Enzyme exhibited high substrate inhibition with cinnamyl (beyond 20 μM) and coniferyl (beyond 100 μM) alcohols. The in silico analysis of CAD protein exhibited four characteristic consensus sequences, GHEXXGXXXXXGXXV; C(100), C(103), C(106), C(114); GXGXXG and C(47), S(49), H(69), L(95), C(163), I(300) involved in catalytic Zn(2+) binding, structural Zn(2+) binding, NADP(+) binding and substrate binding, respectively. Tertiary structure, generated using Modeller 9v5, exhibited a trilobed structure with bulged out structural Zn(2+) binding domain. The catalytic Zn(2+) binding, substrate binding and NADP(+) binding domains formed a pocket protected by two major lobes. The enzyme catalysis, sequence homology and 3-D model, all supported that the cloned CAD belongs to alcohol dehydrogenase family of plants.