1,5-Anhydro- -fructose: regioselective acylation with fatty acids

Regioselective acylation of 1,5-anhydro- -fructose was performed with dodecanoic acid to give 1,5-anhydro-6-O-dodecanoyl- -fructose, chemically in 50% yield and enzymatically in quantitative yield. Quantitative conversions were also obtained using hexadecanoic and octadecanoic acids as acyl donors.     

Biocatalytic acylation of carbohydrates with fatty acids from palm fatty acid distillates

Palm fatty acid distillates (PFAD) are by-products of the palm oil refining process. Their use as the source of fatty acids, mainly palmitate, for the biocatalytic synthesis of carbohydrate fatty acid esters was investigated. Esters could be prepared in high yields from unmodified acyl donors and non-activated free fatty acids obtained from PFAD with an immobilized Candida antarctica lipase preparation. Acetone was found as a compatible non-toxic solvent, which gave the highest conversion yields in a heterogeneous reaction system without the complete solubilization of the sugars. Glucose, fructose, and other acyl acceptors could be employed for an ester synthesis with PFAD. The synthesis of glucose palmitate was optimized with regard to the water activity of the reaction mixture, the reaction temperature, and the enzyme concentration. The ester was obtained with 76% yield from glucose and PFAD after reaction for 74 h with 150 U ml⁻¹ immobilized lipase at 40°C in acetone.     

Enzymatic tRNA acylation by acid and alpha-hydroxy acid analogues of amino acids

Incorporation of unnatural amino acids with unique chemical functionalities has proven to be a valuable tool for expansion of the functional repertoire and properties of proteins as well as for structure-function analysis. Incorporation of alpha-hydroxy acids (primary amino group is substituted with hydroxyl) leads to the synthesis of proteins with peptide bonds being substituted by ester bonds. Practical application of this modification is limited by the necessity to prepare corresponding acylated tRNA by chemical synthesis. We investigated the possibility of enzymatic incorporation of alpha-hydroxy acid and acid analogues (lacking amino group) of amino acids into tRNA using aminoacyl-tRNA synthetases (aaRSs). We studied direct acylation of tRNAs by alpha-hydroxy acid and acid analogues of amino acids and corresponding chemically synthesized analogues of aminoacyl-adenylates. Using adenylate analogues we were able to enzymatically acylate tRNA with amino acid analogues which were otherwise completely inactive in direct aminoacylation reaction, thus bypassing the natural mechanisms ensuring the selectivity of tRNA aminoacylation. Our results are the first demonstration that the use of synthetic aminoacyl-adenylates as substrates in tRNA aminoacylation reaction may provide a way for incorporation of unnatural amino acids into tRNA, and consequently into proteins.     

Kinetics of the hydrolysis of di- and trisaccharides with Aspergillus niger glucoamylases I and II

Near-homogeneous forms of glucoamylases I and II, previously purified from an industrial Aspergillus niger preparation, were used to hydrolyze a number of di- and trisaccharides linked by alpha-D-glucosidic bonds. Maximum rates and Michaelis constants were obtained at various temperatures and pH values with glucoamylase I for the disaccharides beta,alpha-trehalose, kojibiose, nigerose, maltose, and isomaltose and the trisaccharides panose and iso-maltotriose, and with glucoamylase II for maltose, maltotriose, and isomaltotriose. Maximum rates were highest and energies of activation were lowest for maltose, maltotriose, and panose, the only three substrates containing alpha-D-(1, 4)-glucosidic bonds. Michaelis constants were lowest and standard heats of binding were most negative for maltose and maltotriose. The variation of maximum rates and Michaelis constants with varying pH values suggested that two carboxyl groups were involved in substrate binding.     

Heterogeneous fatty acylation of Src family kinases with polyunsaturated fatty acids regulates raft localization and signal transduction.

Fatty acylation of Src family kinases is essential for localization of the modified proteins to the plasma membrane and to plasma membrane rafts. It has been suggested that the presence of saturated fatty acyl chains on proteins is conducive for their insertion into liquid ordered lipid domains present in rafts. The ability of unsaturated dietary fatty acids to be attached to Src family kinases has not been investigated. Here we demonstrate that heterogeneous fatty acylation of Src family kinases occurs and that the nature of the attached fatty acid influences raft-mediated signal transduction. By using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, we show that in addition to 14:0 (myristate), 14:1 and 14:2 fatty acids can be attached to the N-terminal glycine of the Src family kinase Fyn when the growth media are supplemented with these dietary fatty acids. Moreover, we synthesized novel iodinated analogs of oleate and stearate, and we showed that heterogeneous S-acylation can occur on cysteine residues within Fyn as well as Galpha, GAP43, and Ras. Modification of Fyn with unsaturated or polyunsaturated fatty acids reduced its raft localization and resulted in decreased T cell signal transduction. These studies establish that heterogeneous fatty acylation is a widespread occurrence that serves to regulate signal transduction by membrane-bound proteins.     

Enzymatic determination of serum-free fatty acids: a colorimetric method

A simple and sensitive enzymatic method is described for the determination of serumfree fatty acids. The method is based on the activation of free fatty acids by acyl-CoA synthetase (EC 6.2.1.3). The reaction is followed as the production of hydrogen peroxide by using acyl-CoA oxidase/peroxidase/4-aminoantipyrine/phenol system. Results on human sera correlate well with those obtained by our previously described enzymatic method (Shimizu et al. (1979) Anal. Biochem.98, 341–345) and the chemical colorimetric method.     

Heterologous expression of membrane proteins: choosing the appropriate host

Background

Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein.

Methodology/Principal Findings

The expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides) and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells) hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals), functions (transporters, receptors, enzymes) and topologies (between 0 and 13 transmembrane segments). The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts.

Conclusions/Significance

Most of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein.     

Enzymatic synthesis of a capsinoid by the acylation of vanillyl alcohol with fatty acid derivatives catalyzed by lipases

Capsinoids are a novel group of compounds produced by the Capsicum plant. We synthesized a capsinoid by the lipase-catalyzed esterification of vanillyl alcohol with fatty acid derivatives in an organic solvent. The use of seven out of 17 commercially available lipases, especially Novozym 435, was applicable to the synthesis of vanillyl nonanoate, a model compound of capsinoids. The yield of vanillyl nonanoate under the optimum conditions of 50 mM vanillyl alcohol and 50 mM methyl nonanoate in 500 microl of dioxane, using 20 mg of Novozym 435 and 50 mg of 4 A molecular sieves at 25 degrees C, was 86% in 20 h. Several capsinoid homologues having various acyl chain lengths (C6-C18) were synthesized at 64-86% yields from the corresponding fatty acid methyl ester. The natural capsinoids, capsiate and dihydrocapsiate, were obtained by a 400-fold-scale reaction at these optimum conditions in 60% and 59% isolated yields, respectively.     

Enzymatic microassay of serum bile acids: increased sensitivity with an enzyme amplification technique

A new method for the analysis of bile acids in serum is described. Bile acids are oxidized by the 3α-hydroxysteroid dehydrogenase of Pseudomonas testosteroni and after selective destruction of NAD excess the reduced coenzyme is determined by an enzymatic cycling reaction. This amplifying step uses a single enzyme the 3β- or 17β-hydroxysteroid dehydrogenase of P. testosteroni: the microquantity of NADH (formed in the previous reaction) is used alternatively for the reduction of the 17-oxo group and oxidation of the 3β-OH group of dehydroepiandrosterone giving stoichiometrically after each cycle 17β-OH-3-oxo androst-5-ene. The rat of formation of this product is in direct proportion to the initial NADH concentration and is followed continuously by the enzymatic conversion of the product of the amplifying step into the chromophore testosterone (λ_max 248 nm: M_r = 16,600). This step is catalyzed by the 3-ketosteroid Δ5-isomerase of P.-testosteroni. This step, which is very fast and not rate limiting, has the advantage that it shifts the equilibrium of the amplifying system in the right direction. This rapid and sensitive method is adequate for numerous determinations in routine analysis.     

Direct C-glycosylation of indole with unprotected mono-, di-, and trisaccharides: a one-pot synthesis of 1-deoxy-1,1-bis(3-indolyl)alditols

1-Deoxy-1,1-bis(3-indolyl)alditols were synthesized by reacting 2.5equiv of indole with 1equiv of the following seven monosaccharides (D-galactose, D-mannose, D-allose, 2-deoxy-D-arabinohexose (2-deoxy-D-glucose), D-arabinose, L-arabinose, D-xylose), two disaccharides (D-lactose, D-maltose), and a trisaccharide (D-maltotriose) in 1:1 EtOH-H(2)O at room temperature, or at 40 or 50 degrees C, in the presence of 5 mol% scandium(III) trifluoromethanesulfonate [Sc(OTf)(3)], in a one-pot reaction, in 36-95% yields.     

Enzymatic mass determination of high-specific-radioactivity 3H- and 14C-labeled polyunsaturated fatty acids

The soybean lipoxygenase reaction has been applied to calculating the specific activities of 3H- and 14C-labeled polyunsaturated fatty acids of the omega 3 and omega 6 families. The extent of the soybean lipoxygenase reaction with polyunsaturated fatty acids was determined by measuring the increase in absorbance at 234 nm. A salient feature of this application of of the soybean lipoxygenase assay involves the use of a solution which contains highly purified [1-14C]arachidonic acid of known specific radioactivity as a convenient and versatile standard. Because the amount of arachidonic acid in this standard can be easily measured by liquid scintillation counting, the problems of accurately weighing liquid fatty acid standards are avoided.     

A study of the effect on nucleophilic hydrolytic activity of pancreatic elastase, trypsin, chymotrypsin, and leucine aminopeptidase by boronic acids in the presence of arabinogalactan: a subsequent study on the hydrolytic activity of chymotrypsin by boronic acids in the presence of mono-, di-, and trisaccharides

The hydrolytic activity of trypsin, chymotrypsin, elastase, and leucine aminopeptidase, is inhibited by different boronic acids. However, all the enzymes are inhibited by the compound CbzAla(boro)Gly(OH)(2). Therefore, these additives can control the nucleophilic hydrolytic activity of these enzymes.     

Free fatty acids and (Na+,K+)-ATPase: effects on cation regulation, enzyme conformation, and interactions with ethanol

Effects of free fatty acids on parameters of (Na+,K+)-ATPase regulation related to enzyme conformation were examined. Sensitivity to inhibition by free fatty acid increased as the number of double bonds increased. Free fatty acids reduced affinity for K+ or Na+ at their regulatory sites without altering apparent K+ affinity at its high-affinity site, and increased apparent affinity for ATP. The apparent E2/E1 ratio and apparent delta H and delta S for the E1-E2 transition were reduced by fatty acid. High K+ or low temperature reduced the sensitivity of enzyme to inhibition by free fatty acid. In the presence of low K+, arachidonic acid potentiated inhibition of phosphatase activity by ethanol. Arachidonic acid alone had little effect on the rate of ouabain binding, but accelerated ouabain binding in the presence of K+. These data suggest that fatty acids alter (Na+,K+)-ATPase by preventing the univalent cation-mediated transition to E2, the K+-sensitive form of enzyme. (Na+,K+)-ATPase could potentially be influenced in vivo by free fatty acids released by phospholipases or during hypoxia, or by changes in membrane lipid saturation.     

Effects of mono-, di- and tri-hydroxybenzoic acids on the nitrosation of propranolol: structure-activity relationship

Nitrosation of propranolol under standard conditions recommended by the World Health Organization (10mM propranolol hydrochloridre, 40mM sodium nitrite, pH 3.5) was performed in the absence and in the presence of benzoic acid and of twelve mono-, di- and tri-hydroxybenzoic acids, added to the nitrosation mixture in concentrations ranging from 2 to 40mM, in order to examine their effect on the nitrosation reaction. The yield of N-nitrosopropranol (NOP) was reduced by benzoic acid and, with potency decreasing in the following order, by 2,3,4-tri-hydroxybenzoic acid>/=3,4-tri-hydroxybenzoic acid>2,5-di-hydroxybenzoic acid>2,3-di-hydroxybenzoic acid>3-hydroxybenzoic acid>2-hydroxybenzoic acid>3,4,5-tri-hydroxybenzoic acid>4-hydroxybenzoic acid; their inhibiting effect was concentration-dependent. In contrast, 2,4-di-hydroxybenzoic acid, 2,6-di-hydroxybenzoic acid and 2,4,6-tri-hydroxybenzoic acid caused an increase in the yield of NOP that was inversely related to their concentration. 3,5-Di-hydroxybenzoic acid was substantially inactive. These findings indicate that, depending on the positions of carboxyl group and hydroxyl groups on the benzene ring, mono-, di- and tri-hydroxybenzoic acids may inhibit or hasten nitrosation reactions. As compared with benzenediols and benzenetriols [Mutat. Res. 398 (1998) 75], hydroxybenzoic acids inhibit the nitrosation of propranolol to a greater extent and have the advantage of being nonmutagenic and less toxic.     

Inhibition of phospholipase A2 by cis-unsaturated fatty acids: evidence for the binding of fatty acid to enzyme.

Calcium-dependent phospholipases A2 are markedly inhibited in vitro by cis-unsaturated fatty acids (CUFAs) and to a much lesser extent by trans-unsaturated or saturated fatty acids. Thus, CUFAs may function as endogenous suppressors of lipolysis. To better understand the mechanism of inhibition, kinetic analysis, fluorescence spectroscopy and gel permeation chromatography were employed to demonstrate that CUFAs interact with a highly purified Ca(2+)-dependent phospholipase A2 from Naja mossambica mossambica venom. Arachidonate inhibited hydrolysis of both [1-14C]oleate-labelled, autoclaved Escherichia coli and [1-14C]linoleate-labelled phosphatidylethanolamine in an apparent competitive manner. When subjected to gel permeation chromatography, [3H]arachidonate, but not [3H]palmitate, comigrated with the enzyme. Arachidonic and other CUFAs increased the fluorescence intensity of the enzyme almost 2-fold in a dose-dependent fashion (50 microM = 180% of control); methyl arachidonate was without effect. Saturated fatty acids had only a modest effect on enzyme fluorescence (50 microM = 122% of control). Concentrations of arachidonate that inhibited in vitro enzymatic activity by almost 80% did not alter binding of phospholipase A2 to the E. coli substrate. Collectively, these data demonstrate that, while CUFAs selectively bind to the enzyme, they do not influence phospholipase A2-substrate interaction. Inhibition of in vitro phospholipase A2 activity by CUFAs may be mediated by the formation of an enzymatically inactive enzyme-substrate-inhibitor complex.