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At least 757 records · Page 42Linked to original sources
PubMed2003

Effect of dietary p-aminobenzoic acid on murine Plasmodium yoelii infection.

Plasmodia species, unlike humans, can utilize p-aminobenzoic acid (PABA) for the de novo generation of folate. Plasmodial enzymes for the synthesis of PABA via the shikimate pathway are being investigated as novel targets for malaria chemotherapy. We show that, despite the presence of biosynthetic machinery to synthesize PABA, Plasmodium yoelii, a rodent malaria species, requires exogenous dietary PABA for survival. Mice fed low-PABA diets do not die from lethal doses of P. yoelii. The initiation of a PABA-deficient diet after P. yoelii infection is established leads to the clearance of parasites and subsequent resistance to infection by P. yoelii. An intact immune system is not necessary for protection, given that mice with severe combined immunodeficiency were also protected by PABA-deficient diet. Our studies suggest that the PABA content in the diet will affect the host clearance of malaria parasites and may affect the efficacy of treatments that target the shikimate pathway.

4-Aminobenzoic Acid↗
PubMed2003

Regulation of biosynthesis and transport of aromatic amino acids in low-GC Gram-positive bacteria.

Computational comparative techniques were applied to analysis of the aromatic amino acid regulon in Gram-positive bacteria. A new candidate transcription regulation signal of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase and shikimate kinase genes was identified in Streptococcus and Lactococcus species. New T-boxes were found upstream of aromatic amino acid biosynthesis and transport genes in the Bacillus/Clostridium group. The substrate specificity of proteins from the PabA/TrpG family was assigned based on metabolic reconstruction and analysis of regulatory signals and phylogenetic patterns. New candidate tryptophan transporters were identified; their specificity was predicted by analysis of T-box regulatory sites. Comparison of all available genomes shows that regulation of genes of the aromatic amino acid biosynthesis pathway is quite labile and involves at least four regulatory systems, two at the DNA level and two more involving competition of alternative RNA secondary structures for transcription and/or translation regulation at the RNA level.

3-Deoxy-7-Phosphoheptulonate Synthase↗
PubMed1986

The serC-aro A operon of Escherichia coli. A mixed function operon encoding enzymes from two different amino acid biosynthetic pathways.

Sub-cloning experiments aimed at precisely locating the E. coli aroA gene, which encodes the shikimate pathway enzyme 5-enolpyruvylshikimate 3-phosphate synthase, showed that in certain constructions, which remain capable of complementing an auxotrophic aroA mutation, expression of aroA is reduced. DNA sequence analysis revealed that a sequence approx. 1200 base pairs (bp) upstream of aroA is necessary for its expression. An open reading frame was identified in this region which encodes a protein of 362 amino acids with a calculated Mr of 39,834 and which ends 70 bp before the start of the aroA coding sequence. This gene has been identified as serC, the structural gene for 3-phosphoserine aminotransferase, an enzyme of the serine biosynthetic pathway. Both genes are expressed as a polycistronic message which is transcribed from a promotor located 58 bp upstream of serC. Evidence is presented which confirms that the aroA and serC genes constitute an operon which has the novel feature of encoding enzymes from two different amino acid biosynthetic pathways.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed2026

Strategy for enhanced production of A40926B0 in Nonomuraea gerenzanensis using an efficient CRISPR/AsCas12f1 system.

The global emergence of vancomycin-resistant Gram-positive pathogens underscores the urgent need for efficient production of novel lipoglycopeptide antibiotics. Dalbavancin, a last-resort therapeutic agent, relies on its key biosynthetic precursor A40926B0, whose industrial manufacture is severely limited by the low yield of wild-type Nonomuraea gerenzanensis and inefficient genetic tools for this rare actinomycete. Here, we developed a high-efficiency CRISPR/AsCas12f1 genome editing system and applied systematic metabolic engineering to boost A40926B0 biosynthesis. First, conjugation conditions were optimized to elevate the transfer efficiency in N. gerenzanensis D11. The hypercompact AsCas12f1 nuclease showed markedly lower cytotoxicity than SpCas9 and enabled 100% gene deletion efficiency with preferred PAMs (TTTG, CTTG, GTTG). Second, we strengthened the shikimate pathway via multiple genetic strategies: overexpressing feedback-resistant DAHP synthase (aroG fbr ) and chorismate mutase/prephenate dehydrogenase (tyrA fbr ), as well as knocking out pheA. This manipulation blocks the phenylalanine synthetic branch and redirects metabolic flux toward the l-tyrosine branch. Third, we engineered the branched-chain fatty acid (BCFA) pathway via promoter replacement of bkdA2B2C2, LipAB, fabF and deletion of acdH to enhance isododecanoyl side-chain supply. The combinatorial engineering yielded strain B-13, which produced 1740 mg/L A40926B0 in shake flasks. Finally, 50-L fed-batch fermentation with continuous maltodextrin feeding further increased the titer to 1817 mg/L, the highest reported titer to date. This work establishes a robust CRISPR editing tool for N. gerenzanensis and provides valuable engineering references for precursor-oriented strain improvement targeting lipoglycopeptide antibiotics, offering insights for the industrial scale production of A40926B0.

A40926B0↗
PubMed2003

Mechanism of chorismate synthase. Role of the two invariant histidine residues in the active site.

Chorismate synthase catalyzes the last step in the common shikimate pathway leading to aromatic compounds such as the aromatic amino acids. The reaction consists of the 1,4-anti-elimination of the 3-phosphate group and the C-(6proR) hydrogen from 5-enolpyruvylshikimate 3-phosphate to yield chorismate. Although this reaction does not involve a net redox change, the enzyme has an absolute requirement for reduced flavin mononucleotide, which is not consumed during the reaction. Two invariant histidine residues are found in the active site of the enzyme: His(17) and His(106). Using site-directed mutagenesis, both histidines were replaced by alanine, reducing the activity 10- and 20-fold in the H106A and H17A mutant protein, respectively. Based on the characterization of the two single mutant proteins, it is proposed that His(106) serves to protonate the monoanionic reduced FMN, whereas His(17) protonates the leaving phosphate group of the substrate. An enzymatic reaction mechanism in keeping with the experimental results is presented.

Catalytic Domain↗
PubMed2003

Effects of salts on the function and conformational stability of shikimate kinase.

The unfolding of shikimate kinase (SK) from Erwinia chrysanthemi by urea and its subsequent refolding on dilution of the denaturing agent has been studied in detail [Eur. J. Biochem. 269 (2002) 2124]. Comparison of the effects of urea on the enzyme with those of guanidinium chloride (GdmCl) and NaCl indicated that chloride ions significantly weakened the binding of shikimate. This finding prompted a more detailed examination of the effects of salts on the structure, function and stability of the enzyme; the effects of NaCl and Na(2)SO(4) were investigated in detail. These salts have very small effects on the secondary structure as judged by far UV CD circular dichroism (CD), although the near UV CD spectra suggest that some limited changes in the environment of aromatic amino acids may occur. Both salts inhibit SK activity and analysis of the kinetic and substrate binding parameters point to a complex mechanism for the inhibition. Inclusion of salts leads to a marked stabilisation against unfolding of the enzyme by urea. When the enzyme is unfolded by incubation in 4 M urea, addition of NaCl or Na(2)SO(4) leads to a relatively slow refolding of the enzyme as judged by the regain of native-like CD and fluorescence. In addition, the refolded enzyme can bind shikimate, though more weakly than the native enzyme. However, the refolded enzyme does not appear to be capable of binding nucleotides, nor does it possess detectable catalytic activity. The refolding process brought about by addition of salt in the presence of 4 M urea is not associated with any change in the fluorescence of the probe 8-anilino-1-naphthalenesulfonic acid (ANS), indicating that an intermediate formed by hydrophobic collapse is unlikely to be significantly populated. The results point to both specific and general effects of salts on SK. These are discussed in the light of the structural information available on the enzyme.

Dickeya chrysanthemi↗
PubMed2004

The innate immunity of a marine red alga involves oxylipins from both the eicosanoid and octadecanoid pathways.

The oxygenated derivatives of fatty acids, known as oxylipins, are pivotal signaling molecules in animals and terrestrial plants. In animal systems, eicosanoids regulate cell differentiation, immune responses, and homeostasis. In contrast, terrestrial plants use derivatives of C18 and C16 fatty acids as developmental or defense hormones. Marine algae have emerged early in the evolution of eukaryotes as several distinct phyla, independent from the animal and green-plant lineages. The occurrence of oxylipins of the eicosanoid family is well documented in marine red algae, but their biological roles remain an enigma. Here we address the hypothesis that they are involved with the defense mechanisms of the red alga Chondrus crispus. By investigating its association with a green algal endophyte Acrochaete operculata, which becomes invasive in the diploid generation of this red alga, we showed that (1) when challenged by pathogen extracts, the resistant haploid phase of C. crispus produced both C20 and C18 oxylipins, (2) elicitation with pathogen extracts or methyl jasmonate activated the metabolism of C20 and C18 polyunsaturated fatty acids to generate hydroperoxides and cyclopentenones such as prostaglandins and jasmonates, and (3) C20 and C18 hydroperoxides as well as methyl jasmonate did induce shikimate dehydrogenase and Phe ammonialyase activities in C. crispus and conferred an induced resistance to the diploid phase, while inhibitors of fatty acid oxidation reduced the natural resistance of the haploid generation. The dual nature of oxylipin metabolism in this alga suggests that early eukaryotes featured both animal- (eicosanoids) and plant-like (octadecanoids) oxylipins as essential components of innate immunity mechanisms.

Acetates↗
PubMed2000

Plant aromatic L-amino acid decarboxylases: evolution, biochemistry, regulation, and metabolic engineering applications.

A comprehensive survey of the extensive literature relevant to the evolution, physiology, biochemistry, regulation, and genetic engineering applications of plant aromatic L-amino acid decarboxylases (AADCs) is presented. AADCs catalyze the pyridoxal-5'-phosphate (PLP)-dependent decarboxylation of select aromatic L-amino acids in plants, mammals, and insects. Two plant AADCs, L-tryptophan decarboxylase (TDC) and L-tyrosine decarboxylase (TYDC), have attracted considerable attention because of their role in the biosynthesis of pharmaceutically important monoterpenoid indole alkaloids and benzylisoquinoline alkaloids, respectively. Although plant and animal AADCs share extensive amino acid homology, the enzymes display striking differences in their substrate specificities. AADCs from mammals and insects accept a broad range of aromatic L-amino acids, whereas TDC and TYDC from plants exhibit exclusive substrate specificity for L-amino acids with either indole or phenol side chains, but not both. Recent biochemical and kinetic studies on animal AADCs support basic features of the classic AADC reaction mechanism. The catalytic mechanism involves the formation of a Schiff base between PLP and an invariable lysine residue, followed by a transaldimination reaction with an aromatic L-amino acid substrate. Both TDC and TYDC are primarily regulated at the transcriptional level by developmental and environmental factors. However, the putative post-translational regulation of TDC via the ubiquitin pathway, by an ATP-dependent proteolytic process, has also been suggested. Isolated TDC and TYDC genes have been used to genetically alter the regulation of secondary metabolic pathways derived from aromatic amino acids in several plant species. The metabolic modifications include increased serotonin levels, reduced indole glucosinolate levels, redirected shikimate metabolism, increased indole alkaloid levels, and increased cell wall-bound tyramine levels.

Amino Acid Sequence↗
PubMed1992

Cloning, primary structure and regulation of the ARO4 gene, encoding the tyrosine-inhibited 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, two differently regulated 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase (DAHPS; EC 4.1.2.15) isoenzymes carry out the first step in the shikimate pathway. Mutations in both genes are necessary to cause aromatic amino acid (aa) auxotrophy, since one isoenzyme alone is sufficient to produce enough DAHP for normal growth of the cells. The phenylalanine-inhibited DAHPS is encoded by the previously isolated and characterized ARO3 gene. Here, we report the cloning and characterization of the ARO4 gene, encoding the second DAHPS, which is inhibited by tyrosine. The aa sequence of the ARO4 gene product reveals 76% similarity to the ARO3-encoded isoenzyme and 66 and 73% to the three DAHPS isoenzymes from Escherichia coli. ARO4 gene expression is regulated by the general control system of aa biosynthesis. As in the case of the ARO3 gene, a single GCN4-recognition element in the promoter is responsible for derepression of the ARO4 gene under aa starvation conditions. However, in contrast to the situation in the isogene, ARO3, GCN4 does not contribute to the basal level of ARO4 transcription under nonderepressing conditions.

3-Deoxy-7-Phosphoheptulonate Synthase↗
PubMed1995

The gene (aroK) encoding shikimate kinase I from Escherichia coli.

Shikimate kinase I (SKI), encoded by the aroK gene, converts shikimate to shikimate 3-phosphate, an intermediate in the biosynthesis of the aromatic amino acids. This paper provides evidence that the E. coli aroK reading frame is in a different place to that reported previously and presents a predicted SKI sequence of 173 residues and a molecular mass of 19.5 kDa. The translational start point of aroK is some 84 bp downstream of the site proposed earlier and the reading frame ends 169 bp beyond the stop site reported in the previous sequence. The SKI sequence reported here more closely resembles those from other organisms and the predicted molecular mass approximates more closely that determined by experiment.

Amino Acid Sequence↗
PubMed1988

Arginine chemical modification of Petunia hybrida 5-enol-pyruvylshikimate-3-phosphate synthase.

Reaction of Petunia hybrida 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS) with the arginine reagents phenylglyoxal (PGO) and p-hydroxyphenylglyoxal (HPGO) leads to inactivation of the enzyme. Inactivation with HPGO leads to modification of approximately 3 mol of arginine per mole of enzyme. The modification reaction follows pseudo-first-order kinetics with a t1/2 of 1 min at 5 mM p-hydroxyphenylglyoxal in 0.1 M triethanolamine HCl, pH 7.8. By titration of HPGO-modified enzyme with 5,5'-bis(dithio-2-nitrobenzoic acid), the possibility of cysteine modification by the arginine reagent was ruled out. While shikimate 3-phosphate (S3P) afforded partial protection to the enzyme against inactivation by HPGO, complete protection could be obtained by using a mixture of S3P and glyphosate. Under the latter conditions, only 1 mol arginine was modified per mole of enzyme. This pattern of reactivity suggests that two arginines may be involved in the binding of S3P and glyphosate to EPSP synthase. A third reactive arginine appears to be nonessential for EPSPS activity. Labeling of EPSP synthase with [14C]phenylglyoxal, peptic digestion, HPLC mapping, and amino acid sequencing indicate that Arg-28 and Arg-131 are two of the reactive arginines labeled with [14C]PGO.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1971

Mise en evidence chez les vegetaux superieurs d'une association enzymatique bifonctionnelle intervenant dans la biosynthese de l'acide shikimique.

In the extracts of Quercus pedunculata roots, the activities of 5-dehydroquinate hydro-lyase and shikimate: NADP oxydoreductase are eluted in the same fractions whatever separation procedures are used: chromatography on Sephadex G 100, DEAE cellulose and hydroxylapatite, centrifugation in sucrose density gradient, disc electrophoresis. These results are closely related to those obtained on the aggregate of enzymes involved in the shikimate pathway in Neurospora crassa. The molecular weight of the association determined by gel filtration is about 55,000.

Journal Article↗
PubMed2006

Novel benzene ring biosynthesis from C(3) and C(4) primary metabolites by two enzymes.

The shikimate pathway, including seven enzymatic steps for production of chorismate via shikimate from phosphoenolpyruvate and erythrose-4-phosphate, is common in various organisms for the biosynthesis of not only aromatic amino acids but also most biogenic benzene derivatives. 3-Amino-4-hydroxybenzoic acid (3,4-AHBA) is a benzene derivative serving as a precursor for several secondary metabolites produced by Streptomyces, including grixazone produced by Streptomyces griseus. Our study on the biosynthesis pathway of grixazone led to identification of the biosynthesis pathway of 3,4-AHBA from two primary metabolites. Two genes, griI and griH, within the grixazone biosynthesis gene cluster were found to be responsible for the biosynthesis of 3,4-AHBA; the two genes conferred the in vivo production of 3,4-AHBA even on Escherichia coli. In vitro analysis showed that GriI catalyzed aldol condensation between two primary metabolites, l-aspartate-4-semialdehyde and dihydroxyacetone phosphate, to form a 7-carbon product, 2-amino-4,5-dihydroxy-6-one-heptanoic acid-7-phosphate, which was subsequently converted to 3,4-AHBA by GriH. The latter reaction required Mn(2+) ion but not any cofactors involved in reduction or oxidation. This pathway is independent of the shikimate pathway, representing a novel, simple enzyme system responsible for the synthesis of a benzene ring from the C(3) and C(4) primary metabolites.

Aminobenzoates↗
PubMed1985

Selective overproduction of 5-enol-pyruvylshikimic acid 3-phosphate synthase in a plant cell culture which tolerates high doses of the herbicide glyphosate.

Cultured cells of the higher plant Corydalis sempervirens Pers. which had been adapted to growing in the presence of 5 mM glyphosate (N-[phosphonomethyl]-glycine), a herbicide and a potent specific inhibitor of the shikimate pathway enzyme 5-enol-pyruvylshikimate-3-phosphate (EPSP) synthase, had a nearly 40-fold increased level of the extractable activity of EPSP synthase. Activities of five other shikimate pathway enzymes were, however, similar in the adapted and nonadapted cells, and the concentrations of the free aromatic amino acids in the two cell lines were also similar. EPSP synthases purified from glyphosate-adapted, as well as nonadapted cells, had identical physical, kinetic, and immunological properties, which indicated that the glyphosate-sensitive enzyme was overproduced in the adapted culture. Overproduction of EPSP synthase in the adapted culture was unequivocally established by two-dimensional polyacrylamide gel electrophoresis, as well as by one-dimensional sodium dodecyl sulfate-gradient gel electrophoresis and quantitation of EPSP protein by immunoassay after transfer to nitrocellulose membranes. While about 0.06% of the total soluble protein from nonadapted cells was EPSP synthase protein, the proportion was 2.6% in the adapted cells. In vivo pulse-labeling experiments with [35S]methionine established that the adapted cells have an increased rate of EPSP synthase protein synthesis.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed2005

Glyphosate inhibits rust diseases in glyphosate-resistant wheat and soybean.

Glyphosate is a broad-spectrum herbicide used for the control of weeds in glyphosate-resistant crops. Glyphosate inhibits 5-enolpyruvyl shikimate 3-phosphate synthase, a key enzyme in the synthesis of aromatic amino acids in plants, fungi, and bacteria. Studies with glyphosate-resistant wheat have shown that glyphosate provided both preventive and curative activities against Puccinia striiformis f. sp. tritici and Puccinia triticina, which cause stripe and leaf rusts, respectively, in wheat. Growth-chamber studies demonstrated wheat rust control at multiple plant growth stages with a glyphosate spray dose typically recommended for weed control. Rust control was absent in formulation controls without glyphosate, dependent on systemic glyphosate concentrations in leaf tissues, and not mediated through induction of four common systemic acquired resistance genes. A field test with endemic stripe rust inoculum confirmed the activities of glyphosate pre- and postinfestation. Preliminary greenhouse studies also demonstrated that application of glyphosate in glyphosate-resistant soybeans suppressed Asian soybean rust, caused by Phakopsora pachyrhizi.

Agriculture↗
PubMed2004

Volatile organic compound (VOC) analysis and sources of limonene, cyclohexanone and straight chain aldehydes in axenic cultures of Calothrix and Plectonema.

The volatile organic compounds (VOCs) in four standing cultures of axenic benthic cyanobacteria (Calothrix and Plectonema) were studied by GC/MS analysis. To verify the biogenic origin of the VOCs, 13C-labelling experiments were performed, and the differences in labelling were used to differentiate between biogenic and non-biogenic sources. Strongly labelled biogenic compounds were derived from the shikimate (cresol, skatole), isoprenoid (beta-cyclocitral, beta-ionone, geosmin) and fatty acid pathways (5Z-heptadecene, 3-octanone, 1-hexanol). New odour compounds derived from microorgansims were beta-cyclogeraniol, dihydro-beta-ionone and 2-hydroxy-2,6,6-trimethylcyclohexanone. The latter compound had already been found in lake water, but could not be assigned to a particular organism. Straight chain aldehydes (octanal, nonanal, decanal), cyclohexanone, cyclohexanol and limonene were unlabelled beyond their natural isotope ratio. Therefore, the biogenic origin of these compounds is doubtful despite their occurrence in axenic cultures. The observation that limonene was a racemate supports this view. The precursors and reactions that lead to the formation of these non-biogenic compounds are unknown and it may be that they are formed by the analytical procedures rather than then being present in the culture medium.

Aldehydes↗
PubMed1998

Containment of herbicide resistance through genetic engineering of the chloroplast genome.

Glyphosate is a potent herbicide. It works by competitive inhibition of the enzyme 5-enol-pyruvyl shikimate-3-phosphate synthase (EPSPS), which catalyzes an essential step in the aromatic amino acid biosynthetic pathway. We report the genetic engineering of herbicide resistance by stable integration of the petunia EPSPS gene into the tobacco chloroplast genome using the tobacco or universal vector. Southern blot analysis confirms stable integration of the EPSPS gene into all of the chloroplast genomes (5000-10,000 copies per cell) of transgenic plants. Seeds obtained after the first self-cross of transgenic plants germinated and grew normally in the presence of the selectable marker, whereas the control seedlings were bleached. While control plants were extremely sensitive to glyphosate, transgenic plants survived sprays of high concentrations of glyphosate. Chloroplast transformation provides containment of foreign genes because plastid transgenes are not transmitted by pollen. The escape of foreign genes via pollen is a serious environmental concern in nuclear transgenic plants because of the high rates of gene flow from crops to wild weedy relatives.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed2001

Relationship between the enzymatic browning and phenylalanine ammonia-lyase activity of cut lettuce, and the prevention of browning by inhibitors of polyphenol biosynthesis.

Cut lettuce stored at 4 degrees C gradually turned brown on the cut section after several days of storage. Three factors for enzymatic browning, the polyphenol content, polyphenol oxidase activity, and phenylalanine ammonia-lyase (PAL) activity, were examined during the cold storage of cut lettuce. A relationship between the browning and PAL activity was apparent. We tried to prevent this browning by using the two enzyme inhibitors, 2-aminoindane-2-phosphonic acid (AIP), an inhibitor of the phenylpropanoid pathway, and glyphosate, an inhibitor of the shikimate pathway. AIP and glyphosate significantly inhibited the browning of cut lettuce. The polyphenol content and PAL activity were both reduced by the treatment with AIP. These results show that regulating the biosynthesis of polyphenols is essential to prevent the browning of cut lettuce.

Catechol Oxidase↗