Synthesis of sulfur-substituted phosphatidylethanolamines and inhibition of protozoan cyclopropane fatty acid synthase

A phosphalidylethanolamine(it1) containing two 9- thiastearyl groups was prepared by chemical synthesis. This compound and its methyl sulfonium derivative were found to be inhibitors of the cyclopropane fatty acid synthase from parasitic protozoan Crithidia fasciculata.     

Enzymatic desaturation of fatty acids: delta11 desaturase activity on cyclopropane acid probes

The formation of methylenecyclopropanes by enzymatic desaturation of 11-cyclopropylundecanoic acid (1) and its disubstituted derivatives cis- and trans-3-5 has been investigated using the Delta(11) desaturase of Spodoptera littoralis as model enzyme. Gas chromatography coupled to mass spectrometry analyses of methanolyzed lipidic extracts from tissues incubated with each probe revealed that all the cyclopropyl fatty acids were transformed into the corresponding 11-cyclopropylidene acids, except for compound trans-5 (5b), which was not desaturated at C11. The formation of methylenecyclopropane 9 as the only reaction product from 1 indicates that a potential radical intermediate is too short-lived to allow rearrangement reactions. Information on the Delta(11) desaturase substrate binding domain is provided considering the cyclopropyl probes 3-5 as conformationally restricted analogues of the straight-chain substrates.     

The structure of mammalian fatty acid synthase turned back to front

On page 1667 of this issue, Stuart Smith and colleagues [1] demonstrate that the animal fatty acid synthase is a head-to-head dimer rather than the head-to-tail dimer depicted in textbooks. This has important ramifications for the mechanisms of other multifunctional enzymes such as polyketide synthases [2].     

Grape extracts suppress the formation of preneoplastic foci and activity of fatty acid synthase in rat liver

This study was conducted to examine the effects of dietary grape extracts on preneoplastic foci formation in rat hepatocarcinogenesis, and related hepatic enzymes. Male Sprague-Dawley rats were fed basal diet or grape diet containing 15% concentrated grape extracts (68 bricks). The grape diet groups were divided into whole-period grape diet group (DEN-GW; grape diet group fed throughout experimental period) and postinitiation grape diet group (DEN-GP; grape diet group fed from post initiation stage) according to the starting time point of the grape diet. Hepatocarcinogenesis was induced by diethylnitrosamine (DEN; 200 mg/kg bw) and 2/3 partial hepatectomy (DEN-B; DEN-treated basal diet group, DEN-GW, and DEN-GP groups), while the control group treated with saline and sham operation (Control group). The formation of placental glutathione (GSH) S-transferase positive (GST-P+) foci in DEN-GW group was moderately but significantly suppressed, however, not in DEN- GP group. Thiobarbituric acid reactive substances content of DEN-GW group was significantly lower than that of DEN-B group. The activity of fatty acid synthase (FAS) in the grape diet groups was decreased about 1/2 of the DEN-B group. The content of GSH and GSH peroxidase activity were increased by carcinogen treatment, but not modulated by grape diet. The activities of GSH S-transferase, p-nitrophenol hydroxylase, and catalase were not affected by diet or treatment. Conclusively, the grape diet-induced reduction of FAS activity that was expressed highly in neoplastic tissues, might be one of the contributing mechanisms of hepatic cancer prevention.     

Head-to-head coiled arrangement of the subunits of the animal fatty acid synthase

The role of the beta-ketoacyl synthase domains in dimerization of the 2505 residue subunits of the multifunctional animal FAS has been evaluated by a combination of crosslinking and characterization of several truncated forms of the protein. Polypeptides containing only the N-terminal 971 residues can form dimers, but polypeptides lacking only the N-terminal 422 residue beta-ketoacyl synthase domain cannot. FAS subunits can be crosslinked with spacer lengths as short as 6 A, via cysteine residues engineered near the N terminus of the full-length polypeptides. The proximity of the N-terminal beta-ketoacyl synthase domains and their essential role in dimerization is consistent with a revised model for the FAS in which a head-to-head arrangement of two coiled subunits facilitates functional interactions between the dimeric beta-ketoacyl synthase and the acyl carrier protein domains of either subunit.     

Study on Escherichia coli alkaline phosphatase conformation by phosphorimetry in the presence of denaturant

The influence of different denaturants on the phosphorescence spectrum and lifetime decay of Escherichia coli alkaline phosphatase (AP) was investigated. Phosphorescence intensity and lifetime of tryptophan residue (Trp-109) decrease upon addition of guanidine hydrochloride, ethylene diamine tetraacetic acid, and urea or decreasing acidity. The experiments show that AP undergoes different pathways with different denaturants and that the activation energy data, DeltaS degrees (not equal) and deltaH degrees (not equal) further confirm that there is a stable intermediate state between the folded and unfolded AP states in solution.     

Inhibition of Escherichia coli porphobilinogen synthase using analogs of postulated intermediates

BACKGROUND: Porphobilinogen synthase is the second enzyme involved in the biosynthesis of natural tetrapyrrolic compounds, and condenses two molecules of 5-aminolevulinic acid (ALA) through a nonsymmetrical pathway to form porphobilinogen. Each substrate is recognized individually at two different active site positions to be regioselectively introduced into the product. According to pulse-labeling experiments, the substrate forming the propionic acid sidechain of porphobilinogen is recognized first. Two different mechanisms for the first bond-forming step between the two substrates have been proposed. The first involves carbon-carbon bond formation (an aldol-type reaction) and the second carbon-nitrogen bond formation, leading to an iminium ion. RESULTS: With the help of kinetic studies, we determined the Michaelis constants for each substrate recognition site. These results explain the Michaelis-Menten behavior of substrate analog inhibitors - they act as competitive inhibitors. Under standard conditions, however, another set of inhibitors demonstrates uncompetitive, mixed, pure irreversible, slow-binding or even quasi-irreversible inhibition behavior. CONCLUSIONS: Analysis of the different classes of inhibition behavior allowed us to make a correlation between the type of inhibition and a specific site of interaction. Analyzing the inhibition behavior of analogs of postulated intermediates strongly suggests that carbon-nitrogen bond formation occurs first.     

Configurational analysis of cyclopropyl fatty acids isolated from Escherichia coli

[reaction: see text] The absolute configuration of methyl lactobacillate and its 9,10 homologue, both isolated from Escherichia coli B-ATCC 11303, was found to be 11R,12S and 9R,10S, respectively.     

Mechanistic investigations of lipoic acid biosynthesis in Escherichia coli: both sulfur atoms in lipoic acid are contributed by the same lipoyl synthase polypeptide

Lipoyl synthase catalyzes the final step in the de novo biosynthesis of the lipoyl cofactor, which is the insertion of two sulfur atoms into an octanoyl chain that is bound in an amide linkage to a conserved lysine on a lipoyl-accepting protein. We show herein that the sulfur atoms in the lipoyl cofactor are derived from lipoyl synthase itself, and that each lipoyl synthase polypeptide contributes both of the sulfur atoms to the intact cofactor.     

Amino acid backbone specificity of the Escherichia coli translation machinery

Using a pure Escherichia coli translation system, we tested the intrinsic specificity of the protein biosynthetic machinery by determining the relative yields of peptide synthesis for incorporation of a series of acyl-%@mt;sys@%tRNA%@sx@%GAC%@be@%AsnB%@sxx@%%@mx@% 's with varied backbone structures at the sense codon GUU (Val). The results showed that different amino acids on the same tRNA adaptor give significantly different peptide yields and the potential for cross-talk between the amino acid and tRNA body/anticodon in aa-tRNA decoding by the ribosome. They further support the substrate plasticity of the ribosomal biosynthetic machinery and provide immediate candidates for ribosomally encoded polymer synthesis.     

Bacteriocin release protein-mediated secretory expression of recombinant chalcone synthase in Escherichia coli

Flavonoids are secondary metabolites synthesized by plants shown to exhibit health benefits such as anti-inflammatory, antioxidant, and anti-tumor effects. Thus, due to the importance of this compound, several enzymes involved in the flavonoid pathway have been cloned and characterized in Escherichia coli. However, the formation of inclusion bodies has become a major disadvantage of this approach. As an alternative, chalcone synthase from Physcomitrella patens was secreted into the medium using a bacteriocin release protein expression vector. Secretion of P. patens chalcone synthase into the culture media was achieved by co-expression with a psW1 plasmid encoding bacteriocin release protein in E. coli Tuner (DE3) plysS. The optimized conditions, which include the incubation of cells for 20 h with 40 ng/ml mitomycin C at OD₆₀₀ induction time of 0.5 was found to be the best condition for chalcone synthase secretion.     

The damage of outer membrane of Escherichia coli in the presence of TiO2 combined with UV light

The biological consequences of exposure to TiO₂, UV light, and their combined effect were studied on the Escherichia coli (E. coli) cells. The damage of outer membrane was observed for the cells after treatment of TiO₂ or UV light. TiO₂ alone can break down lipopolysacchride (LPS), the outermost layer of the E. coli cells, but was not able to destroy peptidoglycan underneath. The same phenomenon was observed for E. coli under 500 W UV light treatment alone. However, the outer membrane of E. coli could be removed completely in the presence of both TiO₂ and UV light, and the cells became elliptical or round without a mechanically strong network. From the analysis of the concentrations for Ca²⁺ and Mg²⁺, a large amount of Ca²⁺ and Mg²⁺ were detected in the solution of the treated cells by photo-catalysis, and this was attributed to the damage of LPS dispatches. After TiO₂ or UV light treatment, a significant decrease in membrane fluidity of E. coli was found from an increase in fluorescence polarization by a fluorescence probe. The permeability of the treated cells increased to some degree that can be confirmed by quantum dots labeling technique.     

E. coli adhesion to silica in the presence of humic acid

The influence of humic acid on the adhesion of Escherichia coli to silica particles or glass surfaces was investigated. After adsorbing various amounts of humic acid to the particles or surfaces, bacteria were added to the sample and allowed to adhere. For the silica particles the number of bacteria-particle couplets formed were counted from video microscopy images. For the glass surfaces, a differential electrophoresis force was applied, and the force required to detach the bacteria was quantified. These experiments showed a slight increase in the number of couplets formed in the presence of humic acid, and also showed a slight increase in the force required for detachment of the bacteria. Although an increase in adhesion number and strength was measured, the magnitude of the increase was small, indicating that humic acid plays a small role in bacterial adhesion to silica or glass surfaces.     

LC-MS analysis of oils of Monodora myristica and Monodora tenuifolia and isolation of a novel cyclopropane fatty acid

Abstract

Seeds of Monodora myristica and M. tenuifolia were extracted with hexane and the extracts were subjected to column chromatography, LC-MS and NMR analysis. In addition to masses of previously isolated compounds, other masses corresponding to unidentified compounds from the plants were detected. Using 2?D NMR techniques, one of the fractions from M. tenuifolia was characterised as a novel 13-(2-butylcyclopropyl)-6,9-dodecadienoic acid. However, none of the compounds detected in LC-MS corresponded to the ones previously identified by GC-MS.     

The molecular mechanism of dicarboxylic acid transport in Escherichia coli K 12.

It is the purpose of this communication to review the properties of the dicarboxylic acid transport system in Escherichia coli K 12, in particular the role of various dicarboxylate transport proteins, and the disposition of these components in the cytoplasmic membrane. The dicarboxylate transport system is an active process and is responsible for the uptake of succinate, fumarate, and malate. Membrane vesicles prepared from the EDTA, lysozyme, and osmotic shock treatment take up the dicarboxylic acids in the presence of an electron donor. Genetic analysis of various transport mutants indicates that there is only one dicarboxylic acid transport system present in Escherichia coli K 12, and that at least 3 genes, designated cbt, dct A, and dct B, are involved in this transport system. The products corresponding to the 3 genes are: a periplasmic binding protein (PBP) specified by cbt, and 2 membrane integral proteins, SBP 1 and SBP 2, specified by dct B and dct A, respectively. Components SBP 1 and SBP 2 appear to be exposed on both the inner and outer surfaces of the membrane, and lie in close proximity to each other. The substrate recognition sites of SBP 2 and SBP 1 are exposed on the outer and inner surfaces of the membrane respectively. The data presently available suggest that dicarboxylic acids may be translocated across the membrane via a transport channel. A tentative working model on the mechanism of translocation of dicarboxylic acids across the cell envelope by the periplasmic binding protein, and the 2 membrane carrier proteins is presented.     

The kinetics of the solvolysis of propylene and cyclopropane in concentrated aqueous sulfuric acid relative energies of the 2-propyl cation and protonated cyclopropane

The rates of solvolysis of propylene and cyclopropane in 6.49M H₂SO₄ have been measured as a function of temperature. From the data, calculations of the relative heats of formation in solution of the 2-propyl cation and protonated cyclopropane have been made. The heat of formation of protonated cyclopropane has been found to be 6.4 kcal/mol greater than that of the 2-propyl cation. The implications of this result are discussed.