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At least 469 records · Page 26Linked to original sources
PubMed1999

Why superoxide imposes an aromatic amino acid auxotrophy on Escherichia coli. The transketolase connection.

The lack of superoxide dismutase and the consequent elevation of [O2-] imposes, on Escherichia coli, auxotrophies for branched chain, sulfur-containing, and aromatic amino acids. The former two classes of auxotrophies have already been explained, whereas the third is explained herein. Thus O2- is shown to interfere with the production of erythrose-4-phosphate, which is essential for the first step of the aromatic biosynthetic pathway. It does so by oxidizing the 1, 2-dihydroxyethyl thiamine pyrophosphate intermediate of transketolase and inactivating this enzyme.

Bacterial Proteins↗
PubMed2002

Biosynthesis of 1-deoxy-1-imino-D-erythrose 4-phosphate: a defining metabolite in the aminoshikimate pathway.

With respect to the source of the nitrogen atom incorporated into the aminoshikimate pathway, d-erythrose 4-phosphate has been proposed to undergo a transamination reaction resulting in formation of 1-deoxy-1-imino-d-erythrose 4-phosphate. Condensation of this metabolite with phosphoenolpyruvate catalyzed by aminoDAHP synthase would then hypothetically form the 4-amino-3,4-dideoxy-d-arabino-heptulosonic acid 7-phosphate (aminoDAHP), which is the first committed intermediate of the aminoshikimate pathway. However, in vitro formation of aminoDAHP has not been observed. In this account, the possibility is examined that 3-amino-3-deoxy-d-fructose 6-phosphate is the source of the nitrogen atom of the aminoshikimate pathway. Transketolase-catalyzed ketol transfer from 3-amino-3-deoxy-d-fructose 6-phosphate to d-ribose 5-phosphate would hypothetically release 1-deoxy-1-imino-d-erythrose 4-phosphate. Along these lines, a chemoenzymatic synthesis of 3-amino-3-deoxy-d-fructose 6-phosphate was elaborated. Incubation of 3-amino-3-deoxy-d-fructose 6-phosphate in Amycolatopsis mediterranei crude cell lysate with d-ribose 5-phosphate and phosphoenolpyruvate resulted in the formation of aminoDAHP and 3-amino-5-hydroxybenzoic acid. 3-[15N]-Amino-3-deoxy-d-6,6-[2H2]-fructose 6-phosphate was also synthesized and similarly incubated in A. mediterranei crude cell lysate. Retention of both 15N and 2H2 labeling in product aminoDAHP indicates that 3-amino-3-deoxy-d-fructose 6-phosphate is serving as a sequestered form of 1-deoxy-1-imino-d-erythrose 4-phosphate.

3-Deoxy-7-Phosphoheptulonate Synthase↗
PubMed2002

Plant allelochemicals differentially regulate Helicoverpa zea cytochrome P450 genes.

Four cytochrome P450 genes, CYP6B8, CYP6B9, CYP6B27 and CYP6B28, exist in the Helicoverpa zea genome as two pairs of paralogs that evolved from gene duplication and 5'-polar gene conversion events. RT-PCR gel blot analyses have shown that all of these genes are expressed constitutively in midguts of all larval instars, suggesting that they have primary roles in the detoxification of plant allelochemicals. Among these, CYP6B9 is expressed only in midgut tissue whereas its paralog, CYP6B27, is expressed primarily in midgut and secondarily in fat body and ovary. CYP6B28 is expressed in midgut, fat body and, to lesser extents in ovary and integument whereas its paralog, CYP6B8, is expressed in midgut and to some extent in fat body. Comparison of the expression levels induced by eight plant allelochemicals, one drug (phenobarbital), and an insecticide (alpha-cypermethrin) indicates that, for the most part, the four P450s respond individually to these inducers, with all four induced strongly by chlorogenic acid, a shikimate pathway intermediate and a lignin biosynthesis intermediate present in a wide variety of plants, and indole-3-carbinol, a glucobrassicin breakdown product present in the Brassicaceae. The multiple levels at which these P450 genes are apparently diverging (e.g. transcriptional responses, protein sequences) support the suggestion that gene conversion events facilitate gene evolution by allowing duplicated copies greater time to acquire selectable differences in both coding and promoter sequences.

Animals↗
PubMed1990

Chorismate synthase. Pre-steady-state kinetics of phosphate release from 5-enolpyruvylshikimate 3-phosphate.

The pre-steady-state kinetics of phosphate formation from 5-enolpyruvylshikimate 3-phosphate catalysed by Escherichia coli chorismate synthase (EC 4.6.1.4) were studied by a rapid-acid-quench technique at 25 degrees C at pH 7.5. No pre-steady-state 'burst' or 'lag' phase was observed, showing that phosphate is released concomitant with the rate-limiting step of the enzyme. The implications of this result for the mechanism of action of chorismate synthase are discussed.

Binding Sites↗
PubMed2003

Parallel solid-phase synthesis and evaluation of inhibitors of Streptomyces coelicolor type II dehydroquinase.

A series of 1-substituted and 4-substituted benzyl analogues of the known inhibitor (1S,3R,4R)-1,3,4-trihydroxy-5-cyclohexene-1-carboxylic acid has been synthesized and tested as inhibitors of Streptomyces coelicolor type II dehydroquinase. The solid-phase syntheses of 18 new analogues are reported. The most potent inhibitor, 2-nitrobenzyloxy analogue 5i, has K(i) of 8 microM, more than 30 times lower than the K(M) of the substrate and approximately 4 times more potent than the original inhibitor. The binding modes of the synthesized analogues in the active site were studied by molecular docking with GOLD 2.0.

Benzyl Compounds↗
PubMed2002

Characterization of the early stage aminoshikimate pathway in the formation of 3-amino-5-hydroxybenzoic acid: the RifN protein specifically converts kanosamine into kanosamine 6-phosphate.

The biosynthesis of 3-amino-5-hydroxybenzoic acid (AHBA), precursor of the ansamycin and mitomycin antibiotics, proceeds by the aminoshikimate pathway from 3,4-dideoxy-4-amino-D-arabino-heptulosonic acid 7-phosphate (aminoDAHP). Identification of RifN, product of one of three genes from the rifamycin biosynthetic gene cluster known to be essential for aminoDAHP formation, as a specific kanosamine (3-deoxy-3-amino-D-glucose) 6-kinase establishes kanosamine and its 6-phosphate as specific intermediates in AHBA formation. This suggests a hypothetical reaction sequence for aminoDAHP formation, and thus for the early steps of AHBA biosynthesis, starting from UDP-D-glucose and introducing the nitrogen by oxidation and transamination at C-3.

Actinomycetales↗
PubMed1999

The phosphoenolpyruvate/phosphate translocator is required for phenolic metabolism, palisade cell development, and plastid-dependent nuclear gene expression.

The Arabidopsis chlorophyll a/b binding protein (CAB) gene underexpressed 1 (cue1) mutant underexpresses light-regulated nuclear genes encoding chloroplast-localized proteins. cue1 also exhibits mesophyll-specific chloroplast and cellular defects, resulting in reticulate leaves. Both the gene underexpression and the leaf cell morphology phenotypes are dependent on light intensity. In this study, we determine that CUE1 encodes the plastid inner envelope phosphoenolpyruvate/phosphate translocator (PPT) and define amino acid residues that are critical for translocator function. The biosynthesis of aromatics is compromised in cue1, and the reticulate phenotype can be rescued by feeding aromatic amino acids. Determining that CUE1 encodes PPT indicates the in vivo role of the translocator in metabolic partitioning and reveals a mesophyll cell-specific requirement for the translocator in Arabidopsis leaves. The nuclear gene expression defects in cue1 suggest that a light intensity-dependent interorganellar signal is modulated through metabolites dependent on a plastid supply of phosphoenolpyruvate.

Arabidopsis↗
PubMed2001

Effects and mechanisms of triacetylshikimic acid on platelet adhesion to neutrophils induced by thrombin and reperfusion after focal cerebral ischemia in rats.

AIM: To investigate the effect of triacetylshikimic acid (TSA) on the platelet adhesion to neutrophils and P-selectin expression on activated platelet membrane induced by thrombin and reperfusion after focal cerebral ischemia. METHODS: The platelet adhesion to neutrophils was evaluated by rosette assay, and P-selectin expression on platelet membrane was determined by flow cytometry. RESULTS: TSA 10 - 1000 micromol/L markedly inhibited thrombin(0.4 kU/L)-induced platelet adhesion to neutrophils. The platelet adhesion to neutrophils induced by a 21-h reperfusion after middle cerebral artery occlusion for 3 h was also inhibited in a dose-dependent manner by TSA 50 - 200 mg/kg given by ig immediately and at 60 min again after the onset of cerebral ischemia. TSA was also shown to decrease the P-selectin expression on platelet surface induced by thrombin in washed platelet and by adenosine diphosphate (ADP) 5 micromol/L in whole blood. CONCLUSION: Reperfusion after cerebral ischemia and thrombin induced platelet adhesion to neutrophils, which could be reduced by TSA probably due to its inhibition of P-selectin expression on activated platelets.

Animals↗
PubMed2001

Spectroscopic and kinetic characterization of the bifunctional chorismate synthase from Neurospora crassa: evidence for a common binding site for 5-enolpyruvylshikimate 3-phosphate and NADPH.

Chorismate synthase catalyzes the anti-1,4-elimination of the phosphate group and the C-(6proR) hydrogen from 5-enolpyruvylshikimate 3-phosphate to yield chorismate, a central building block in aromatic amino acid biosynthesis. The enzyme has an absolute requirement for reduced FMN, which in the case of the fungal chorismate synthases is supplied by an intrinsic FMN:NADPH oxidoreductase activity, i.e. these enzymes have an additional catalytic activity. Therefore, these fungal enzymes have been termed "bifunctional." We have cloned chorismate synthase from the common bread mold Neurospora crassa, expressed it heterologously in Escherichia coli, and purified it in a three-step purification procedure to homogeneity. Recombinant N. crassa chorismate synthase has a diaphorase activity, i.e. it catalyzes the reduction of oxidized FMN at the expense of NADPH. Using NADPH as a reductant, a reduced flavin intermediate was observed under single and multiple turnover conditions with spectral features similar to those reported for monofunctional chorismate synthases, thus demonstrating that the intermediate is common to the chorismate synthase-catalyzed reaction. Furthermore, multiple turnover experiments in the presence of oxygen have provided evidence that NADPH binds in or near the substrate (5-enolpyruvylshikimate 3-phosphate) binding site, suggesting that NADPH binding to bifunctional chorismate synthases is embedded in the general protein structure and a special NADPH binding domain is not required to generate the intrinsic oxidoreductase activity.

Binding Sites↗
PubMed1990

Observation by 13C NMR of the EPSP synthase tetrahedral intermediate bound to the enzyme active site.

Direct observation of the tetrahedral intermediate in the EPSP synthase reaction pathway was provided by 13C NMR by examining the species bound to the enzyme active site under internal equilibrium conditions and using [2-13C]PEP as a spectroscopic probe. The tetrahedral center of the intermediate bound to the enzyme gave a unique signal appearing at 104 ppm. Separate signals were observed for free EPSP (152 ppm) and EPSP bound to the enzyme in a ternary complex with phosphate (161 ppm). These peak assignments account for our quantitation of the species bound to the enzyme and liberated upon quenching with either triethylamine or base. A comparison of quenching with acid, base, or triethylamine was conducted; the intermediate could be isolated by quenching with either triethylamine or 0.2 N KOH, allowing direct quantitation of the species bound to the enzyme. After long times of incubation during the NMR measurement, a signal at 107 ppm appeared. The compound giving rise to this resonance was isolated and identified as an EPSP ketal [Leo et al. (1990) J. Am. Chem. Soc. (in press)]. The rate of formation of the EPSP ketal was very slow, 3.3 X 10(-5) s-1, establishing that it is a side product of the normal enzymatic reaction, probably arising as a breakdown product of the tetrahedral intermediate. A slow formation of pyruvate was also observed and is attributable to the enzymatic hydrolysis of EPSP, with 5% of the enzyme sites occupied by EPSP and hydrolyzing EPSP at a rate of 4.7 X 10(-4) s-1. To look for additional signals that might arise from a covalent adduct which has been postulated to arise from reaction of enzyme with PEP, an NMR experiment was performed with an analogue of S3P lacking the 4- and 5-hydroxyl groups.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1995

Mutagenesis of active site residues in type I dehydroquinase from Escherichia coli. Stalled catalysis in a histidine to alanine mutant.

Chemical modification experiments have previously implicated four amino acid residues in the mechanism of type I dehydroquinase from Escherichia coli. To further test their importance, these residues were mutated, and the resulting mutants were expressed, purified, and characterized. When the highly conserved, Schiff base-forming lysine residue was mutated (K170A) the resulting enzyme showed a approximately 10(6)-fold reduction in catalytic activity, but was still able to bind both substrate and product, as shown by a novel fluorescence-based ligand-binding assay. This is consistent with Lys-170 playing a central role in catalysis and shows that, although forming a covalent bond with the substrate, it is not essential for ground state binding of substrate or product. Conversely, substituting leucine for the conserved, iodoacetate-reactive methionine residue (M205L) had little effect on kcat or Km. Diethylpyrocarbonate experiments had previously implicated either His-143 or His-146 as the putative active site general base. Substituting alanine for each shows that H146A retains full catalytic activity while H143A shows a 10(6)-fold loss of activity. As with the K170A mutant, H143A can bind ligand, and in addition to the predicted role of this residue as the proton-abstracting general base, our data suggest that it is also involved in the formation and breakdown of Schiff base intermediates. Isoelectric focusing, electrospray ionization mass spectrometry, and fluorescence spectroscopy show that the H143A mutant preferentially stabilizes the formation of the product Schiff base, and that this results in burst kinetics reminiscent of p-nitrophenyl acetate hydrolysis by chymotrypsin. The most striking illustration of this stabilization is the fact that the H143A mutant is isolated from overexpressing cells with a significant proportion of the enzyme monomers covalently bound to the product, 3-dehydroshikimate, via a Schiff base linkage. Our data suggest that the H143A mutant is able to slowly transform substrate to product but that the hydrolytic release of the product is stalled. The proposed dual role of His-143 in the mechanism of type I dehydroquinase may explain why the elimination reaction catalyzed by the enzyme proceeds with syn stereochemistry.

Alanine↗
PubMed1992

Origins of secondary metabolism.

Secondary metabolites generally benefit their producers as poisons that protect them against competitors, predators or parasites. They are produced from universally present precursors (most often acetyl-CoA, amino acids or shikimate) by specific enzymes that probably arose by the duplication and divergence of genes originally coding for primary metabolism. Most secondary metabolites are restricted to single major taxa on the universal phylogenetic tree and so probably originated only once. But different secondary metabolic pathways have originated from different ancestral enzymes at radically different times in evolution. Secondary metabolites are most abundantly produced by microorganisms in crowded habitats and by plants, fungi and sessile animals like sponges, where chemical defence and attack rather than physical escape or fighting are at a premium. The first secondary metabolites were probably antibiotics produced in microbial mats over 3500 million years ago. These first ecosystems probably consisted entirely of eubacteria: archaebacteria and eukaryotes arose much later. As a phylogenetic context for considering the earliest origins of antibiotics I summarize a cladistic analysis of the explosive eubacterial primary diversification. This suggests that the most primitive surviving cells are the photosynthetic heliobacteria. Study of these and of the nearly as primitive chloroflexibacteria, spirochaetes and deinobacteria may provide the best evidence on the origins of secondary and primary metabolism.

Anti-Bacterial Agents↗
PubMed2006

Production of indole diterpenes by Aspergillus alliaceus.

Production of two related indole diterpenes (differing by a dimethyl leucine side chain) by Aspergillus alliaceus was improved through several pilot scale fermentations. Media were optimized through focus primarily on initial increases, as well as mid-cycle additions, of carbon and nitrogen sources. Fermentation conditions were improved by varying ventilation conditions using various combinations of air flowrate and back-pressure set points. Production improvements were quantified based on total indole diterpene concentration as well as the ratio of the major-to-minor by-product components. Those changes with a positive substantial impact primarily on total indole diterpene concentration included early cycle glycerol shots and enhanced ventilation conditions (high air flowrate, low back-pressure). Those changes with a significant impact primarily on ratio included higher initial cerelose, soybean oil, monosodium glutamate, tryptophan, or ammonium sulfate concentrations, higher broth pH, and enhanced ventilation conditions. A few changes (higher initial glycerol and monosodium glutamate concentrations) resulted in less notable and desirable titer or ratio changes when implemented individually, but they were adopted to more fully realize the impact of other improvements or to simplify processing. Overall, total indole diterpene titers were improved at the 600 L pilot scale from 125-175 mg/L with a ratio of about 2.1 to 200-260 mg/L with a ratio of about 3.3-4.5. Thus, the ability to optimize total indole diterpene titer and/or ratio readily exists for secondary metabolite production using Aspergillus cultures.

Aspergillus↗
PubMed1979

Mass spectrometry in the elucidation of shikimate biotransformation products in the rat.

The biotransformation products of shikimate in the rat have been identified by electron impact mass spectrometry. Analysis of the metabolites produced after oral administration of shikimate resulted in the identification of hippurate, 3,4,5,6-tetrahydrohippurate, hexahydrohippurate, benzoyl and cyclohexylcarbonyl-beta-D-glucuronides and two isomeric 3,4-dihydroxycyclohexanecarboxylates. Results showed that shikimate itself is not metabolized by rat tissues and that all metabolites produced were dependent upon initial metabolic transformations by gastrointestinal microrganisms. The various hippurate derivatives and glucuronide conjugates appear to arise via a conversion of shikimate to cyclohexanecarboxylate, which is then further metabolized by the rat tissues.

Animals↗