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PubMedDate not supplied

Effect of glyphosate on plant cell metabolism. 31P and 13C NMR studies.

The effect of glyphosate (N-phosphonomethyl glycine; the active ingredient of Roundup herbicide) on plant cells metabolism was analysed by 31P and 13C NMR using suspension-cultured sycamore (Acer pseudoplatanus L) cells. Cells were compressed in the NMR tube and perfused with an original arrangement enabling a tight control of the circulating nutrient medium. Addition of 1 mM glyphosate to the nutrient medium triggered the accumulation of shikimate (20-30 mumol g-1 cell wet weight within 50 h) and shikimate 3-phosphate (1-1.5 mumol g-1 cell wet weight within 50 h). From in vivo spectra it was demonstrated that these two compounds were accumulated in the cytoplasm where their concentrations reached potentially lethal levels. On the other hand, glyphosate present in the cytoplasmic compartment was extensively metabolized to yield aminomethylphosphonic acid which also accumulated in the cytoplasm. Finally, the results presented in this paper indicate that although the cell growth was stopped by glyphosate the cell respiration rates and the level of energy metabolism intermediates remained unchanged.

Carbon↗

PubMed1994

Functionalized 3,5-dihydroxybenzoates as potent novel inhibitors of EPSP synthase.

Aromatic analogues of the EPSP synthase enzyme substrate (S3P), reaction intermediate (1), and product (EPSP) were synthesized from 3,5-dihydroxybenzoic acid and were evaluated as inhibitors of E. coli EPSP synthase. These simple, synthetically accessible aromatic analogues are highly effective competitive inhibitors versus S3P with an apparent Ki for the tetrahedral intermediate analogue 4 of 160 +/- 40 nM. This demonstrates that a simple benzene ring is a quite suitable substitute for the complex shikimate ring in the design of EPSP synthase inhibitors.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗

PubMed2003

The structure of chorismate synthase reveals a novel flavin binding site fundamental to a unique chemical reaction.

The crystal structure of chorismate synthase (CS) from Streptococcus pneumoniae has been solved to 2.0 A resolution in the presence of flavin mononucleotide (FMN) and the substrate 5-enolpyruvyl-3-shikimate phosphate (EPSP). CS catalyses the final step of the shikimate pathway and is a potential therapeutic target for the rational design of novel antibacterials, antifungals, antiprotozoals, and herbicides. CS is a tetramer with the monomer possessing a novel beta-alpha-beta fold. The interactions between the enzyme, cofactor, and substrate reveal the structural reasons underlying the unique catalytic mechanism and identify the amino acids involved. This structure provides the essential initial information necessary for the generation of novel anti-infective compounds by a structure-guided medicinal chemistry approach.

Amino Acid Sequence↗

PubMed1996

Hydroxycinnamoyltransferases Involved in the Accumulation of Caffeic Acid Esters in Gametophytes and Sporophytes of Equisetum arvense.

Four hydroxycinnamoyltransferases from Equisetum arvense L. were studied that catalyze the formation of mono-O-caffeoyl-meso-tartrate, di-O-caffeoyl-meso-tartrate, 5-O-caffeoylshikimate (dactylifrate), and 5-O-caffeoylquinate (chlorogenate). The enzymes were classified as coenzyme A (CoA)-ester-dependent acyltransferases (EC 2.3.1), i.e. hydroxycinnamoyl-CoA:meso-tartrate hydroxycinnamoyltransferase (CTT), hydroxycinnamoyl-CoA:caf-feoyl-meso-tartrate hydroxycinnamoyltransferase (CCT), hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase (CST), and hydroxycinnamoyl-CoA:quinate hydroxycinnamoyltransferase. The CTT, CCT, and CST were partially purified and separated from E. arvense gametophytes by hydrophobic interaction chromatography on Fractogel TSK Butyl-650 followed by molecular exclusion on fast protein liquid chromatography-Superdex-75 with 87-, 62-, and 130- fold enrichments and 12, 8, and 11% yields, respectively. The enzyme activities obtained with caffeoyl-CoA were 95 (CTT), 74 (CCT), and 200 [mu]kat (CST) kg-1 protein. The apparent native relative molecular weight values were found to be approximately 45,000 (CTT), 52,000 (CCT), and 50,000 (CST). Each enzyme showed highest activities at pH 7.5, the CCT and CST in Tris-HCl (1.2 and 1.0 M) and the CTT in imidazole-HCl (1.25 M). Enzyme activities were stimulated more than 3-fold by 100 mM ascorbate. The apparent energies of activation (kilojoules mol-1) were calculated to be 56 (CTT), 69 (CST), and 76 (CCT). The enzymes accepted cinnamoyl-CoA and various hydroxycinnamoyl-CoAs. The time course of the transferase activities along with that of a fourth one, hydroxycinnamoyl-CoA:quinate hydroxycinnamoyltransferase, and the pattern of product accumulation were determined during a 1-year growth period of the E. arvense sporophytes.

Journal Article↗

PubMed1999

Molecular characterization and analysis of the biosynthetic gene cluster for the antitumor antibiotic mitomycin C from Streptomyces lavendulae NRRL 2564.

BACKGROUND: The mitomycins are natural products that contain a variety of functional groups, including aminobenzoquinone- and aziridine-ring systems. Mitomycin C (MC) was the first recognized bioreductive alkylating agent, and has been widely used clinically for antitumor therapy. Precursor-feeding studies showed that MC is derived from 3-amino-5-hydroxybenzoic acid (AHBA), D-glucosamine, L-methionine and carbamoyl phosphate. A genetically linked AHBA biosynthetic gene and MC resistance genes were identified previously in the MC producer Streptomyces lavendulae NRRL 2564. We set out to identify other genes involved in MC biosynthesis. RESULTS: A cluster of 47 genes spanning 55 kilobases of S. lavendulae DNA governs MC biosynthesis. Fourteen of 22 disruption mutants did not express or overexpressed MC. Seven gene products probably assemble the AHBA intermediate through a variant of the shikimate pathway. The gene encoding the first presumed enzyme in AHBA biosynthesis is not, however, linked within the MC cluster. Candidate genes for mitosane nucleus formation and functionalization were identified. A putative MC translocase was identified that comprises a novel drug-binding and export system, which confers cellular self-protection on S. lavendulae. Two regulatory genes were also identified. CONCLUSIONS: The overall architecture of the MC biosynthetic gene cluster in S. lavendulae has been determined. Targeted manipulation of a putative MC pathway regulator led to a substantial increase in drug production. The cloned genes should help elucidate the molecular basis for creation of the mitosane ring system, as well efforts to engineer the biosynthesis of novel natural products.

Amino Acid Sequence↗

PubMed1995

Creation of a Metabolic Sink for Tryptophan Alters the Phenylpropanoid Pathway and the Susceptibility of Potato to Phytophthora infestans.

The creation of artificial metabolic sinks in plants by genetic engineering of key branch points may have serious consequences for the metabolic pathways being modified. The introduction into potato of a gene encoding tryptophan decarboxylase (TDC) isolated from Catharanthus roseus drastically altered the balance of key substrate and product pools involved in the shikimate and phenylpropanoid pathways. Transgenic potato tubers expressing the TDC gene accumulated tryptamine, the immediate decarboxylation product of the TDC reaction. The redirection of tryptophan into tryptamine also resulted in a dramatic decrease in the levels of tryptophan, phenylalanine, and phenylalanine-derived phenolic compounds in transgenic tubers compared with nontransformed controls. In particular, wound-induced accumulation of chlorogenic acid, the major soluble phenolic ester in potato tubers, was found to be two- to threefold lower in transgenic tubers. Thus, the synthesis of polyphenolic compounds, such as lignin, was reduced due to the limited availability of phenolic monomers. Treatment of tuber discs with arachidonic acid, an elicitor of the defense response, led to a dramatic accumulation of soluble and cell wall-bound phenolics in tubers of untransformed potato plants but not in transgenic tubers. The transgenic tubers were also more susceptible to infection after inoculation with zoospores of Phytophthora infestans, which could be attributed to the modified cell wall of these plants. This study provides strong evidence that the synthesis and accumulation of phenolic compounds, including lignin, could be regulated by altering substrate availability through the introduction of a single gene outside the pathway involved in substrate supply. This study also indicates that phenolics, such as chlorogenic acid, play a critical role in defense responses of plants to fungal attack.

Journal Article↗

PubMedDate not supplied

Analysis of genetic diversity at the arom locus in isolates of Pneumocystis carinii.

The DNA sequences of a portion of the 5-enolpyruvyl shikimate phosphate synthase domain of the arom gene, encoding the pentafunctional AROM protein, were determined from isolates of Pneumocystis carinii from five mammalian host species (rat, human, ferret, rabbit and mouse). High levels of genetic divergence were found among P. carinii derived from different host species, 7-22% at the DNA sequence level, and 7-26% at the derived amino acid sequence level. Two separate and distinct sequences were isolated from infected ferret lungs. Low levels of divergence were seen in human-derived organisms.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗

PubMed2001

Molecular characterization of tomato 3-dehydroquinate dehydratase-shikimate:NADP oxidoreductase.

Analysis of cDNAs encoding the bifunctional 3-dehydroquinate dehydratase-shikimate:NADP oxidoreductase (DHQase-SORase) from tomato (Lycopersicon esculentum) revealed two classes of cDNAs that differed by 57 bp within the coding regions, but were otherwise identical. Comparison of these cDNA sequences with the sequence of the corresponding single gene unequivocally proved that the primary transcript is differentially spliced, potentially giving rise to two polypeptides that differ by 19 amino acids. Quantitative real-time polymerase chain reaction revealed that the longer transcript constitutes at most 1% to 2% of DHQase-SORase transcripts. Expression of the respective polypeptides in Escherichia coli mutants lacking the DHQase or the SORase activity gave functional complementation only in case of the shorter polypeptide, indicating that skipping of a potential exon is a prerequisite for the production of an enzymatically active protein. The deduced amino acid sequence revealed that the DHQase-SORase is most likely synthesized as a precursor with a very short (13-amino acid) plastid-specific transit peptide. Like other genes encoding enzymes of the prechorismate pathway in tomato, this gene is elicitor-inducible. Tissue-specific expression resembles the patterns obtained for 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase 2 and dehydroquinate synthase genes. This work completes our studies of the prechorismate pathway in that cDNAs for all seven enzymes (including isozymes) of the prechorismate pathway from tomato have now been characterized.

3' Untranslated Regions↗

PubMed2006

3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase is regulated for the accumulation of polysaccharide-linked hydroxycinnamoyl esters in rice (Oryza sativa L.) internode cell walls.

Polysaccharide-linked hydroxycinnamoyl esters (PHEs) over-accumulate in the internodes of a rice (Oryza sativa L.) mutant, Fukei 71 (F71). This accumulation is accompanied by over-expression of phenylalanine ammonialyase (PAL). In this study, we show that only one member of the 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS) family expresses in close correlation with PAL. Furthermore, substrate availability to DAHPS is promoted by down-regulating the expression of plastidic pyruvate kinase (PKp), a competitor of DAHPS. Since the over-production of PHEs is caused by D50 gene disruption, these results suggest that specific enzymes in the phenylpropanoid and shikimate pathways are coordinately up-regulated. In addition, the results indicate that carbon-flow into the shikimate pathway is modified for the synthesis of PHEs, and is probably controlled by D50.

3-Deoxy-7-Phosphoheptulonate Synthase↗

PubMed1993

The genes aroA and trnQ are located upstream of psbO in the chromosome of Synechocystis 6803.

We have identified the existence of two genes, trnQ and aroA, located upstream of the psbO gene in Synechocystis sp. PCC 6803. The trnQ gene encodes a glutamine-specific transfer RNA (tRNA(Gln)) and the sequence given is the first reported for any cyanobacterium. The gene seems to exist as a single copy since its deletion results in non-viable mutation. The aroA gene encodes for 5-enolpyruvylshikimate 3-phosphate synthase and its discovery in the genome of Synechocystis 6803 is the first genetic evidence for the existence of the shikimate biosynthetic pathway in cyanobacteria. Interestingly, the partial sequence shares close homologies with the sequences of aroA from Gram-positive bacteria.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗

PubMed1995

5-Enolpyruvylshikimate-3-phosphate synthase of Bacillus subtilis is an allosteric enzyme. Analysis of Arg24-->Asp, Pro105-->Ser and His385-->Lys mutations suggests a hidden phosphoenolpyruvate-binding site.

5-Enolpyruvylshikimate-3-phosphate synthase of Bacillus subtilis has been cloned, expressed and purified to near homogeneity. Clustal alignment of the amino acid sequences from different bacteria revealed several conserved residues located in the N-terminal, middle and C-terminal domains. The role of conserved Arg24, Pro105, and His385 residues has been examined by site-directed mutagenesis. Steady-state kinetic analysis of the native synthase exhibited allosteric behaviour, a feature thought to be unique amongst bacterial and plant 5-enolpyruvylshikimate-3-phosphate synthase enzymes investigated so far. Both substrates, phosphoenolpyruvate (P-pyruvate) and shikimate 3-phosphate have multiple interaction sites. There are two sites for P-pyruvate binding, catalytic and non-catalytic. Glyphosate (N-phosphonomethyl glycine) competes for binding at the catalytic site and does not interact at the secondary site. Glyphosate in the absence of ammonium ions increases cooperativity of P-pyruvate binding and favors dimerization of the enzyme through an interaction between P-pyruvate-binding sites. The ammonium-ion-activated 5-enolpyruvylshikimate-3-phosphate synthase displays no cooperativity with respect to P-pyruvate. Absence of ammonium ions decreases affinity for substrates and introduces cooperativity. Cooperativity was also introduced in the enzyme by point mutations, Arg24-->Asp and His385-->Lys. The latter mutant of the native enzyme exists as a dimer and aggregates to a tetrameric form in the presence of glyphosate. The occurrence of multimeric forms of the synthase has been demonstrated by staining for the enzyme activity on the native gel and by resolving purified enzyme preparations on a sucrose density gradient. A model describing the alteration in the aggregation status of the enzyme by the inhibitor, activator and the substrates has been proposed.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗

PubMed1994

Evolution of transcription-regulating proteins by enzyme recruitment: molecular models for nitrogen metabolite repression and ethanol utilisation in eukaryotes.

Studies on the quinic acid utilisation gene (qut) cluster in Aspergillus nidulans showed that the genes encoding transcriptional activator and repressor proteins evolved by co-opting duplicated copies of genes encoding metabolic enzymes. In order to test the hypothesis that this was a general route for the genesis of regulatory proteins, the origins of the major control protein mediating nitrogen metabolite repression (an example of inter-pathway regulation) and ethanol utilisation (an example of intra-pathway regulation) in filamentous fungi were sought. The regulatory proteins mediating nitrogen metabolite repression were deduced to have originated in a duplication of genes encoding the anthranilate synthase complex which is active in the shikimate pathway. The major protein regulating ethanol utilisation was deduced to have its origin in the fusion of duplicated genes encoding the aldehyde and alcohol dehydrogenases (ALDA and ALCA). These data strongly support the view that transcriptional regulatory proteins evolve by the recruitment of functional domains provided by metabolic enzymes.

Aspergillus nidulans↗

PubMed1989

The biosynthesis of phytoalexins in Dianthus caryophyllus L. cell cultures: induction of benzoyl-CoA:anthranilate N-benzoyltransferase activity.

It has been shown that cell cultures of Dianthus caryophyllus L. c.v. Eleganz accumulate N-benzoyl-4-methoxyanthranilic acid, previously identified as the phytoalexin methoxydianthramide B, in response to treatment either with a crude elicitor isolated from the cell walls of Phytophthora megasperma f.sp. glycinea or with a commercial yeast extract. Cell-free extracts from the induced cells efficiently catalyzed the N-benzoylation of anthranilate in the presence of benzoyl-CoA. The partially purified transferase was shown to be specific for anthranilate with almost no activity toward 4-hydroxyanthranilate, whereas acyl donors other than benzoyl-CoA such as salicyloyl-, cinnamoyl-, or 4-coumaroyl-CoA were also accepted. Elicitor treatment of the cells additionally induced an S-adenosyl-L-methionine:N-benzoyl-4-hydroxyanthranilate 4-O-methyltransferase activity. We propose, therefore, that methoxydianthramide B is derived from N-benzoylanthranilic acid via N-benzoyl-4-hydroxyanthranilic acid. Dark-grown cells contained little N-benzoyltransferase activity (approx 8 mu kat/kg), which increased roughly ninefold within 6 h following the addition of the elicitor. In addition, phenylalanine ammonia-lyase activity of the cells increased about twofold under these conditions to a maximum (approx 40 mu kat/kg) at 5 h. The rapid induction of both enzyme activities suggests that the shikimate pathway is of crucial importance in the disease resistance response of carnation cells.

Acyltransferases↗

PubMed2002

Molecular model of shikimate kinase from Mycobacterium tuberculosis.

Tuberculosis (TB) resurged in the late 1980s and now kills approximately 3 million people a year. The reemergence of tuberculosis as a public health threat has created a need to develop new anti-mycobacterial agents. The shikimate pathway is an attractive target for herbicides and anti-microbial agents development because it is essential in algae, higher plants, bacteria, and fungi, but absent from mammals. Homologs to enzymes in the shikimate pathway have been identified in the genome sequence of Mycobacterium tuberculosis. Among them, the shikimate kinase I encoding gene (aroK) was proposed to be present by sequence homology. Accordingly, to pave the way for structural and functional efforts towards anti-mycobacterial agents development, here we describe the molecular modeling of M. tuberculosis shikimate kinase that should provide a structural framework on which the design of specific inhibitors may be based.

Adenosine Diphosphate↗

PubMed2002

Genes coding for a new pathway of aerobic benzoate metabolism in Azoarcus evansii.

A new pathway for aerobic benzoate oxidation has been postulated for Azoarcus evansii and for a Bacillus stearothermophilus-like strain. Benzoate is first transformed into benzoyl coenzyme A (benzoyl-CoA), which subsequently is oxidized to 3-hydroxyadipyl-CoA and then to 3-ketoadipyl-CoA; all intermediates are CoA thioesters. The genes coding for this benzoate-induced pathway were investigated in the beta-proteobacterium A. evansii. They were identified on the basis of N-terminal amino acid sequences of purified benzoate metabolic enzymes and of benzoate-induced proteins identified on two-dimensional gels. Fifteen genes probably coding for the benzoate pathway were found to be clustered on the chromosome. These genes code for the following functions: a putative ATP-dependent benzoate transport system, benzoate-CoA ligase, a putative benzoyl-CoA oxygenase, a putative isomerizing enzyme, a putative ring-opening enzyme, enzymes for beta-oxidation of CoA-activated intermediates, thioesterase, and lactone hydrolase, as well as completely unknown enzymes belonging to new protein families. An unusual putative regulator protein consists of a regulator protein and a shikimate kinase I-type domain. A deletion mutant with a deletion in one gene (boxA) was unable to grow with benzoate as the sole organic substrate, but it was able to grow with 3-hydroxybenzoate and adipate. The data support the proposed pathway, which postulates operation of a new type of ring-hydroxylating dioxygenase acting on benzoyl-CoA and nonoxygenolytic ring cleavage. A beta-oxidation-like metabolism of the ring cleavage product is thought to lead to 3-ketoadipyl-CoA, which finally is cleaved into succinyl-CoA and acetyl-CoA.

Aerobiosis↗

PubMed1982

Biochemical diversity for biosynthesis of aromatic amino acids among the cyanobacteria.

We examined the enzymology and regulatory patterns of the aromatic amino acid pathway in 48 strains of cyanobacteria including representatives from each of the five major grouping. Extensive diversity was found in allosteric inhibition patterns of 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase, not only between the major groupings but also within several of the generic groupings. Unimetabolite inhibition by phenylalanine occurred in approximately half of the strains examined; in the other strains unimetabolite inhibition by tyrosine and cumulative, concerted, and additive patterns were found. The additive patterns suggest the presence of regulatory isozymes. Even though both arogenate and prephenate dehydrogenase activities were found in some strains, it seems clear that the arogenate pathway to tyrosine is a common trait that has been highly conserved among cyanobacteria. No arogenate dehydratase activities were found. In general, prephenate dehydratase activities were activated by tyrosine and inhibited by phenylalanine. Chorismate mutase, arogenate dehydrogenase, and shikimate dehydrogenase were nearly always unregulated. Most strains preferred NADP as the cofactor for the dehydrogenase activities. The diversity in the allosteric inhibition patterns for 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase, cofactor specificities, and the presence or absence of prephenate dehydrogenase activity allowed the separation of subgroupings within several of the form genera, namely, Synechococcus, Synechocystis, Anabaena, Nostoc, and Calothrix.

3-Deoxy-7-Phosphoheptulonate Synthase↗

PubMed1988

Biosynthesis of Territrems by Aspergillus terreus.

Different radioactive precursors were added to 8-day potato-dextrose liquid cultures of Aspergillus terreus 23-1. Territrems were isolated from chloroform extracts of the cultures at day 14 and purified by thin-layer chromatography and high-pressure liquid chromatography. The territrem B obtained was treated with alkaline hydrogen peroxide, and 3, 4, 5-trimethoxy benzoic acid was isolated from an ethyl acetate extract of the reaction mixture and purified by thin-layer chromatography and high-pressure liquid chromatography. By comparison of the specific radioactivities of territrem B and its cleaved aromatic product (disintegrations per minute per micromole of compound), it was demonstrated that the radioactivity of territrem B was located mainly on its aromatic moiety when [U-C]shikimate, l-[methyl-C]methionine, and l-[methyl-H]methionine were precursors; however, the radioactivity of territrem B was located mainly on its nonaromatic moiety when [2-C]mevalonate was the precursor. Mevinolin, a specific inhibitor of beta-hydroxyl beta-methyl glutaryl coenzyme A reductase, was shown to inhibit production of territrems by A. terreus 23-1. When [U-C]acetate was used as a precursor, mevinolin inhibited the incorporation of radioactive carbon into territrem but mevinolin did not inhibit incorporation of radioactive carbon from [2-C]mevalonate into territrem.

Journal Article↗

PubMed1992

Sequence organization and regulation of the Bacillus subtilis menBE operon.

Menaquinone (MK) plays a central role in the respiratory chain of Bacillus subtilis. The biosynthesis of MK requires the formation of a naphthoquinone ring via a series of specific reactions branching from the shikimate pathway. "Early" MK-specific reactions catalyze the formation of o-succinylbenzoate (OSB) from isochorismate, and "late" reactions convert OSB to dihydroxynaphthoate, by utilizing an OSB-coenzyme A intermediate. We have cloned and sequenced the B. subtilis menE and menB genes encoding, respectively, OSB-coenzyme A synthase and dihydroxynaphthoate synthase. The MenB open reading frame encodes a potential polypeptide of 261 amino acid residues with a predicted size of 28.5 kDa, while the MenE open reading frame could encode a 24.4-kDa polypeptide of 220 amino acid residues. Probable promoter sequences were identified by high-resolution primer extension assays. Organization of these genes and regulatory regions was found to be menBp menB menEp menE. Expression of menE was dependent on both menEp and menBp, indicating an operonlike organization. A region of dyad symmetry capable of forming a stable RNA secondary structure was found between menB and menE. Culture cycle-dependent expression of menB and menE was measured by steady-state transcript accumulation. For both genes, maximal accumulation was found to occur within an hour after the end of exponential growth. The menBp and menEp promoters have sequences compatible with recognition by the major vegetative form of B. subtilis RNA polymerase, E sigma A. Both promoter regions also were found to contain homologies to a sequence motif previously identified in the menCDp region and in promoters for several B. subtilis tricarboxylic acid cycle genes.

Amino Acid Sequence↗