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Diverse rigidified shikimic acids derivatives, which are stable mimetics of the high-energy conformation of shikimic acid, have been synthesized to enhance inhibitor efficacy against shikimate kinase enzyme (SK), an attractive target for antibiotic drug discovery. The synthesis of the reported conformationally restricted shikimic acid derivatives was carried out by ring-closing metathesis of allyloxy vinyl derivatives as the key step. The rigidification of the ligand conformation was used to maximize the effectiveness of the substituents introduced in the ether carbon bridge of the scaffold by pre-orienting their interaction with key residues and enzyme domains that are essential for catalysis and enzyme motion. Molecular Dynamics simulation studies on the enzyme/ligand complexes revealed marked differences in the positioning of the ligand substituent in the active site of the two enzymes studied (SK from Mycobacterium tuberculosis and Helicobacter pylori) and this explains their greater efficacy against one of the enzymes. This enhancement is due to the distinct induced-fit motion of the two homologous enzymes. A 20-fold improvement against the H. pylori enzyme was achieved by the introduction of a CH2OEt group in the rigid ether bridge of the reported shikimic acid analogs

Like all higher organisms, plants have evolved in the context of a microbial world, shaping both their evolution and their contemporary ecology. Interactions between plant roots and soil microorganisms are critical for plant fitness in natural environments. Given this co-evolution and the pivotal importance of plant-microbial interactions, it has been hypothesized, and a growing body of literature suggests, that plants may regulate the composition of their rhizosphere to promote the growth of microorganisms that improve plant fitness in a given ecosystem. Here, using a combination of comparative genomics and exometabolomics, we show that pre-programmed developmental processes in plants (Avena barbata) result in consistent patterns in the chemical composition of root exudates. This chemical succession in the rhizosphere interacts with microbial metabolite substrate preferences that are predictable from genome sequences. Specifically, we observed a preference by rhizosphere bacteria for consumption of aromatic organic acids exuded by plants (nicotinic, shikimic, salicylic, cinnamic and indole-3-acetic). The combination of these plant exudation traits and microbial substrate uptake traits interact to yield the patterns of microbial community assembly observed in the rhizosphere of an annual grass. This discovery provides a mechanistic underpinning for the process of rhizosphere microbial community assembly and provides an attractive direction for the manipulation of the rhizosphere microbiome for beneficial outcomes.

Lignin poses a major challenge in the processing of plant biomass for agro-industrial applications. For bioengineering purposes, there is a pressing interest in identifying and characterizing the enzymes responsible for the biosynthesis of lignin. Hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl transferase (HCT; EC 2.3.1.133) is a key metabolic entry point for the synthesis of the most important lignin monomers: coniferyl and sinapyl alcohols. In this study, we investigated the substrate promiscuity of HCT from a bryophyte (Physcomitrella) and from five representatives of vascular plants (Arabidopsis, poplar, switchgrass, pine and Selaginella) using a yeast expression system. We demonstrate for these HCTs a conserved capacity to acylate with p-coumaroyl-CoA several phenolic compounds in addition to the canonical acceptor shikimate normally used during lignin biosynthesis. Using either recombinant HCT from switchgrass (PvHCT2a) or an Arabidopsis stem protein extract, we show evidence of the inhibitory effect of these phenolics on the synthesis of p-coumaroyl shikimate in vitro, which presumably occurs via a mechanism of competitive inhibition. A structural study of PvHCT2a confirmed the binding of a non-canonical acceptor in a similar manner to shikimate in the active site of the enzyme. Finally, we exploited in Arabidopsis the substrate flexibility of HCT to reduce lignin content and improve biomass saccharification by engineering transgenic lines that overproduce one of the HCT non-canonical acceptors. Our results demonstrate conservation of HCT substrate promiscuity and provide support for a new strategy for lignin reduction in the effort to improve the quality of plant biomass for forage and cellulosic biofuels.

In recent years, glyphosate-tolerant legumes have been used as cover crops for weed management in tropical areas of Mexico. Mexican cologania (Cologania broussonetii (Balb.) DC.) is an innate glyphosate-tolerant legume with a potential as a cover crop in temperate areas of the country. In this work, glyphosate tolerance was characterized in two Mexican cologania (a treated (T) and an untreated (UT)) populations as being representatives of the species, compared in turn to a glyphosate-susceptible hairy fleabane (S) (Conyza bonariensis (L.) Cronq.) population. Experiments revealed that T and UT Mexican cologania populations had a higher tolerance index (TI), and a lower shikimic acid accumulation and foliar retention than the hairy fleabane S population. Absorption and translocation, leaf morphology and metabolism studies were only carried out in the Mexican cologania T population and the hairy fleabane S population. The latter absorbed 37% more 14C-glyphosate compared to the Mexican cologania T at 96 h after treatment (HAT). Mexican cologania T translocated less herbicide from the treated leaf to the remainder of the plant than hairy fleabane S. The Mexican cologania T presented a greater epicuticular wax coverage percentage than the hairy fleabane S. This morphological characteristic contributed to the low glyphosate absorption observed in the Mexican cologania. In addition, the Mexican cologania T metabolized glyphosate mainly into AMPA, formaldehyde and sarcosine. These results indicate that the high glyphosate tolerance observed in Mexican cologania is mainly due to the poor penetration and translocation of glyphosate into the active site, and the high glyphosate degradation into non-toxic substances.

Gallic acid (GA), a key intermediate in the synthesis of plant hydrolysable tannins, is also a primary anti-inflammatory, cardio-protective agent found in wine, tea, and cocoa. In this publication, we reveal the identity of a gene and encoded protein essential for GA synthesis. Although it has long been recognized that plants, bacteria, and fungi synthesize and accumulate GA, the pathway leading to its synthesis was largely unknown. Here we provide evidence that shikimate dehydrogenase (SDH), a shikimate pathway enzyme essential for aromatic amino acid synthesis, is also required for GA production. Escherichia coli (E. coli) aroE mutants lacking a functional SDH can be complemented with the plant enzyme such that they grew on media lacking aromatic amino acids and produced GA in vitro. Transgenic Nicotiana tabacum lines expressing a Juglans regia SDH exhibited a 500% increase in GA accumulation. The J. regia and E. coli SDH was purified via overexpression in E. coli and used to measure substrate and cofactor kinetics, following reduction of NADP(+) to NADPH. Reversed-phase liquid chromatography coupled to electrospray mass spectrometry (RP-LC/ESI-MS) was used to quantify and validate GA production through dehydrogenation of 3-dehydroshikimate (3-DHS) by purified E. coli and J. regia SDH when shikimic acid (SA) or 3-DHS were used as substrates and NADP(+) as cofactor. Finally, we show that purified E. coli and J. regia SDH produced GA in vitro.

Glyphosate resistance was found in Lolium rigidum Gaudin (Rigid ryegrass, LOLRI) in South Africa. Suspected glyphosate-resistant L. rigidum populations were collected and grown under greenhouse conditions. The plants were sprayed with a range of doses of glyphosate 35 days after planting and shoot dry biomass was determined 17 days after herbicide treatment. Based on the dose-response experiment conducted in the greenhouse, one population of L. rigidum suspected to be resistant to glyphosate was approximately 5.3 fold more resistant than susceptible population. The other population was 2.8 fold more resistant than susceptible population. Difference between the two suspected resistant populations was 1.9 fold. All plants were treated with glyphosate (1000 g a.i. ha-1) and shikimic acid was extracted 2, 4 and 6 days after treatment. The plants of susceptible populations accumulated more shikimic acid than other two populations., Rezistentnost na glifosat je potvrđena kod Lolium rigidum u Južnoj Africi. Semena rezistentne, očekivano glifosat-rezistentne i osetljive populacije L. rigidum su sakupljena u usevu pšenice sa različitih lokaliteta na području Južnoafričke Republike. Testiranje rezistentnosti na glifosat je izvedena u kontrolisanim uslovima staklare Univerziteta u Pretoriji (Južnoafrička Republika). Tridesetpet dana nakon setve (u plastičnim sudovima) biljke su tretirane suspenzijom od 125, 250, 500, 1000, 2000, 4000, 8000 i 12000 ml ha-1 glifosata, a suva masa je izmerena 17 dana nakon primene herbicida. Za fitovanje krive i utvrđivanje efektivne doze (dose-response-test) korišćena je jednačina ne-linearne regresione analize (R softver, drc paket). Na osnovu dobijenih rezultata utvrđena je 5,3 puta veća otpornost na glifosat kod rezistentne populacije L. rigidum (RLR) u poređenju sa osetljivom populacijom (OLR). Kod pretpostavljeno rezistentne populacije (PRP) konstatovana je 2,8 puta veća otpornost u odnosu na osetljivu populaciju. Razlika između pretpostavljeno rezistentne i rezistentne populacije je bila 1,9 puta. Za utvđivanje efekta glifosata na sadržaj šikiminske kiseline biljke su tretirane suspenzijom od 1000 g a.m. ha-1, a sadržaj šikiminske kiseline (HPLC metodom) je meren 2, 4 i 6 dana posle primene herbicida. Sadržaj šikiminske kiseline je bio veći kod osetljive populacije u poređenju sa druge dve testirane populacije L. rigidum. Dakle, potvrđena je rezistentnost RLR populacije, odnosno osetljivost OLR populacije, što obavezuje farmere da sprovode elemente antirezistentne strategije da bi usporili i sprečili ekspanziju rezistentnih populacija L. rigidum na području Južnoafričke Republike. Takođe, ta iskustva treba preneti i na druga područja u svetu da bi se sprečilo/usporilo širenje rezistentnosti korova na glifosat.

Changes in leaf anatomy were examined in suspectedly resistant populations of C. canadensis and C. bonariensis after application of 2, 4 and 8 L ha-1 of the product Touch down (500 g a.i. glyphosate L-1). Samples for the analysis (LM, TEM) were collected 3, 7 and 24 h after herbicide application. Changes in chlorophyll and shikimic acid contents were also examined in the populations tested. Changes in the anatomy of leaves were not detected in the investigated populations 3 and 7 h after herbicide application. However, 24 h after application of different herbicide rates, differences were observed in leaf anatomy. Injuries were detected in C. bonariensis population after application of 2 L ha-1, and in C. canadensis population after treatment with the highest glyphosate rate (8 L ha-1). Changes were detected on cell walls, chloroplast lamellae and chloroplasts themselves. Based on chlorophyll and shikimic acid contents, significant statistical differences were detected between the treated and control plants., Praćene su promene u anatomskoj građi listova pretpostavljeno rezistentih populacija C. canadensis i C. bonariensis nakon primene 2, 4 i 8 L ha-1 Touch down (500 g a.m. glifosat L-1). Za analizu anatomske građe listova (LM, TEM) materijal je uzorkovan nakon 3, 7 i 24 h posle primene herbicida. Takođe, praćene su promene u sadržaju hlorofila i šikiminske kiseline kod testiranih populacija. Promene u anatomskoj građi listova nisu konstatovane kod ispitivanih populacija nakon 3 i 7 časova posle primene herbicida. Međutim, 24 časa nakon primene različitih količina uočene su razlike u anatomskoj građi listova. Oštećenja su bila uočljiva kod pop. C. bonariensis nakon primene 2 L ha-1, a kod pop. C. canadensis tek nakon primene najveće količine glifosata (8 L ha-1). Promene su uočene na ćelijskom zidu, lamelama hloroplasta, kao i na samim hloroplastama. Na osnovu sadržaja hlorofila i šikiminske kiseline takođe su utvrđene statistički značajne razlike između tretiranih i kontrolnih biljaka.

3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7P synthase) catalyses the condensation reaction between phosphoenolpyrtivate (PEP) and the four-carbon monosaccharide D-erythrose 4-phosphate (D-E4P). 3-Deoxy-D-manno-octulosonate 8-phosphate synthase (KDO8P synthase) catalyses a closely related reaction of PEP with the five-carbon monosaccharide D-arabinose 5-phosphate (D-A5P). These enzymes are two functionally unrelated enzymes that share many mechanistic and structural features. D-Threose 4-phosphate (D-T4P), L-threose 4-phosphate (L-T4P), D-arabinose 5-phosphate (D-A5P), D-lyxose 5-phosphate (D-L5P), and L-xylose 5-phosphate (L-X5P) have been prepared synthetically or enzymatically to provide insights into aspects of metal requirement and substrate specificity. These compounds were different stereoisomers of natural substrates D-E4P and D-A5P. The results presented in this thesis show that D-T4P and L-T4P (C2 and C3 stereoisomers of D-E4P) are substrates for the DAH7P synthases from E. coli and P. furiosus. For N. meningitidis KDO8P synthase, natural substrate D-A5P and L-X5P (the C4 epimer of D-A5P) were substrates, whereas D-L5P, the C3 epimer of D-A5P, was not. These observations show that the configuration of the C2 and C3 hydroxyl groups is not important for DAH7P synthase reaction, but having the correct configuration at these positions is critical for KDO8P synthase. The analysis of the interaction of D-T4P and L-T4P with DAH7P synthase, and D-A5P, D-R5P, and L-X5P reveals previously unrecognised mechanistic differences between the DAH7P synthase-catalysed reaction and that catalysed by the closely related enzyme, KDO8P synthase.

The short-term effects of the herbicide glyphosate (1.25-10 mM) on the growth, nitrogen fixation, carbohydrate metabolism, and shikimate pathway were investigated in leaves and nodules of nodulated lupine plants. All glyphosate treatments decreased nitrogenase activity rapidly (24 h) after application, even at the lowest and sublethal dose used (1.25 mM). This early effect on nitrogenase could not be related to either damage to nitrogenase components (I and II) or limitation of carbohydrates supplied by the host plant. In fact, further exposure to increasing glyphosate concentrations (5 mM) and greater time after exposure (5 days) decreased nodule starch content and sucrose synthase (SS; EC 2.4.1.13) activity but increased sucrose content within the nodule. These effects were accompanied by a great inhibition of the activity of phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31). There were remarkable and rapid effects on the increase of shikimic and protocatechuic (PCA) acids in nodules and leaves after herbicide application. On the basis of the role of shikimic acid and PCA in the regulation of PEPC, as potent competitive inhibitors, this additional effect provoked by glyphosate on 5-enolpyruvylshikimic-3-phosphate synthase enzyme (EPSPS; EC 2.5.1.19) inhibition would divert most PEP into the shikimate pathway, depriving energy substrates to bacteroids to maintain nitrogen fixation. These findings provide a new explanation for the effectiveness of glyphosate as a herbicide in other plant tissues, for the observed differences in tolerance among species or cultivars, and for the transitory effects on glyphosate-resistant transgenic crops under several environmental conditions.

Four strategies which involve synthetic elaboration of the shikimate nucleus are discussed. Methyl shikimate was differentially protected as the 3,4-0-cyclopentylidene ketal and as the 5-0-tert-butyldiphenylsilyl ether. The methyl ester was then reduced with diisobutylaluminum hydride and the resulting allylic alcohol was oxidized to aldehyde with pyridinium chlorochromate. The cyclopentylidene protecting group was removed to liberate the 3- and 4- hydroxyl groups which were acylated with maleic anhydride and succinic anhydride. The acylated aldehydes, as well as aldehyde were converted to their respective dimethlhydrazones.

Cultures of Streptomyces sp. 3022a were grown in the presence of C14-labelled substrates and incorporation of radioactivity into chloramphenicol measured. D-Glucose, labelled in carbons 1 or 2 or uniformly, was an efficient precursor of the p-nitrophenylserinol moiety and of the phenylpropanoid amino acids of the mycelium. Since phenylalanine and tyrosine were incorporated into the mycelium the biosynthetic route to the phenylpropanoid portion of chloramphenicol evidently does not pass through either of these amino acids but branches at an earlier step. (Author), Incooperation with Prairie Regional Lab., Saskatoon (Saskatchewan).