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Summary: Plant secondary metabolism represents an important and ancient form of defense against pathogens. Phytopathogens secrete effectors to suppress plant defenses and promote infection. However, it is largely unknown, how fungal effectors directly manipulate plant secondary metabolism.Here, we characterized a fungal defense‐suppressing effector CfEC28 from Colletotrichum fructicola. Gene deletion assays showed that ∆CfEC28‐mutants differentiated appressoria normally on plant surface but were almost nonpathogenic due to increased number of plant papilla accumulation at attempted penetration sites. CfEC28 interacted with a family of chloroplast‐localized 3‐deoxy‐d‐arabinose‐heptulonic acid‐7‐phosphate synthases (DAHPSs) in apple. CfEC28 inhibited the enzymatic activity of an apple DAHPS (MdDAHPS1) and suppressed DAHPS‐mediated secondary metabolite accumulation through blocking the manganese ion binding region of DAHPS. Dramatically, transgene analysis revealed that overexpression of MdDAHPS1 provided apple with a complete resistance to C. fructicola.We showed that a novel effector CfEC28 can be delivered into plant chloroplasts and contributes to the full virulence of C. fructicola by targeting the DAHPS to disrupt the pathway linking the metabolism of primary carbohydrates with the biosynthesis of aromatic defense compounds.Our study provides important insights for understanding plant–microbe interactions and a valuable gene for improving plant disease resistance. [ABSTRACT FROM AUTHOR] more...

Unlabelled Image • Quinate is an alternative source of carbon for the shikimate pathway and is converted into tyrosine and phenylalanine. • In glyphosate-susceptible plants, the application of one-quarter of the recommended dose was as lethal as the recommended dose if quinate was sprayed one day after. • In glyphosate-resistant plants, quinate application after glyphosate did not change the lethality provoked by the herbicide but induced metabolic changes in the shikimate pathway. • The results raise the possibility of the application of quinate after glyphosate to improve the efficacy of the herbicide in the control of A. palmeri. [ABSTRACT FROM AUTHOR] more...

The shikimate pathway is a metabolic route for the biosynthesis of aromatic amino acids (AAAs) ( i.e . phenylalanine, tyrosine, and tryptophan). A key enzyme of shikimate pathway (5-enolpyruvylshikimate-3-phosphate synthase, EPSPS) is the target of the widely used herbicide glyphosate. Quinate is a compound synthesized in plants through a side branch of the shikimate pathway. Glyphosate provokes quinate accumulation and exogenous quinate application to plants shows a potential role of quinate in the toxicity of the herbicide glyphosate. Based on this, we hypothesized that the role of quinate accumulation in the toxicity of the glyphosate would be mediated by a deregulation of the shikimate pathway. In this study the effect of the glyphosate and of the exogenous quinate was evaluated in roots of pea plants by analyzing the time course of a full metabolic map of several metabolites of shikimate and phenylpropanoid pathways. Glyphosate application induced an increase of the 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHPS, first enzyme of the shikimate pathway) protein and accumulation of metabolites upstream of the enzyme EPSPS. No common effects on the metabolites and regulation of shikimate pathway were detected between quinate and glyphosate treatments, supporting that the importance of quinate in the mode of action of glyphosate is not mediated by a common alteration of the regulation of the shikimate pathway. Contrary to glyphosate, the exogenous quinate supplied was probably incorporated into the main trunk from the branch pathway and accumulated in the final products, such as lignin, concomitant with a decrease in the amount of DAHPS protein. [ABSTRACT FROM AUTHOR] more...

Environmental stresses such as high light intensity and temperature cause induction of the shikimate pathway, aromatic amino acids (AAA) pathways, and of pathways downstream from AAAs. The induction leads to production of specialized metabolites that protect the cells from oxidative damage. The regulation of the diverse AAA derived pathways is still not well understood. To gain insight on that regulation, we increased AAA production in red grape Vitis vinifera cv. Gamay Red cell suspension, without inducing external stress on the cells, and characterized the metabolic effect of this induction. Increased AAA production was achieved by expressing a feedback-insensitive bacterial form of 3-deoxy- D-arabino-heptulosonate 7-phosphate synthase enzyme (AroG*) of the shikimate pathway under a constitutive promoter. The presence of AroG* protein led to elevated levels of primary metabolites in the shikimate and AAA pathways including phenylalanine and tyrosine, and to a dramatic increase in phenylpropanoids. The AroG* transformed lines accumulated up to 20 and 150 fold higher levels of resveratrol and dihydroquercetin, respectively. Quercetin, formed from dihydroquercetin, and resveratrol, are health promoting metabolites that are induced due to environmental stresses. Testing the expression level of key genes along the stilbenoids, benzenoids and phenylpropanoid pathways showed that transcription was not affected by AroG*. This suggests that concentrations of AAAs, and of phenylalanine in particular, are rate-limiting in production of these metabolites. In contrast, increased phenylalanine production did not lead to elevated concentrations of anthocyanins, even though they are also phenylpropanoid metabolites. This suggests a control mechanism of this pathway that is independent of AAA concentration. Interestingly, total anthocyanin concentrations were slightly lower in AroG* cells, and the relative frequencies of the different anthocyanins changed as well. more...

Picroside-I (P-I) is a member of the iridoid glycosides family of natural products, which are used to treat liver disorders. The growing medicinal need for this benign compound has stimulated the present study to identify genes important for the biosynthesis of P-I. In this study, molecular screens have been generated using gene expression patterns obtained by quantitative RT-PCR which have extended the knowledge of genes associated with P-I biosynthesis. A total of 13 genes encoding the rate limiting enzymes of different pathways, were analyzed by qRT-PCR in Picrorhiza kurroa shoots collected at 0, 10, 20, 30 and 40 days. The results showed that five of the genes (HK, DXPS, ISPD, HMGR and PMK) are supposed to be essential for P-I biosynthesis up to 20 days while DAHPS and G-10-H, in conjunction might assist P-I biosynthesis between 20 and 30 days of P. kurroa growth. This is apparently the first report on the molecular aspects of different pathways integrated in P-I biosynthesis. Moreover, principal component analysis prediction also corroborated the genes selection by identifying genes signatures for different samples (collected at different time intervals) and supported the link between samples and gene expression patterns. Overall, this study capitalizes on dynamic gene expression patterns obtained in response to the P-I stimuli under different stages of P. kurroa growth which are likely to define the regulatory steps in P-I biosynthesis. [ABSTRACT FROM AUTHOR] more...

Environmental stresses such as high light intensity and temperature cause induction of the shikimate pathway, aromatic amino acids (AAA) pathways, and of pathways downstream from AAAs. The induction leads to production of specialized metabolites that protect the cells from oxidative damage. The regulation of the diverse AAA derived pathways is still not well understood. To gain insight on that regulation, we increased AAA production in red grape Vitis vinifera cv. Gamay Red cell suspension, without inducing external stress on the cells, and characterized the metabolic effect of this induction. Increased AAA production was achieved by expressing a feedback-insensitive bacterial form of 3-deoxy- D-arabino-heptulosonate 7-phosphate synthase enzyme (AroG*) of the shikimate pathway under a constitutive promoter. The presence of AroG* protein led to elevated levels of primary metabolites in the shikimate and AAA pathways including phenylalanine and tyrosine, and to a dramatic increase in phenylpropanoids. The AroG* transformed lines accumulated up to 20 and 150 fold higher levels of resveratrol and dihydroquercetin, respectively. Quercetin, formed from dihydroquercetin, and resveratrol, are health promoting metabolites that are induced due to environmental stresses. Testing the expression level of key genes along the stilbenoids, benzenoids, and phenylpropanoid pathways showed that transcription was not affected by AroG*. This suggests that concentrations of AAAs, and of phenylalanine in particular, are rate-limiting in production of these metabolites. In contrast, increased phenylalanine production did not lead to elevated concentrations of anthocyanins, even though they are also phenylpropanoid metabolites. This suggests a control mechanism of this pathway that is independent of AAA concentration. Interestingly, total anthocyanin concentrations were slightly lower in AroG* cells, and the relative frequencies of the different anthocyanins changed as well. [ABSTRACT FROM AUTHOR] more...

3-Deoxy- d - arabino- heptulosonate 7-phosphate synthase (DAH7PS) catalyses the first committed step of the shikimate pathway, which produces the aromatic amino acids as well as many other aromatic metabolites. DAH7PS catalyses an aldol-like reaction between phosphoenolpyruvate and erythrose 4-phosphate. Three phosphoenolpyruvate mimics, ( R )-phospholactate, ( S )-phospholactate and vinyl phosphonate [( E )-2-methyl-3-phosphonoacrylate], were found to competitively inhibit DAH7PS from Neisseria meningitidis , which is the pathogen responsible for bacterial meningitis. The most potent inhibitor was the vinyl phosphonate with a K i value of 3.9 ± 0.4 μM. We report for the first time crystal structures of these compounds bound in the active site of a DAH7PS enzyme which reveals that the inhibitors bind to the active site of the enzyme in binding modes that mimic those of the predicted oxocarbenium and tetrahedral intermediates of the enzyme-catalysed reaction. Furthermore, the inhibitors accommodate the binding of a key active site water molecule. Together, these observations provide strong evidence that this active site water participates directly in the DAH7PS reaction, enabling the facial selectivity of the enzyme-catalysed reaction sequence to be delineated. [ABSTRACT FROM AUTHOR] more...

Highlights: [•] The first enzyme of the shikimate pathway from Thermotoga maritima, DAH7PS, is inhibited by l-Tyr. [•] Substitution of residue Ser31 severely reduced the response to l-Tyr. [•] When Ser31 and His29 were interchanged to give variant His29Ser/Ser31His, l-Phe was a more potent inhibitor than l-Tyr. [ABSTRACT FROM AUTHOR] more...

Neisseria meningitidis is the causative agent of meningitis and meningococcal septicemia is a major cause of disease worldwide, resulting in brain damage and hearing loss, and can be fatal in a large proportion of cases. The enzyme 3-deoxy- d- arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyzes the first reaction in the shikimate pathway leading to the biosynthesis of aromatic metabolites including the aromatic acids l-Trp, l-Phe, and l-Tyr. This pathway is absent in humans, meaning that enzymes of the pathway are considered as potential candidates for therapeutic intervention. As the entry point, feedback inhibition of DAH7PS by pathway end products is a key mechanism for the control of pathway flux. The structure of the single DAH7PS expressed by N. meningitidis was determined at 2.0 Å resolution. In contrast to the other DAH7PS enzymes, which are inhibited only by a single aromatic amino acid, the N. meningitidis DAH7PS was inhibited by all three aromatic amino acids, showing greatest sensitivity to l-Phe. An N. meningitidis enzyme variant, in which a single Ser residue at the bottom of the inhibitor-binding cavity was substituted to Gly, altered inhibitor specificity from l-Phe to l-Tyr. Comparison of the crystal structures of both unbound and Tyr-bound forms and the small angle X-ray scattering profiles reveal that N. meningtidis DAH7PS undergoes no significant conformational change on inhibitor binding. These observations are consistent with an allosteric response arising from changes in protein motion rather than conformation, and suggest ligands that modulate protein dynamics may be effective inhibitors of this enzyme. [ABSTRACT FROM AUTHOR] more...

Abstract: 3-Deoxy-d-arabino-heptulosonate-7-phosphate synthase (DAHPS), (EC 2.5.1.54) catalyzes the first step of the shikimate pathway, the route for the biosynthesis of aromatic compounds in plants and microbes. In Actinosynnema pretiosum, the aroF gene (GenBank: AF056968.1) encodes DAHPS to condensate phosphoenolpyruvate (PEP) and d-erythrose 4-phosphate (E4P) to generate DAHP. In this study, a recombinant pET28a-aroF plasmid was constructed and A. pretiosum DAHPS was successfully expressed in soluble form by co-expression with chaperonins GroEL/GroES in Escherichia coli. The purification and kinetic characterization of the expressed protein were then investigated. The DAHPS originated from A. pretiosum demonstrated a pronounced substrate inhibition by PEP but was not sensitive to E4P. The purified enzyme was completely inactivated by EDTA but potently activated by several bivalent metal ions, especially Mn2+ and Co2+. [Copyright &y& Elsevier] more...

3-Deoxy-d-arabino-heptulosonate-7-phosphate synthase (DAHPS) catalyzes the first reaction of the aromatic biosynthetic pathway in bacteria, fungi, and plants, the condensation of phosphoenolpyruvate (PEP) and d-erythrose-4-phosphate (E4P) with the formation of DAHP. Crystals of DAHPS from Thermotoga maritima (DAHPSTm) were grown in the presence of PEP and metal cofactor, Cd2+, and then soaked with E4P at 4 °C where the catalytic activity of the enzyme is negligible. The crystal structure of the “frozen” reaction complex was determined at 2.2 Å resolution. The subunit of the DAHPSTm homotetramer consists of an N-terminal ferredoxin-like (FL) domain and a (β/α)8-barrel domain. The active site located at the C-end of the barrel contains Cd2+, PEP, and E4P, the latter bound in a non-productive conformation. The productive conformation of E4P is suggested and a catalytic mechanism of DAHPS is proposed. The active site of DAHPSTm is nearly identical to the active sites of the other two known DAHPS structures from Escherichia coli (DAHPSEc) and Saccharomyces cerevisiae (DAHPSSc). However, the secondary, tertiary, and quaternary structures of DAHPSTm are more similar to the functionally related enzyme, 3-deoxy-d-manno-octulosonate-8-phosphate synthase (KDOPS) from E. coli and Aquiflex aeolicus, than to DAHPSEc and DAHPSSc. Although DAHPSTm is feedback-regulated by tyrosine and phenylalanine, it lacks the extra barrel segments that are required for feedback inhibition in DAHPSEc and DAHPSSc. A sequence similarity search revealed that DAHPSs of phylogenetic family Iβ possess a FL domain like DAHPSTm while those of family Iα have extra barrel segments similar to those of DAHPSEc and DAHPSSc. This indicates that the mechanism of feedback regulation in DAHPSTm and other family Iβ enzymes is different from that of family Iα enzymes, most likely being mediated by the FL domain. [Copyright &y& Elsevier] more...

OBJECTIVES: Shikimate pathway, the pathway required for the synthesis of aromatic amino acids for microbial survival is very tightly controlled by feedback inhibition. The overproduction of aromatiXc amino acids requires the removal of this regulation. 3-deoxy-D-arabino-heptulosonate-7-phosphate-synthase (DAHPS) is the first enzyme of this pathway that is under feedback regulation. The three type-1 DAHPS enzymes in E. coli undergo feedback inhibition by phenylalanine, tyrosine, and tryptophan, respectively. In Corynebacterium glutamicum, there are two DAHPS enzymes: type-1 and type-2. Although the inhibition mechanism of the type-1 enzyme in C. glutamicum has been elucidated, it has been reported that this enzyme is not necessary for the cells. On the other hand, although phenylalanine and tyrosine were shown to inhibit the type-2 DAHPS enzyme, its mechanism is not clear. In this work, the data obtained from MD simulations of phenylalanine-regulated E.coli DAHPS will be used to predict residues to remove to remove feedback inhibition of type-2 DAHPS in C.glutamicum, is aimed. MATERIALS and METHODS: In this study, first, the amino acid sequences of E. coli and C. glutamicum DAHPS were compared with double and multiple sequence alignment tools. Using the information of feedback-regulation-resistant variants of E.coli phenylalanine-regulated DAHPS enzyme, molecular dynamics simulations of these variants and wild-type of the enzyme were performed after 50,000 minimization steps performed under NPT conditions at 1 bar pressure and 310 K temperature bu using the GROMACS program and CHARMM27 using the force-field function for 250 ns With the completion of the simulations, Root Mean Square Deviation (RMSD) and Root Mean Square Fluctuation (RMSF), Principal Component Analysis (PCA), Dynamic Cross Correlation Matrices (DCCM), RMSIP and further analysis will be performed. RESULTS: It was observed that the feedback inibition of DAHPS enzyme found in E.coli and encoded by AroG was removed by S180F, P150L and D146N mutations. For this reason, MD simulations and analyzes were performed on the structure with inhibitor and PDB code of 1KFL. Analysis results shows that S180F, P150L and D146N variants of DAHPS comes to equilibrium after 70ns and they shows significant difference from WT-DAHPS. The S180F variant of DAHPS enzyme is the most distinctive variant according to the RMSIP analysis. After further analyses, the prediction of the residue for C.glutamicum will be proceeded. CONCLUSIONS: Suggestions for mutations on the inhibitor binding site to make the C. glutamicum type-2 enzyme insensitive to feedback regulation will be proposed. [ABSTRACT FROM AUTHOR] more...

The leaf blade is the main photosynthetic organ and its morphology is related to light energy capture and conversion efficiency. We isolated a novel rice Dynamic Narrow-Rolled Leaf 1 (dnrl1) mutant showing reduced width of leaf blades, rolled leaves and lower chlorophyll content. The narrow-rolled leaf phenotype resulted from the reduced number of small longitudinal veins per leaf, smaller size and irregular arrangement of bulliform cells compared with the wild-type. DNRL1 was mapped to chromosome 7 and encoded a putative 3-deoxy-7-phosphoheptulonate synthase (DAHPS) which catalyzes the conversion of phosphoenolpyruvate and D-erythrose 4-phosphate to DAHP and phosphate. Sequence analysis revealed that a single base substitution (T–A) was detected in dnrl1, leading to a single amino acid change (L376H) in the coding protein. The mutation led to a lower expression level of DNRL1 as well as the lower activity of DAHPS in the mutant compared with the wild type. Genetic complementation and over-expression of DNRL1 could rescue the narrow-rolled phenotype. DNRL1 was constitutively expressed in all tested organs and exhibited different expression patterns from other narrow-rolled leaf genes. DNRL1-GFP located to chloroplasts. The lower level of chlorophyll in dnrl1 was associated with the downregulation of the genes responsible for chlorophyll biosynthesis and photosynthesis. Furthermore, dnrl1 showed significantly reduced levels of aromatic amino acids including Trp, Phe and Tyr. We conclude that OsDAHPS, encoded by DNRL1, plays a critical role in leaf morphogenesis by mediating the biosynthesis of amino acids in rice. [ABSTRACT FROM AUTHOR] more...