Evidence for a conserved relationship between an acceptor stem and a tRNA for aminoacylation.

The anticodon-independent aminoacylation of RNA hairpin helices that reconstruct tRNA acceptor stems has been demonstrated for at least 10 aminoacyl-tRNA synthetases. For Escherichia coli cysteine tRNA synthetase, the specificity of aminoacylation of the acceptor stem is determined by the U73 nucleotide adjacent to the amino acid attachment site. Because U73 is present in all known cysteine tRNAs, we investigated the ability of the E. coli cystein enzyme to aminoacylate a heterologous acceptor stem. We show here that a minihelixCys based on the acceptor-T psi C stem of yeast tRNACys is a substrate for the E. coli enzyme, and that aminoacylation of this minihelix is dependent on U73. Additionally, we identify two base pairs in the acceptor stem that quantitatively convert the E. coli acceptor stem to the yeast acceptor stem. The influence of U73 and these two base pairs is completely retained in the full-length tRNA. This suggests a conserved relationship between the acceptor stem alone and the acceptor stem in the context of a tRNA for aminoacylation with cysteine. However, the primary determinant in the species-specific aminoacylation of the E. coli and yeast cysteine tRNAs is a tertiary base pair at position 15:48 outside of the acceptor stem. Although E. coli tRNACys has an unusual G15:G48 tertiary base pair, yeast tRNACys has a more common G15:C48 that prevents efficient aminoacylation of yeast tRNACys by the E. coli enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and characteristics of an aspartame analogue, L-asparaginyl L-3-phenyllactic acid methyl ester

Aspartame (L-aspartyl L-phenylalanine methyl ester) isan artificial sweetener as shown in Fig.1 (A) [1]. Studieson its structure and function showed that its N-terminalL-aspartyl residue could only be replaced by aminomalonyl[2] or L-asparaginyl [3] residue. When its peptide bondwas replaced by an ester bond [Fig. 1(B)] or the hydrogenof amide in the peptide bond replaced by a methyl group[Fig. 1(C)], its sweetness was lost [4]. According to thecrystal structure of aspartame, between the …     

Synthesis of leukotriene A4 methyl ester via acetylene intermediates

Rac-leukotriene A4 methyl ester has been synthesized from propargylic alcohol and 1-heptyne. The synthetic strategy involves the assembly of carbon chain by acetylenide anion condensations and the introduction of (Z)-double bonds by the triple bond hydrogenation.     

The relationship between adjuvant and mitogenic effects of amphotericin methyl ester

The antifungal polyene amphotericin B (AmB) and its methyl ester derivative (AME) both show potent murine immunostimulant as well as B-cell activating effects. Under certain experimental conditions, AME is a much more potent polyclonal B-cell activator (PBA) than AmB. Notable features of the murine B-cell stimulation induced by AME include: (i) High concentrations of AME (50-100 microgram/ml) are required and even at this level exhibit little or no spleen cell toxicity. (ii) Several lines of evidence suggest that the B-cell activating properties of AME are not involved in the cellular mechanism of adjuvant activity in vivo. (iii) There is a strong correlation between the magnitude of the in vitro PBA effects and the in vivo adjuvant effects of AME in a survey of different mouse strains. This evidence suggests that there is genetic control of the murine lymphoid cell-stimulatory effects of AME and that a small number of genes determines the responsive phenotype.     

Synthesis of the leukotriene A4 methyl ester via acetylene intermediates

The strategy of acyclic eicosanoid synthesis via polyacetylenic intermediates is examplified by the synthesis of the racemic leukotriene A4 methyl ester. Leukotriene synthons, namely, trideca-1,4,7-triyne and methyl 6-formyl-5,6-trans-epoxyhexanoate, were synthesised using propargylic alcohol (thrice) and 1-heptyne as starting materials. In the course of the synthesis all new carbon-carbon bonds were created through acetylenide anion condensations and (Z)-double bonds are introduced by triple bond hydrogenations. The strategy provides a straightforward and stereospecific synthetic pathway.     

Synthesis of BC-ring model of globostellatic acid X methyl ester, an anti-angiogenic substance from marine sponge

Concise synthesis of BC-ring model compounds of 13E,17E-globostellatic acid X methyl ester, an anti-angiogenic triterpene derivative from Indonesian marine sponge, was achieved through ynolate olefination and allylic oxidation as key steps. The model compound 5, which was synthesized within 10 reaction steps from commercially available Hajos-Parrish ketone, showed anti-proliferative activity against HUVECs with moderate selectivity.     

Synthesis of N-carbobenzoxy-l-aspartyl-l-phenylalanine methyl ester catalyzed by thermolysin variants with improved activity

Thermolysin is industrially used for the synthesis of N-carbobenzoxy-l-aspartyl-l-phenylalanine methyl ester (ZDFM), a precursor of an artificial sweetener, aspartame, from N-carbobenzoxy-l-aspartic acid (ZD) and l-phenylalanine methyl ester (FM). We have reported five thermolysin variants [D150A (Asp150 is replaced with Ala), D150E, D150W, I168A, and N227H] with improved activity generated by site-directed mutagenesis of the residues located at the active site [Kusano et al. J Biochem 2009;145:103–13]. In this study, we analyzed the ZDFM synthesis reaction catalyzed by these variants. Steady-state kinetic analysis revealed that in the ZDFM synthesis reaction at pH 7.5, at 25 °C, the molecular activity k_cat values of the variants were 1.6–3.8 times higher than that of the wild-type thermolysin (WT), while their Michaelis constant K_m values for ZD and FM were almost the same as those of WT. With the initial concentrations of enzyme, ZD, and FM of 0.1 μM, 5 mM, and 5 mM, respectively, the synthesis of ZDFM catalyzed by these variants reached the maximum level at 4 h while that catalyzed by WT did at 12 h. These results suggest that the five thermolysin variants examined are more suitable than WT for use in ZDFM synthesis.     

Mechanism of the activation step of the aminoacylation reaction: a significant difference between class I and class II synthetases

In the present work we report, for the first time, a novel difference in the molecular mechanism of the activation step of aminoacylation reaction between the class I and class II aminoacyl tRNA synthetases (aaRSs). The observed difference is in the mode of nucleophilic attack by the oxygen atom of the carboxylic group of the substrate amino acid (AA) to the αP atom of adenosine triphosphate (ATP). The syn oxygen atom of the carboxylic group attacks the α-phosphorous atom (αP) of ATP in all class I aaRSs (except TrpRS) investigated, while the anti oxygen atom attacks in the case of class II aaRSs. The class I aaRSs investigated are GluRS, GlnRS, TyrRS, TrpRS, LeuRS, ValRS, IleRS, CysRS, and MetRS and class II aaRSs investigated are HisRS, LysRS, ProRS, AspRS, AsnRS, AlaRS, GlyRS, PheRS, and ThrRS. The variation of the electron density at bond critical points as a function of the conformation of the attacking oxygen atom measured by the dihedral angle ψ (C(α)-C') conclusively proves this. The result shows that the strength of the interaction of syn oxygen and αP is stronger than the interaction with the anti oxygen for class I aaRSs. This indicates that the syn oxygen is the most probable candidate for the nucleophilic attack in class I aaRSs. The result is further supported by the computation of the variation of the nonbonded interaction energies between αP atom and anti oxygen as well as syn oxygen in class I and II aaRSs, respectively. The difference in mechanism is explained based on the analysis of the electrostatic potential of the AA and ATP which shows that the relative arrangement of the ATP with respect to the AA is opposite in class I and class II aaRSs, which is correlated with the organization of the active site in respective aaRSs. A comparative study of the reaction mechanisms of the activation step in a class I aaRS (Glutaminyl tRNA synthetase) and in a class II aaRS (Histidyl tRNA synthetase) is carried out by the transition state analysis. The atoms in molecule analysis of the interaction between active site residues or ions and substrates are carried out in the reactant state and the transition state. The result shows that the observed novel difference in the mechanism is correlated with the organizations of the active sites of the respective aaRSs. The result has implication in understanding the experimentally observed different modes of tRNA binding in the two classes of aaRSs.     

Synthesis of haptens and development of an indirect enzyme-linked immunosorbent assay for tris(2,3-dibromopropyl) isocyanurate

A competitive indirect enzyme-linked immunosorbent assay (ciELISA) was developed for detection of tris(2,3-dibromopropyl) isocyanurate (TBC). Polyclonal antibodies against TBC were raised from synthesized haptens and then screened against various coating antigens. After optimization of the immunoassay conditions, the linear range and IC₅₀ value of the assay were 0.30–100 and 5.17 μg/L, respectively, with little cross-reactivity (?2%). Recovery of various samples (water, serum, soil) was tested and the values ranged from 68% to 110%. This ciELISA was also applied to determine TBC in the riverside soil of the Liuyang River, and the results were compared with the data obtained by UHPLC–MS/MS. The experimental assay results confirmed that this proposed immunoassay is a specific, sensitive, and reliable method for determination and monitoring of TBC.     

Synthesis and biological properties of 16(S)-amino-PGF2 alpha methyl ester

A synthesis of 16-amino-derivatives of PGF2 alpha is reported. Introduction of an amino group into position 16 of PGF2 alpha has decreased the sensitivity of the compound to metabolic degradation. 16(S)-amino-PGF2 alpha methyl ester shows high abortifacient activity with reduced diarrhoeic side effect.     

Enzymatic removal of a cephalosporin methyl ester blocking group

Over 7000 microorganisms were screened to find an enzyme source for the hydrolysis of a C₄ methyl ester blocking group on 7-aminodesacetoxycephalosporanic acid (7-ADCA). Only one culture, Streptomyces capillispira Mertz and Higgens nov. sp., produced an enzyme that catalysed the reaction. Enzyme synthesis in a defined mineral salts medium was repressed by NH₃ and amino acids. Under optimum fermentation conditions, the maximum rate of substrate hydrolysis was 6 × 10^?10 mol min^?1 mg^?1 cell. The enzyme was recovered from the mycelia and partially purified by gel filtration. Kinetic studies by pH-stat titration indicated that the pH optimum was 7.5–8.5, the temperature optimum was 25–30°C, and the substrate K_m value was 2.3 mg ml^?1. The reaction products, 7-ADCA and methanol, were weak competitive inhibitors of the enzyme with K₁ values of 6.63 and 0.188 mg ml^?1, respectively. The enzyme also hydrolysed cefaclor and cephalexin methyl esters but did not hydrolyse cephalosporin ethyl esters. With further improvements in enzyme yields and stability, enzymatic deblocking of cephalosporins could provide an alternative to chemical deblocking processes.     

Synthesis and cytotoxicity of a novel iridoid glucoside derived from aucubin

The novel iridoid glycoside 2 was prepared in six steps (15% overall yield) from natural aucubin (1) and fully characterized. Compound 2, which comprises the same conjugated cyclopentenone pharmacophore as known antitumor oxylipins and prostaglandins, displayed significant antiproliferative in vitro activity towards leukemia L1210 cells. The Michael addition of nucleophilic thiols to compound 2 occurred on a different position compared to classical delta7-prostaglandin A1 methyl ester. The resulting adducts 7a and 7b were fully characterized, and their MS fragmentation patterns were elucidated.     

CA074 methyl ester: a proinhibitor for intracellular cathepsin B.

The specificity of compound CA074 [N-(L-3-trans-propylcarbamoyloxirane-2-carbonyl)-L-isoleucyl-L-pro line] for the inactivation of cathepsin B was quantified in in vitro measurements with cysteine endopeptidases from cattle, it being found that the compound is a very rapid inactivator of cathepsin B (rate constant 112,000 M-1.s-1), with barely detectable action on cathepsins H, L, and S or m-calpain. Conversion of the proline carboxyl group of the inhibitor to the methyl ester virtually abolished the effect on cathepsin B, and a possible explanation for the importance of the carboxyl is presented on the basis of the tertiary structure of cathepsin B. It was found that CA074 methyl ester (1 microM, 3 h) caused selective inactivation of the intracellular cathepsin B of human gingival fibroblasts in culture, in contrast to other available agents, and we suggest that CA074 methyl ester will be of value in the elucidation of the biological functions of cathepsin B.     

Synthesis and application of organomercury haptens for enzyme-linked immunoassay of inorganic and organic mercury

Two organomercury haptens were synthesized via the classical oxymercuration reaction. An intramolecular oxymercuration reaction was the strategy employed to prepare a structurally simple, but chemically robust, organomercury hapten that was conjugated to chicken immunoglobulin G (IgG). The resulting immunogen afforded mouse anti-mercury antibodies that were evaluated in an enzyme-linked immunosorbent assay (ELISA). Antibodies demonstrating high titers were obtained, and various immunoassay parameters were investigated. The sensitivity and selectivity of the resulting antibodies were evaluated by exploring different cross-coupling chemistries and solid-phase synthetic variations. A second hapten was prepared with the intermolecular oxymercuration reaction, and the resulting compound, once coupled to carrier protein, afforded a solid-phase conjugate that revealed the versatility of the mouse anti-mercury antibody. The anti-mercury antibody developed in this study was capable of detecting both mercury(II) salts and organomercury compounds.     

Conservation of a tRNA core for aminoacylation

The core region of Escherichia coli tRNA(Cys)is important for aminoacylation of the tRNA. This core contains an unusual G15:G48 base pair, and three adenosine nucleotides A13, A22 and A46 that are likely to form a 46:[13:22] adenosine base triple. We recently observed that the 15:48 base pair and the proposed 46:[13:22] triple are structurally and functionally coupled to contribute to aminoacylation. Inspection of a database of tRNA sequences shows that these elements are only found in one other tRNA, the Haemophilus influenzae tRNA(Cys). Because of the complexity of the core, conservation of sequence does not mean conservation of function. We here tested whether the conserved elements in H. influenzae tRNA(Cys)were also important for aminoacylation of H. influenzae tRNA(Cys). We cloned and purified a recombinant H. influenzae cysteine-tRNA synthe-tase and showed that it depends on 15:48 and 13, 22 and 46 in a relationship analogous to that of E. coli cysteine-tRNA synthetase. The functional conservation of the tRNA core is correlated with sequence conservation between E.coli and H.influenzae cysteine-tRNA synthetases. As the genome of H. influenzae is one of the smallest and may approximate a small autonomous entity in the development of life, the dependence of this genome on G15:G48 and its coupling with the proposed A46:[A13:A22] triple for aminoacylation with cysteine suggests an early role of these motifs in the evolution of decoding genetic information.     

Acquisition of an insertion peptide for efficient aminoacylation by a halophile tRNA synthetase

Enzymes of halophilic organisms contain unusual peptide motifs that are absent from their mesophilic counterparts. The functions of these halophile-specific peptides are largely unknown. Here we have identified an unusual peptide that is unique to several halophile archaeal cysteinyl-tRNA synthetases (CysRS), which catalyze attachment of cysteine to tRNA(Cys) to generate the essential cysteinyl-tRNA(Cys) required for protein synthesis. This peptide is located near the active site in the catalytic domain and is highly enriched with acidic residues. In the CysRS of the extreme halophile Halobacterium species NRC-1, deletion of the peptide reduces the catalytic efficiency of aminoacylation by a factor of 100 that largely results from a defect in kcat, rather than the Km for tRNA(Cys). In contrast, maintaining the peptide length but substituting acidic residues in the peptide with neutral or basic residues has no major deleterious effect, suggesting that the acidity of the peptide is not important for the kcat of tRNA aminoacylation. Analysis of general protein structure under physiological high salt concentrations, by circular dichroism and by fluorescence titration of tRNA binding, indicates little change due to deletion of the peptide. However, the presence of the peptide confers tolerance to lower salt levels, and fluorescence analysis in 30% sucrose reveals instability of the enzyme without the peptide. We suggest that the stability associated with the peptide can be used to promote proper enzyme conformation transitions in various stages of tRNA aminoacylation that are associated with catalysis. The acquisition of the peptide by the halophilic CysRS suggests an enzyme adaptation to high salinity.     

Selectivity in enzyme catalysed reaction: Preferential hydrolysis of saturated methyl ester over the corresponding unsaturated ester by pig liver esterase

Summary Saturated methyl ester is preferentially hydrolysed in presence of corresponding ,-unsaturated ester by Pig Liver Esterasse. This selectivity is known for aliphatic, aromatic and heterocyclic esters.Chemo, regio and stereoselective transformations are an important goal towards synthetic efficiency (Bartman and Trost, 1984). Among the various methods that are employed to achieve such reactions, enzymatic transformations are in the forefront of current research activities (Jones, 1986) because of several advantages over the chemical methods. These include extremely high degree of selectivity, easier isolation of products, specially with immobilized enzymes and their reusability. In the past decade this class of biocatalysts has provided a plathora of valuable selective transformations (Boland et.al, 1991). Pig Liver Esterase (PLE) is one such enzyme which has been thoroughly studied and used for asymmetric (Zhu and Tedford, 1990) and regioselective (Adachi et.al, 1986) hydrolysis of esters. Several chiral building blocks for the synthesis of complex natural products (Imori et.al., 1983) have been synthesized through PLE. In this communication, we report the preferential hydrolysis of a saturated ester over its unsaturated counterpart. The preference is total and works well for aliphatic, aromatic as well as heterocyclic systems.     

X-Pro peptides: Synthesis and solution conformation of benzyloxycarbonyl-(Aib-Pro)4methyl ester. Evidence for a novel helical structure

The synthesis of the octapeptide, benzyloxycarbonyl-(α-aminoisobutyryl-L-prolyl)₄-methyl ester [Z-(Aib-Pro)₄-OMe] and an analysis of its solution conformation is reported. The octapeptide is shown to possess three strong intramolecular hydrogen bonds on the basis of studies of the solvent and temperature dependence of NH chemical shifts and rates of hydrogen–deuterium exchange. ¹³C studies are consistent with a structure involving only trans Aib-Pro bonds, while ir experiments support a hydrogen-bonded conformation. The Aib 3, 5, and 7 NH groups are shown to participate in hydrogen bonding. A 3₁₀ helical conformation compatible with the spectroscopic data is suggested. The proposed conformation consists of three type III β-turns with Aib and Pro at the corners and stabilized by 4 → 1 intramolecular hydrogen bonds.