Ubiquinone synthetic pathway in flagellation of Salmonella typhimurium.

Flagellation of Salmonella typhimurium was found to require a functional pathway for ubiquinone synthesis as well as growth in the presence of aliphatic or aromatic carboxylic acids. Selection for constitutive flagellation eliminated the requirement for growth in the presence of added carboxylic acids.     

Structural requirements for nucleophilic substrates of carboxypeptidase Y

The composition and structural aspects of the amino and carboxylic acid groups required for incorporation into peptides by transpeptidation and inhibition of hydrolysis in carboxypeptidase Y-catalyzed reactions were studied. Separation of these two groups by even one carbon prevents incorporation by transpeptidation and does not inhibit incorporation of other amino acids into model peptides. Substitution of phosphonic or sulfonic acids for the carboxylic acid group also results in loss of incorporation by transpeptidation. Only the sulfonic acid analog of glycine causes inhibition of hydrolysis and this inhibition is lost when serine is included in the reaction. d-Serine is not incorporated by carboxypeptidase Y, and its presence in the reaction mixture does not inhibit the incorporation of the L-isomer.     

Accumulation of p-hydroxybenzoic acid in hairy roots of Daucus carota

We report here the accumulation of p-hydroxybenzoic acid in Agrobacterium rhizogenes-induced hairy root cultures of Daucus carota. This phenolic acid finds application in food, pharmaceutical and polymer industries. Metabolic profiling of phenolics by HPLC/ESI-MS from these hairy roots showed a considerable amount of p-hydroxybenzoic acid accumulation both in cytosol and in the cell wall. Analyses of HCl and NaOH treated soluble phenolic fractions resulted in the elution of peaks with same retention time and similar UV-absorption spectra as observed with p-hydroxybenzoic acid standard. This suggests that p-hydroxybenzoic acid is present in the cytosol as free-form (unconjugated). A correlation has been drawn between the accumulation of soluble and wall-bound phenolic acids on a time-course basis. An apparent absence of any p-hydroxybenzoic acid-glucoside supports this observation, which in turn encourages the idea of its incorporation in the cell wall in an alkaline-labile form.     

Microbial synthesis of p-hydroxybenzoic acid from glucose.

A series of recombinant Escherichia coli strains have been constructed and evaluated for their ability to synthesize p-hydroxybenzoic acid from glucose under fed-batch fermentor conditions. The maximum concentration of p-hydroxybenzoic acid synthesized was 12 g/L and corresponded to a yield of 13% (mol/mol). Synthesis of p-hydroxybenzoic acid began with direction of increased carbon flow into the common pathway of aromatic amino acid biosynthesis. This was accomplished in all constructs with overexpression of a feedback-insensitive isozyme of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthase. Expression levels of enzymes in the common pathway of aromatic amino acid biosynthesis were also increased in all constructs to deliver increased carbon flow from the beginning to the end of the common pathway. A previously unreported inhibition of 3-dehydroquinate synthase by L-tyrosine was discovered to be a significant impediment to the flow of carbon through the common pathway. Chorismic acid, the last metabolite of the common pathway, was converted into p-hydroxybenzoic acid by ubiC-encoded chorismate lyase. Constructs differed in the strategy used for overexpression of chorismate lyase and also differed as to whether mutations were present in the host E. coli to inactivate other chorismate-utilizing enzymes. Use of overexpressed chorismate lyase to increase the rate of chorismic acid aromatization was mitigated by attendant decreases in the specific activity of DAHP synthase and feedback inhibition caused by p-hydroxybenzoic acid. The toxicity of p-hydroxybenzoic acid towards E. coli metabolism and growth was also evaluated.     

Optimization of redox reactions employing whole cell biocatalysis

The ability of Saccharomyces cerevisiae to catalyse the reduction reaction of carboxylic acids into alcohols is described. Earlier reports have led to the characterization of the reduction of carbonyl groups into alcohols mediated by the enzyme alcohol dehydrogenase. We investigated the ability of this organism to catalyse the said conversion using the carboxylic acids, acetic acid and butyric acid. In the absence of any previous characterization, whole cell catalysis proved effective. The uptake of these acids from the medium was estimated using a plate assay method involving litmus-agar. The plate assay was found to be a convenient and extremely adaptable method for quantitation of acids in organic as well as aqueous medium. The comparison of existing paradigms in pure protein catalysis with whole cells catalysis proved anomalous. We report that it is solvent toxicity rather than hydrophobic index that correlates with the activity observed in non-aqueous conditions for whole cell biocatalysis. Reduction of acetic acid as well as butyric acid occurred, with efficiency of reaction with butyric acid being marginally higher. The reduction therefore occurs for both the short chain carboxylic acids used in this study. We therefore illustrate the reduction route of acids into alcohols and propose a model two-step pathway for the reaction. Process optimization may be further attempted to enhance the presently moderate reaction efficiencies. Steps made in the direction by studying the pH dependency and use of sacrificial substrate have yielded encouraging results.     

A novel biosynthetic pathway providing precursors for fatty acid biosynthesis and secondary metabolite formation in myxobacteria

Short chain carboxylic acids are well known as the precursors of fatty acid and polyketide biosynthesis. Iso-fatty acids, which are important for the control of membrane fluidity, are formed from branched chain starter units (isovaleryl-CoA and isobutyryl-CoA), which in turn are derived from the degradation of leucine and valine, respectively. Branched chain carboxylic acids are also employed as starter molecules for the biosynthesis of secondary metabolites, e.g. the therapeutically important anthelmintic agent avermectin or the electron transport inhibitor myxothiazol. During our studies on myxothiazol biosynthesis in the myxobacterium, Stigmatella aurantiaca, we detected a novel biosynthetic route to isovaleric acid. After cloning and inactivation of the branched chain keto acid dehydrogenase complex, which is responsible for the degradation of branched chain amino acids, the strain is still able to produce iso-fatty acids and myxothiazol. Incorporation studies employing deuterated leucine show that it can only serve as precursor in the wild type strain but not in the esg mutant. Feeding experiments using (13)C-labeled precursors show that isovalerate is efficiently made from acetate, giving rise to a labeling pattern in myxothiazol that provides evidence for a novel branch of the mevalonate pathway involving the intermediate 3,3-dimethylacryloyl-CoA. 3,3-Dimethylacrylic acid was synthesized in deuterated form and fed to the esg mutant, resulting in strong incorporation into myxothiazol and iso-fatty acids. Similar experiments employing Myxococcus xanthus revealed that the discovered biosynthetic route described is present in other myxobacteria as well.     

Hepatic microsomal oxygenation of aldehydes to carboxylic acids

Hepatic microsomal oxygenation of aldehydes to carboxylic acids was investigated. Aldehydes (veratrum aldehyde, cinnamic aldehyde, myrtenal, cuminaldehyde, 3-phenylpropionaldehyde, perillaldehyde and 9-anthraldehyde) were incubated with hepatic microsomes of mice in the presence of an NADPH-generating system under 18O2 (97 atom%). The incorporation of oxygen-18 into carboxylic acids formed was determined by gas chromatography-mass spectrometry. Oxygen-18 was incorporated into the carboxylic acids formed from all aldehyde substrates examined. Hepatic microsomal formation of 3,4-dimethoxybenzoic acid and cumic acid from veratrum aldehyde and cuminaldehyde, respectively, was inhibited by CO and SKF 525-A. These results indicate that the oxygenation of aldehydes which may be catalyzed by cytochrome P450 is a common reaction in the biotransformation of xenobiotic aldehydes.     

Metabolism of polyunsaturated fatty acids by rabbit reticulocytes

Rabbit reticulocytes obtained by repeated bleeding metabolize exogenous [1-14C]linoleic acid and [1-14C]arachidonic acid by three different pathways. 1. Incorporation into cellular lipids: 50% of the fatty acids metabolized are incorporated into phospholipids, mainly phosphatidylcholine (32.8%) but also into phosphatidylethanolamine (12%), whereas about 10% of the radioactivity was found in the neutral lipids (mono- di- and triacylglycerols, but not cholesterol esters). 2. Formation of lipoxygenase products: 30% of the fatty acids metabolized are converted via the lipoxygenase pathway mainly to hydroxy fatty acids. Their formation is strongly inhibited by lipoxygenase inhibitors such as 5,8,11,14-eicosatetraynoic acid or nordihydroguaiaretic acid. Inhibition of the lipoxygenase pathway results in an increase of the incorporation of the fatty acids into cellular lipids. 15-Hydroxy-5,8,11,13(Z,Z,Z,E)eicosatetraenoic acid and 13-hydroxy-9,11(Z,E)-octadecadienoic acid are incorporated by reticulocytes into cellular lipids and also are metabolized via beta-oxidation. The metabolism of arachidonic acid and linoleic acid is very similar except for a higher incorporation of linoleic acid into neutral lipids. 3. beta-Oxidation of the exogenous fatty acids: about 10% of the polyenoic fatty acids are metabolized via beta-oxidation to 14CO2. Addition of 5,8,11,14-eicosatetraynoic acid strongly increased the 14CO2 formation from the polyenoic fatty acids whereas antimycin A completely abolished beta-oxidation. Erythrocytes show very little incorporation of unsaturated fatty acids into phospholipids and neutral lipids. Without addition of calcium and ionophore A23187 lipoxygenase metabolites could not be detected.     

Preferential incorporation of eicosanoid precursor fatty acids into human umbilical vein endothelial cell phospholipids

We have examined the preferential incorporation of specific fatty acids into phospholipid classes of cultured human umbilical vein endothelial cells. Pulse-labeling of human umbilical vein endothelial cell phospholipids with radiolabeled fatty acids and inhibition of radiolabeled fatty acid incorporation by competition with excess, unlabeled fatty acids in pair-wise combinations revealed two distinct classes of esterification systems into human umbilical vein endothelial cell phospholipids. The eicosanoid precursor fatty acids, including arachidonate, 8,11,14-eicosatrienoate (ETA) and 5,8,11,14,17-eicosapentaenoate (EPA), exhibited high affinity incorporation into total phospholipids, whereas other fatty acids, including docosahexaenoate and monohydroxy eicosatetraenoates, showed low affinity incorporation. The relative degree of incorporation of eicosanoid precursor fatty acids into phospholipid classes was phosphatidylcholine (PC) greater than phosphatidylethanolamine (PE) greater than phosphatidylinositol (PI) greater than phosphatidylserine (PS). The specific activity of [14C]arachidonic acid-labeled PI was two times higher than that of any other radiolabeled phospholipids. When competitive incorporation of eicosanoid precursor fatty acids into phospholipid classes was studied, they were found to be acylated into different phospholipid classes at different rates. Although eicosanoid precursor fatty acids were not preferentially incorporated into PC, arachidonic acid was preferentially incorporated into the other phospholipids and exhibited particular selectivity in comparison with the other eicosanoid precursor fatty acids for incorporation into PI. These results demonstrate that human umbilical vein endothelial cells possess selective incorporation mechanisms for specific fatty acids into various phospholipids via the deacylation-reacylation pathway.     

Growth and nitrogenase activity of Azotobacter vinelandii in the presence of several phenolic acids

Growth and nitrogenase activity (acetylene reduction) of Azotobacter vinelandii in chemically defined N-free media were studied in the presence of p-hydroxybenzoic, vanillic, p-coumaric, and ferulic acids at concentrations from 0.01 to 1% (w/v). Growth and nitrogenase activity were only detected when the microorganism was cultured on p-hydroxybenzoic acid either as sole carbon source or mixed with other phenolic acids, suggesting that p-hydroxybenzoic acid could be utilized as a carbon source by A. vinelandii for growing under certain environmental conditions.     

Polycyclic aromatic hydrocarbons as plausible prebiotic membrane components

Aromatic molecules delivered to the young Earth during the heavy bombardment phase in the early history of our solar system were likely to be among the most abundant and stable organic compounds available. The Aromatic World hypothesis suggests that aromatic molecules might function as container elements, energy transduction elements and templating genetic components for early life forms. To investigate the possible role of aromatic molecules as container elements, we incorporated different polycyclic aromatic hydrocarbons (PAH) in the membranes of fatty acid vesicles. The goal was to determine whether PAH could function as a stabilizing agent, similar to the role that cholesterol plays in membranes today. We studied vesicle size distribution, critical vesicle concentration and permeability of the bilayers using C(6)-C(10) fatty acids mixed with amphiphilic PAH derivatives such as 1-hydroxypyrene, 9-anthracene carboxylic acid and 1,4 chrysene quinone. Dynamic Light Scattering (DLS) spectroscopy was used to measure the size distribution of vesicles and incorporation of PAH species was established by phase-contrast and epifluorescence microscopy. We employed conductimetric titration to determine the minimal concentration at which fatty acids could form stable vesicles in the presence of PAHs. We found that oxidized PAH derivatives can be incorporated into decanoic acid (DA) vesicle bilayers in mole ratios up to 1:10 (PAH:DA). Vesicle size distribution and critical vesicle concentration were largely unaffected by PAH incorporation, but 1-hydroxypyrene and 9-anthracene carboxylic acid lowered the permeability of fatty acid bilayers to small solutes up to 4-fold. These data represent the first indication of a cholesterol-like stabilizing effect of oxidized PAH derivatives in a simulated prebiotic membrane.     

Determination of isokinetic ratios necessary for equimolar incorporation of carboxylic acids in the solid-phase synthesis of mixture-based combinatorial libraries

The methods used to study the relative reaction rates of 45 different aliphatic and aromatic carboxylic acids when coupled to resin-bound amino acid amides is described. Competition experiments involving the coupling of incoming carboxylic acids to resin-bound amino acid amides were performed. The relative composition of each N-acylated amino acid amide in the resulting mixtures was compared to controls prepared by physically mixing equal aliquots of individual compounds in order to study the relative reaction rates of the incoming carboxylic acids. The ratios of the incoming carboxylic acids were then iteratively adjusted to yield as close to equimolar products as possible. As expected, the steric and electronic nature of the incoming carboxylic acids was found to influence their relative reaction rates. The steric hindrance of the resin-bound amino acid appears to have a proportional effect on the reaction rates of the incoming carboxylic acids. N-acylated amino acid amides in the final mixtures, prepared using the final isokinetic ratios, were found to be approximately equimolar.     

Free and glucosylated phenolics, phenol β-glucosyltransferase activity and membrane permeability in cucumber roots affected by derivatives of cinnamic and benzoic acids

Seven-day-old seedlings of cucumber (Cucumis sativus L.) cv. Wisconsin were treated with 0.01, 0.1 and 0.5 mM solutions of derivatives of cinnamic acid (ferulic and p-coumaric acids) and benzoic acid (p-hydroxybenzoic and vanillic acids) as stress factors. In cucumber roots phenolics (free and glucosylated), phenol β-glucosyltransferase (E.C. 2.4.1.35) activity as well as membrane permeability were examined. The most intensive glucosylation took place in the first hour of stress duration in roots treated with 0.01 mM ferulic and p-coumaric acids and with 0.01 and 0.1 mM p-hydroxybenzoic and vanillic acids. At these concentrations a high phenol β-glucosyltransferase activity was found. The deterioration of capacity for phenolic glucosylation as well as the decrease of the phenol β-glucosyltransferase was observed at the higher concentrations. It was associated with increased membrane permeability.     

The effect of medium composition on the structure and physical state of sophorolipids produced by Candida bombicola ATCC 22214

Candida bombicola was grown using a variety of lipophilic carbon substrates. Most of the hydrocarbon and carboxylic acid substrates resulted in a mixture of sophorolipids consisting of free acids and the more desirable lactones. The ratio of diacylated lactone to free acid in these mixtures was a maximum when produced using hexadecane and heptadecane. All of the other lipophilic substrates resulted in significant amounts of free acids being produced. These lactone products were unique in that they precipitated as crystals, which were easily separated from the culture medium. All of the other products were isolated as oils as is usually reported in the literature. Finally, the amounts of these crystals recovered were significantly higher than those observed for any of the oily products. It was possible to determine the degree of direct incorporation of the lipophilic substrates into the sophorolipids for a homologous series of alkanes. The amount of direct incorporation increased with increasing chain length to a maximum for pentadecane, hexadecane and heptadecane. As the length of the alkane substrate increased further, the amount of direct incorporation then decreased until there was no apparent incorporation for eicosane.     

Studies on the cyclophorase system. XXI. Condensation reactions involving glycine

The cyclophorase-mitochondrial system of rabbit liver contains an active hippuric-synthesizing enzyme. In p-aminohippurate formation, the aromatic component can be replaced by a wide variety of cyclic carboxylic acids, while only compounds giving rise to glycine such as sarcosine, glutathione, serine or guanidoacetic acid can substitute for the amino acid component. The incorporation of formate into serine has been shown by tracer experiments.     

Comparative analysis of different Pseudomonas strains that degrade cinnamic acid.

Strains of Pseudomonas stutzeri (CINNS) and Pseudomonas putida (CINNP and CINNW) isolated from soil with cinnamic acid as the sole carbon source were found to be simultaneously adapted to grow on phenylpropionic and p-hydroxybenzoic acids. In cinnamic acid-grown cultures, phenylpropionic acid was isolated. A catabolic plasmid of approximately equal to 75 kilobase pairs encoding the metabolism of cinnamic acid was found in strains CINNP and CINNS.     

Comparative analysis of different Pseudomonas strains that degrade cinnamic acid

Strains of Pseudomonas stutzeri (CINNS) and Pseudomonas putida (CINNP and CINNW) isolated from soil with cinnamic acid as the sole carbon source were found to be simultaneously adapted to grow on phenylpropionic and p-hydroxybenzoic acids. In cinnamic acid-grown cultures, phenylpropionic acid was isolated. A catabolic plasmid of approximately equal to 75 kilobase pairs encoding the metabolism of cinnamic acid was found in strains CINNP and CINNS.     

An efficient conversion of carboxylic acids to one-carbon degraded aldehydes via 2-hydroperoxy acids

After the formation of dianions of a carboxylic acid with lithium diisopropylamide, oxygen was bubbled into the solution to produce 2-hydroperoxy acid. Then the reaction mixture was acidified with a 2 N HCl solution and subsequently elevated to 50 degrees C to afford the aldehyde with the loss of one carbon atom. Even saturated (C(10)-C(20)) and unsaturated (C(18:1)) carboxylic acids were converted into the odd aldehydes (C(9)-C(19), C(17:1)) in high yields. This conversion was found to be an efficient method for the preparation of carboxylic acids (Cn) to one-carbon degraded aldehydes (Cn-1) via 2-hydroperoxy acids.     

Characterization of the Escherichia coli AaeAB efflux pump: a metabolic relief valve?

Treatment of Escherichia coli with p-hydroxybenzoic acid (pHBA) resulted in upregulation of yhcP, encoding a protein of the putative efflux protein family. Also upregulated were the adjacent genes yhcQ, encoding a protein of the membrane fusion protein family, and yhcR, encoding a small protein without a known or suggested function. The function of the upstream, divergently transcribed gene yhcS, encoding a regulatory protein of the LysR family, in regulating expression of yhcRQP was shown. Furthermore, it was demonstrated that several aromatic carboxylic acid compounds serve as inducers of yhcRQP expression. The efflux function encoded by yhcP was proven by the hypersensitivity to pHBA of a yhcP mutant strain. A yhcS mutant strain was also hypersensitive to pHBA. Expression of yhcQ and yhcP was necessary and sufficient for suppression of the pHBA hypersensitivity of the yhcS mutant. Only a few aromatic carboxylic acids of hundreds of diverse compounds tested were defined as substrates of the YhcQP efflux pump. Thus, we propose renaming yhcS, yhcR, yhcQ, and yhcP, to reflect their role in aromatic carboxylic acid efflux, to aaeR, aaeX, aaeA, and aaeB, respectively. The role of pHBA in normal E. coli metabolism and the highly regulated expression of the AaeAB efflux system suggests that the physiological role may be as a "metabolic relief valve" to alleviate toxic effects of imbalanced metabolism.     

Ethylene biosynthesis: 3. Evidence concerning the fate of C1-N1 of 1-aminocyclopropane carboxylic acid

Evidence has been obtained that the product of ethylene biosynthesis from 1-aminocyclopropane carboxylic acid (ACC), aside from CO₂, is cyanide. This has been accomplished by synthesis of a carbon-13-labeled ACC, feeding to apple tissue, and isolation of the free amino acids. By enzymatic degradation to CO₂, followed by mass spectrometry, label is shown to be incorporated into the 4-carbon of asparagine. This occurs via well-known pathways for the metabolism of cyanide, and is confirmed by incorporation experiments using labeled cyanide. In conjunction with previous stereochemical studies, evidence for a sequential single-electron transfer pathway for ethylene biosynthesis has been considerably strengthened.