Rational design of Rhizopus oryzae lipase with modified stereoselectivity toward triradylglycerols

The binding site of sn-1(3)-regioselective Rhizopus oryzae lipase (ROL) has been engineered to change the stereoselectivity of hydrolysis of triacylglycerol substrates and analogs. Two types of prochiral triradylglycerols were considered: 'flexible' substrates with ether, benzylether or ester groups, and 'rigid' substrates with amide or phenyl groups, respectively, in the sn-2 position. The molecular basis of sn-1(3) stereoselectivity of ROL was investigated by modeling the interactions between substrates and ROL, and the model was confirmed by experimental determination of the stereoselectivity of wild-type and mutated ROL. For the substrates, the following rules were derived: (i) stereopreference of ROL toward triradylglycerols depends on the substrate structure. Substrates with 'flexible' sn-2 substituents are preferably hydrolyzed at sn-1, 'rigid' substrates at sn-3. (ii) Stereopreference of ROL toward triradylglycerols can be predicted by analyzing the geometry of the substrate docked to ROL: if the torsion angle phiO3-C3 of glycerol is more than 150 degrees, the substrate will preferably be hydrolyzed in sn-1, otherwise in sn-3. For ROL, the following rules were derived: (i) residue 258 affects stereoselectivity by steric interactions with the sn-2 substituent rather than polar interactions. To a lower extent, stereoselectivity is influenced by mutations further apart (L254) from residue 258. (ii) With 'rigid' substrates, increasing the size of the binding site (mutations L258A and L258S) shifts stereoselectivity of hydrolysis toward sn-1, decreasing its size (L258F and L258F/L254F) toward sn-3.      

Lipase-Catalyzed Esterification

Lipases are versatile catalysts. In addition to their natural reaction of fat hydrolysis, lipases catalyze a plethora of other reactions such as esterification, amidation, and transesterification of esters as well as organic carbonates. Moreover, lipases accept a wide variety of substrates while maintaining their regioselectivity and stereoselectivity. Lipases are highly stable even under adverse conditions such as organic solvents, high temperatures, and so forth. Applications of lipases include production of food additives, chiral intermediates, and pharmaceutical products. Among these, synthesis of various chiral intermediates in pharmaceutical industry and cocoa butter substitutes is being commercially exploited currently.

Lipase-catalyzed esterification and transesterification in anhydrous media (e.g., organic solvents and supercritical fluids) has been an area of major research activity in the past decade or so. Absence of water eliminates the competing hydrolysis reaction. Moreover, substrate specificity, regioselectivity, and stereoselectivity of the enzyme can be controlled by varying the reaction medium. Although organic solvents, which are generally used for lipase-catalyzed reactions, are nearly anhydrous; they contain water in trace quantities. This water content can be controlled over a range and has a profound effect on the activity of lipases. Water not only affects the enzyme but also acts as a competing nucleophile. Enzyme activity has been correlated with thermodynamic activity of water in the medium rather than with the concentration of water. Because lipases are not soluble in most organic solvents, the method of preparation of the enzyme has a strong influence on the enzymatic activity. The major factors are the pH of the aqueous solution in which the enzyme last existed, additives used during preparation, and method of removal of water (e.g., freeze-drying, evaporation, extraction of enzyme into solvent, etc.). Immobilization of lipases allows easy recovery and reuse of the enzyme. Various immobilization techniques have been studied for lipases and some of them have been shown to enhance the activity and stability of the enzyme. Enzyme stability is an important parameter determining the commercial feasibility of the enzymatic process. Various factors, such as temperature, reaction medium, water concentration, as well as the method of preparation, affect the stability of the lipases.

This review deals with fundamental as well as practical aspects of lipase catalysis. A discussion has been presented on various factors affecting lipase activity and stability. Moreover, a brief account of current and potential applications of lipases has been given.     

脂肪酶在催化合成光学活性农药中的应用

脂肪酶作为天然的手性催化剂，具有高立体选择性、作用底物广泛、催化反应条件温和的特点，在不对称合成、光学活性化合物及天然产物的合成领域具有广泛的用途。本文介绍了脂肪酶的催化特性及在合成不谱活性农药中的应用。     

Activity of myocardial lipase using natural edible oils as substrates

Rat heart homogenates were tested for their lipolytic activity toward synthetic and natural substrates such as edible oils. Triolein was hydrolyzed very efficiently by myocardial lipase whereas trierucin was not cleaved by the enzyme. Among the natural substrates, safflower oil, which has the highest degree of unsaturation, was hydrolyzed to a greater extent than the other oils. Mustard oil rich in erucic acid formed a poor substrate for the myocardial lipase.     

Novel fluorescent phosphonic acid esters for discrimination of lipases and esterases

Lipases and esterases are responsible for carboxylester hydrolysis inside and outside cells and are useful biocatalysts for (stereo)selective modification of synthetic substrates. Here we describe novel fluorescent suicide inhibitors that differ in structure and polarity for screening and discrimination of lipolytic enzymes in enzyme preparations. The inhibitors covalently react with the enzymes to form fluorescent lipid-protein complexes that can be resolved by gel electrophoresis. The selectivities of the inhibitors were determined by using different (phospho)lipase, esterase and cholesterol esterase preparations. The results indicate that formation of an inhibitor-enzyme complex is highly dependent on the chemical structure of the inhibitor. We identified inhibitors with very low specificity, and other derivatives that were highly specific for certain subgroups of lipolytic enzymes such as lipases and cholesterol esterases. A combination of these substrate-analogous activity probes represents a useful toolbox for rapid identification and classification of serine hydrolase enzymes.     

Studies of substrate specificities of lipases from different sources

Although lipases are widely applied in a wide variety of reactions, available information on the factors that are responsible for the substrate specificities of lipases from different sources is scarce. In this paper, nine lipase‐producing microorganism strains were isolated from oil‐containing samples. The properties of these enzymes, including pH optima, temperature optima, thermal stability, and pH stability, vary significantly with the different sources from which these lipases were isolated. The substrate specificities of the nine lipases, including fatty acid and positional specificities, were also studied. The fatty acid specificities of the nine lipases were observably different toward 15 substrates with different carbon chain lengths, different saturation degrees and different side chains. The lipases from S₃ Penicillium citrinum (PCL), MJ₁ Aspergillus niger (ANL), MJ₂ Aspergillus oryzae (AOL), YM Bacillus coughing (BCL), S₉ Geotrichum candidum (GCL), and S₁₁ Candida lypolytica (CLL) showed the strongest specificities to short‐chain esters, and the other lipases showed strong selectivity for medium‐ or long‐chain and branched esters. The positional specificities were examined by analyzing the hydrolytic products of triolein catalyzed by the nine lipases, using TLC. The lipases can be mainly classified into two groups by their specificities for the ester bond at position 2 of triglycerides; one group can selectively hydrolyze the ester bond at position 2 of the triglycerides, the other group cannot. All these nine lipases were divided into four groups by hierarchical clustering analysis on the basis of the results of fatty acid and positional specificities.     

Mechanistic Insights from Substrate Preference in Unsaturated Glucuronyl Hydrolase

Natural and synthetic unsaturated glucuronides were tested as substrates for Clostridium perfringens unsaturated glucuronyl hydrolase to probe its mechanism and to guide inhibitor design. Of the natural substrates, a chondroitin disaccharide substrate with sulfation of the primary alcohol on carbon 6 of its N‐acetylgalactosamine moiety was found to have the highest turnover number of any substrate reported for an unsaturated glucuronyl hydrolase, with k_cat=112 s^?1. Synthetic aryl glycoside substrates with electron‐withdrawing aglycone substituents were cleaved more slowly than those with electron‐donating substituents. Similarly, an unsaturated glucuronyl fluoride was found to be a particularly poor substrate, with k_cat/K_m=44 nM ^?1 s^?1—a very unusual result for a glycoside‐cleaving enzyme. These results are consistent with a transition state with positive charge at carbon 5 and the endocyclic oxygen, as anticipated in the hydration mechanism proposed. However, several analogues designed to take advantage of strong enzyme binding to such a transition state showed little to no inhibition. This result suggests that further work is required to understand the true nature of the transition state stabilised by this enzyme.     

Kinetic properties of Penicillium cyclopium lipases studied with vinyl esters

Penicillium cyclopium produces two lipases with different substrate specificities. Lipase I is predominantly active on triacylglycerols whereas lipase II hydrolyzes mono- and diacylglycerols but not triacylglycerols. In this study, we compared the kinetic properties of P. cyclopium lipases and human pancreatic lipase, a classical triacylglycerol lipase, by using vinyl esters as substrates. Results indicate that P. cyclopium lipases I and II and human pancreatic lipase hydrolyze solutions of vinyl propionate or vinyl butyrate at high relative rates compared with emulsions of the same esters, although, in all cases, maximal activity is reached in the presence of emulsified particles, at substrate concentrations above the solubility limit. It appears that partially water-soluble short-chain vinyl esters are suitable substrates for comparing the activity of lipolytic enzymes of different origin and specificity toward esters in solution and in emulsion.     

Novel trifluoromethyl ketones as potent gastric lipase inhibitors

Novel inhibitors of human digestive lipases, lipophilic trifluoromethyl ketones, were developed. These analogues of the natural triacylglycerol substrates of lipases were designed to contain the carbonyl group of the trifluoromethyl ketone functionality in place of the carbonyl group of the scissile ester bond at the sn-1 position. The ester bond at the sn-3 position was replaced by an ether bond, while the secondary hydroxy group was either esterified or etherified. The inhibitors were prepared starting from solketal. The inhibition of human pancreatic and gastric lipases by the trifluoromethyl ketones was studied by the monolayer technique. 5,5,5-Trifluoro-1-(dodecyloxymethyl)-4-oxopentyl decanoate is the best synthetic inhibitor of human gastric lipase ever reported (inhibition constant alpha(50)=0.003).     

Distinction between esterases and lipases: a kinetic study with vinyl esters and TAG

The better to characterize enzymes hydrolyzing carboxyl ester bonds (carboxyl ester hydrolases), we have compared the kinetic behavior of various lipases and esterases against solutions and emulsions of vinyl esters and TAG. Shortchain vinyl esters are hydrolyzed at comparable rates by esterases and lipases and have higher limits of solubility in water than corresponding TAG. Therefore, they are suited to study the influence of the physical state of the substrate on carboxyl ester hydrolase activity within a large concentration range. Enzymes used in this study are TAG lipases from microorganisms, lipases from human and guinea pig pancreas, pig liver esterase, and acetylcholinesterase. This study also includes cutinase, a fungal enzyme that displays functional properties between esterases and lipases. Esterases display maximal activity against solutions of short-chain vinyl esters (vinyl acetate, vinyl propionate, and vinyl butyrate) and TAG (triacetin, tripropionin, and tributyrin). Half-maximal activity is reached at ester concentrations far below the solubility limit. The transition from solution to emulsion at substrate concentrations exceeding the solubility limit has no effect on esterase activity. Lipases are active on solutions of short-chain vinyl esters and TAG but, in contrast to esterases, they all display maximal activity against emulsified substrates and half-maximal activity is reached at substrate concentrations near the solubility limit of the esters. The kinetics of hydrolysis of soluble substrates by lipases are either hyperbolic or deviate from the Michaelis-Menten model and show no or weak interfacial activation. The presence of molecular aggregates in solutions of short-chain substrates, as evidenced by a spectral dye method, likely accounts for the activity of lipases against soluble esters. Unlike esterases, lipases hydrolyze emulsions of water-insoluble medium- and long-chain vinyl esters and IAG such as vinyl laurate, trioctanoin, and olive oil. In conclusion, comparisons of the kinetic behavior of carboxyl ester hydrolases against solutions and emulsions of vinyl esters and TAG allows the distinction between lipases and esterases. In this respect, it clearly appears that guinea pig pancreatic lipase and cutinase are unambiguously classified as lipases.     

Expanding the Scope of Sortase‐Mediated Ligations by Using Sortase Homologues

Sortase‐catalyzed transacylation reactions are widely used for the construction of non‐natural protein derivatives. However, the most commonly used enzyme for these strategies (sortase A from Staphylococcus aureus) is limited by its narrow substrate scope. To expand the range of substrates compatible with sortase‐mediated reactions, we characterized the in vitro substrate preferences of eight sortase A homologues. From these studies, we identified sortase A enzymes that recognize multiple substrates that are unreactive toward sortase A from S. aureus. We further exploited the ability of sortase A from Streptococcus pneumoniae to recognize an LPATS substrate to perform a site‐specific modification of the N‐terminal serine residue in the naturally occurring antimicrobial peptide DCD‐1L. Finally, we unexpectedly observed that certain substrates (LPATXG, X=Nle, Leu, Phe, Tyr) were susceptible to transacylation at alternative sites within the substrate motif, and sortase A from S. pneumoniae was capable of forming oligomers. Overall, this work provides a foundation for the further development of sortase enzymes for use in protein modification.     

Determination of lipase specificity

Specificity of lipases is controlled by the molecular properties of the enzyme, structure of the substrate and factors affecting binding of the enzyme to the substrate. Types of specificity are as follows. I. Substrate: (a) different rates of lipolysis of TG, DG, and MG by the same enzyme; (b) separate enzymes from the same source for TG, DG and MG. II. Positional: (a) primary esters; (b) secondary esters; and (c) all three esters or nonspecific hydrolysis. III. Fatty acid, preference for similar fatty acids. IV. Stereospecificity: faster hydrolysis of one primarysn ester as compared to the other. V. Combinations of I–IV. Lipases with these specificities are: Ia, pancreatic; Ib, postheparin plasma. IIa, pancreatic; IIb,Geotrichum candidum, for fatty acids withcis-9-unsaturation, and IIc,Candida cylindracea. III,G. candidum for unsaturates. IV.sn-1, postheparin plasma andsn-3 human and rat lingual lipases. V. Rat lingual lipase. Methods for determination involve digestion of natural fats of known structure and synthetic acylglycerols followed by analysis of the lipolysis products. All of the types of specificity have been detected with use of synthetic acylglycerols. Detection of stereospecificity requires enantiomeric acylglycerols which are difficult to synthesize, so other methods have been developed. These involve the generation of 1,2-(2,3) DG and resolution of the enantiomers. Trioleoylglycerol or racemic TG can be used as substrates. If the lipase is stereospecific, then either thesn-1,2- or 2,3-enantiomer will predominate. The relative amounts of the enantiomers can be determined by measurement of specific rotation, and nuclear magnetic resonance spectra. The DG can also be separated by conversion to phospholipids and hydrolysis with phospholipases A-2 or C. Applications of these procedures are discussed and data on the specificity of various lipases presented. Scientific Contribution No. 988, Storrs Agricultural Experiment Station, University of Connecticut, Storrs, CT 06268. Trioleoylglycerol is 18∶1−18∶1−18∶1, etc. 1,2-dioleoyl-3-palmitoyl-sn-glycerol issn-18∶1−18∶1−16∶0, with thesn-1 ester to the left. If the TG is racemic,rac is omitted.     

Artificial Light Regulation of an Allosteric Bienzyme Complex by a Photosensitive Ligand

The artificial regulation of proteins by light is an emerging subdiscipline of synthetic biology. Here, we used this concept to photocontrol both catalysis and allostery within the heterodimeric enzyme complex imidazole glycerol phosphate synthase (ImGP‐S). ImGP‐S consists of the cyclase subunit HisF and the glutaminase subunit HisH, which is allosterically stimulated by substrate binding to HisF. We show that a light‐sensitive diarylethene (1,2‐dithienylethene, DTE)‐based competitive inhibitor in its ring‐open state binds with low micromolar affinity to the cyclase subunit and displaces its substrate from the active site. As a consequence, catalysis by HisF and allosteric stimulation of HisH are impaired. Following UV‐light irradiation, the DTE ligand adopts its ring‐closed state and loses affinity for HisF, restoring activity and allostery. Our approach allows for the switching of ImGP‐S activity and allostery during catalysis and appears to be generally applicable for the light regulation of other multienzyme complexes.     

Enhancing the Efficiency and Regioselectivity of P450 Oxidation Catalysts by Unnatural Amino Acid Mutagenesis

The development of effective strategies for modulating the reactivity and selectivity of cytochrome P450 enzymes represents a key step toward expediting the use of these biocatalysts for synthetic applications. We have investigated the potential of unnatural amino acid mutagenesis to aid efforts in this direction. Four unnatural amino acids with diverse aromatic side chains were incorporated at 11 active‐site positions of a substrate‐promiscuous CYP102A1 variant. The resulting “uP450s” were then tested for their catalytic activity and regioselectivity in the oxidation of two representative substrates: a small‐molecule drug and a natural product. Large shifts in regioselectivity resulted from these single mutations, and in particular, for para‐acetyl‐Phe substitutions at positions close to the heme cofactor. Screening this mini library of uP450s enabled us to identify P450 catalysts for the selective hydroxylation of four aliphatic positions in the target substrates, including a C(sp³)?H site not oxidized by the parent enzyme. Furthermore, we discovered a general activity‐enhancing effect of active‐site substitutions involving the unnatural amino acid para‐amino‐Phe, which resulted in P450 catalysts capable of supporting the highest total turnover number reported to date on a complex molecule (34 650). The functional changes induced by the unnatural amino acids could not be reproduced by any of the 20 natural amino acids. This study thus demonstrates that unnatural amino acid mutagenesis constitutes a promising new strategy for improving the catalytic activity and regioselectivity of P450 oxidation catalysts.     

Mutant Lipase‐Catalyzed Kinetic Resolution of Bulky Phenyl Alkyl sec‐Alcohols: A Thermodynamic Analysis of Enantioselectivity

The size of the stereoselectivity pocket of Candida antarctica lipase B limits the range of alcohols that can be resolved with this enzyme. These steric constrains have been changed by increasing the size of the pocket by the mutation W104A. The mutated enzyme has good activity and enantioselectivity toward bulky secondary alcohols, such as 1‐phenylalkanols, with alkyl chains up to eight carbon atoms. The S enantiomer was preferred in contrast to the wild‐type enzyme, which has R selectivity. The magnitude of the enantioselectivity changes in an interesting way with the chain length of the alkyl moiety. It is governed by interplay between entropic and enthalpic contributions and substrates with long alkyl chains are resolved best with E values higher than 100. The enantioselectivity increases with temperature for the small substrates, but decreases for the long ones.     

Modeling and prediction of enantioselectivity of lipases by covalent docking method

以SYBYL软件包中的FlexX对接模块为研究工具,模拟预测了4种脂肪酶对66对手性底物的水解及转酯反应的对映体选择性。模拟过程中,将四面体中间态底物类似物共价对接到脂肪酶的活性位点,以3个必需氢键的形成为筛选条件,去除不符合标准的酶-底物对接构象。对接结果表明：当催化反应的E值较小时（E＜100）,酶与R/S两种底物的结合自由能差不足以准确预测酶的优先反应构型;而当底物的E≥100,并且主链碳原子数目较少时,该方法对脂肪酶对映体选择性的预测率明显提高,达到81.5%。由于模拟中,酶与绝大多数底物形成了含有3个氢键的反应型构象,符合理论催化模型,同时由于该方法计算速度快,因而该方法可用于高通量模拟预测脂肪酶的潜在底物。     

Essential dynamics of lipase binding sites: the effect of inhibitors of different chain length

The biochemical activity of enzymes, such as lipases, is often associated with structural changes in the enzyme resulting in selective and stereospecific reactions with the substrate. To investigate the effect of a substrate and its chain length on the dynamics of the enzyme, we have performed molecular dynamics simulations of the native Rhizomucor miehei lipase (Rml) and lipase-dialkylphosophate complexes, where the length of the alkyl chain ranges from two to 10 carbon atoms. Simulations were performed in water and trajectories of 400 ps were used to analyse the essential motions in these systems. Our results indicate that the internal motions of the Rml and Rml complexes occur in a subspace of only a few degrees of freedom. A high flexibility is observed in solvent-exposed segments, which connect beta-sheets and helices. In particular, loop regions Gly35-Lys50 and Thr57-Asn63 fluctuate extensively in the native enzyme. Upon activation and binding of the inhibitor, involving the displacement of the active site loop, these motions are considerably suppressed. With increasing chain length of the inhibitor, the fluctuations in the essential subspace increase, levelling off at a chain length of 10, which corresponds to the size of the active-site groove.      

Structural basis for the properties of two single-site proline mutants of CYP102A1 (P450BM3)

The crystal structures of the haem domains of Ala330Pro and Ile401Pro, two single‐site proline variants of CYP102A1 (P450_BM3) from Bacillus megaterium, have been solved. In the A330P structure, the active site is constricted by the relocation of the Pro329 side chain into the substrate access channel, providing a basis for the distinctive C? H bond oxidation profiles given by the variant and the enhanced activity with small molecules. I401P, which is exceptionally active towards non‐natural substrates, displays a number of structural similarities to substrate‐bound forms of the wild‐type enzyme, notably an off‐axial water ligand, a drop in the proximal loop, and the positioning of two I‐helix residues, Gly265 and His266, the reorientation of which prevents the formation of several intrahelical hydrogen bonds. Second‐generation I401P variants gave high in vitro oxidation rates with non‐natural substrates as varied as fluorene and propane, towards which the wild‐type enzyme is essentially inactive. The substrate‐free I401P haem domain had a reduction potential slightly more oxidising than the palmitate‐bound wild‐type haem domain, and a first electron transfer rate that was about 10 % faster. The electronic properties of A330P were, by contrast, similar to those of the substrate‐free wild‐type enzyme.     

Biocatalytic Synthesis of Nitriles through Dehydration of Aldoximes: The Substrate Scope of Aldoxime Dehydratases

Nitriles, which are mostly needed and produced by the chemical industry, play a major role in various industry segments, ranging from high‐volume, low‐price sectors, such as polymers, to low‐volume, high‐price sectors, such as chiral pharma drugs. A common industrial technology for nitrile production is ammoxidation as a gas‐phase reaction at high temperature. Further popular approaches are substitution or addition reactions with hydrogen cyanide or derivatives thereof. A major drawback, however, is the very high toxicity of cyanide. Recently, as a synthetic alternative, a novel enzymatic approach towards nitriles has been developed with aldoxime dehydratases, which are capable of converting an aldoxime in one step through dehydration into nitriles. Because the aldoxime substrates are easily accessible, this route is of high interest for synthetic purposes. However, whenever a novel method is developed for organic synthesis, it raises the question of substrate scope as one of the key criteria for application as a “synthetic platform technology”. Thus, the scope of this review is to give an overview of the current state of the substrate scope of this enzymatic method for synthesizing nitriles with aldoxime dehydratases. As a recently emerging enzyme class, a range of substrates has already been studied so far, comprising nonchiral and chiral aldoximes. This enzyme class of aldoxime dehydratases shows a broad substrate tolerance and accepts aliphatic and aromatic aldoximes, as well as arylaliphatic aldoximes. Furthermore, aldoximes with a stereogenic center are also recognized and high enantioselectivities are found for 2‐arylpropylaldoximes, in particular. It is further noteworthy that the enantiopreference depends on the E and Z isomers. Thus, opposite enantiomers are accessible from the same racemic aldehyde and the same enzyme.     

Characterization of functional residues in the interfacial recognition domain of lecithin cholesterol acyltransferase (LCAT)

Lecithin cholesterol acyltransferase (LCAT) is an interfacialenzyme active on both high-density (HDL) and low-density lipoproteins(LDL). Threading alignments of LCAT with lipases suggest thatresidues 50–74 form an interfacial recognition site andthis hypothesis was tested by site-directed mutagenesis. The(56–68) deletion mutant had no activity on any substrate.Substitution of W61 with F, Y, L or G suggested that an aromaticresidue is required for full enzymatic activity. The activityof the W61F and W61Y mutants was retained on HDL but decreasedon LDL, possibly owing to impaired accessibility to the LDLlipid substrate. The decreased activity of the single R52A andK53A mutants on HDL and LDL and the severer effect of the doublemutation suggested that these conserved residues contributeto the folding of the LCAT lid. The membrane-destabilizing propertiesof the LCAT 56–68 helical segment were demonstrated usingthe corresponding synthetic peptide. An M65N–N66M substitutiondecreased both the fusogenic properties of the peptide and theactivity of the mutant enzyme on all substrates. These resultssuggest that the putative interfacial recognition domain ofLCAT plays an important role in regulating the interaction ofthe enzyme with its organized lipoprotein substrates.