EPSP Synthase-Depleted Cells Are Aromatic Amino Acid Auxotrophs in Mycobacterium smegmatis.

The epidemiological importance of mycobacterial species is indisputable, and the necessity to find new molecules that can inhibit their growth is urgent. The shikimate pathway, required for the synthesis of important bacterial metabolites, represents a set of targets for inhibitors of Mycobacterium tuberculosis growth. The aroA-encoded 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) enzyme catalyzes the sixth step of the shikimate pathway. In this study, we combined gene disruption, gene knockdown, point mutations (D61W, R134A, E321N), and kinetic analysis to evaluate aroA gene essentiality and vulnerability of its protein product, EPSPS, from Mycolicibacterium (Mycobacterium) smegmatis (MsEPSPS). We demonstrate that aroA-deficient cells are auxotrophic for aromatic amino acids (AroAAs) and that the growth impairment observed for aroA-knockdown cells grown on defined medium can be rescued by AroAA supplementation. We also evaluated the essentiality of selected MsEPSPS residues in bacterial cells grown without AroAA supplementation. We found that the catalytic residues R134 and E321 are essential, while D61, presumably important for protein dynamics and suggested to have an indirect role in catalysis, is not essential under the growth conditions evaluated. We have also determined the catalytic efficiencies (Kcat/Km) of recombinant wild-type (WT) and mutated versions of MsEPSPS (D61W, R134A, E321N). Our results suggest that drug development efforts toward EPSPS inhibition may be ineffective if bacilli have access to external sources of AroAAs in the context of infection, which should be evaluated further. In the absence of AroAA supplementation, aroA from M. smegmatis is essential, its essentiality is dependent on MsEPSPS activity, and MsEPSPS is vulnerable. IMPORTANCE We found that cells from Mycobacterium smegmatis, a model organism safer and easier to study than the disease-causing mycobacterial species, when depleted of an enzyme from the shikimate pathway, are auxotrophic for the three aromatic amino acids (AroAAs) that serve as building blocks of cellular proteins: l-tryptophan, l-phenylalanine, and l-tyrosine. That supplementation with only AroAAs is sufficient to rescue viable cells with the shikimate pathway inactivated was unexpected, since this pathway produces an end product, chorismate, that is the starting compound of essential pathways other than the ones that produce AroAAs. The depleted enzyme, the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), catalyzes the sixth step of shikimate pathway. Depletion of this enzyme inside cells was performed by disrupting or silencing the EPSPS-encoding aroA gene. Finally, we evaluated the essentiality of specific residues from EPSPS that are important for its catalytic activity, determined with experiments of enzyme kinetics using recombinant EPSPS mutants.

A novel triple amino acid substitution in the EPSPS found in a high-level glyphosate-resistant Amaranthus hybridus population from Argentina.

BACKGROUND: The evolution of herbicide-resistant weeds is one of the most important concerns of global agriculture. Amaranthus hybridus L. is a competitive weed for summer crops in South America. In this article, we intend to unravel the molecular mechanisms by which an A. hybridus population from Argentina has become resistant to extraordinarily high levels of glyphosate. RESULTS: The glyphosate-resistant population (A) exhibited particularly high parameters of resistance (GR50 = 20 900 g ai ha-1 , Rf = 314), with all plants completing a normal life cycle even after 32X dose application. No shikimic acid accumulation was detected in the resistant plants at any of the glyphosate concentrations tested. Molecular and genetic analyses revealed a novel triple substitution (TAP-IVS: T102I, A103V, and P106S) in the 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS) enzyme of population A and an incipient increase on the epsps relative copy number but without effects on the epsps transcription levels. The novel mechanism was prevalent, with 48% and 52% of the individuals being homozygous and heterozygous for the triple substitution, respectively. In silico conformational studies revealed that TAP-IVS triple substitution would generate an EPSPS with a functional active site but with an increased restriction to glyphosate binding. CONCLUSION: The prevalence of the TAP-IVS triple substitution as the sole mechanism detected in the highly glyphosate resistant population suggests the evolution of a new glyphosate resistance mechanism arising in A. hybridus. This is the first report of a naturally occurring EPSPS triple substitution and the first glyphosate target-site resistance mechanism described in A. hybridus. © 2018 Society of Chemical Industry.

From tolerance to resistance: mechanisms governing the differential response to glyphosate in Chloris barbata.

BACKGROUND: Susceptibility and the mechanism (s) governing tolerance/resistance to glyphosate were characterized in two putative-glyphosate-resistant Chloris barbata populations (R1 and R2), collected in Persian lime orchards from Colima State, Mexico, comparing them with one non-treated population (referred to as S). RESULTS: Glyphosate doses required to reduce fresh weight or cause mortality by 50% were 4.2-6.4 times higher in resistant populations than in the S population. The S population accumulated 4.3 and 5.2 times more shikimate than the R2 and R1 populations, respectively. There were no differences in 14 C-glyphosate uptake between R and S populations, but the R plants translocated at least 12% less herbicide to the rest of plant and roots 96 h after treatment. Insignificant amounts of glyphosate were metabolized to aminomethyl phosphonate and glyoxylate in both R and S plants. The 5-enolpyruvylshikimate-3-phosphate synthase gene of the R populations contained the Pro106-Ser mutation, giving them a resistance 12 (R2) and 14.7 (R1) times greater at target-site level compared with the S population. CONCLUSION: The Pro106-Ser mutation governs the resistance to glyphosate of the R1 and R2 C barbata populations, but the impaired translocation could contribute to the resistance. These results confirm the first case of glyphosate resistance evolved in this species. © 2018 Society of Chemical Industry.

Unraveling the Addition-Elimination Mechanism of EPSP Synthase through Computer Modeling.

Enolpyruvyl transfer from phosphoenolpyruvate (PEP) to the hydroxyl group of shikimate-5-OH-3-phosphate (S3P) is catalyzed by 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase in a reaction that involves breaking the C-O bond of PEP. Catalysis involves an addition-elimination mechanism with the formation of a tetrahedral intermediate (THI). Experiments have elucidated the mechanism of THI formation and breakdown. However, the catalytic action of EPSP synthase and the individual roles of catalytic residues Asp313 and Glu341 remains unclear. We have used a hybrid quantum mechanical/molecular mechanical (QM/MM) approach to explore the free energy surface in a reaction catalyzed by EPSP synthase. The Glu341 was the most favorable acid/base catalyst. Our results indicate that the protonation of PEP C3 precedes the nucleophilic attack on PEP C2 in the addition mechanism. Also, the breaking of the C-O bond of THI to form an EPSP cation intermediate must occur before proton transfer from PEP C3 to Glu341 in the elimination mechanism. Analysis of the FES supports cationic intermediate formation during the reaction catalyzed by EPSP synthase. Finally, the computational model indicates a proton transfer shift (Hammond shift) from Glu341 to C3 for an enzyme-based reaction with the shifted transition state, earlier than in the reference reaction in water.

EPSP synthase flexibility is determinant to its function: computational molecular dynamics and metadynamics studies.

Flexibility is involved in a wide range of biological processes, such as protein assembly and binding recognition. EPSP synthase is an enzyme that must undergo a large conformational change to accommodate its ligands into its binding cavity. However, although the structure of EPSP synthase has been determined, its plasticity has not been explored in depth. Therefore, in this work, we extensively examined the influence of the flexibility of Mycobacterium tuberculosis EPSP (MtEPSP) synthase on the function of this protein using classical and replica-exchange metadynamics simulations. We were able to identify five well-populated conformational clusters for MtEPSP synthase: two corresponding to open, one to ajar, and two to closed conformations. We also pinpointed three hydrophobic regions that are responsible for guiding transitions among these states. Taken together, the new findings presented here indicate how the hydrophobic regions modulate the flexibility of MtEPSP synthase, and they highlight the importance of considering these dynamic features in drug design projects employing this enzyme as a target. Graphical abstract The flexibility of EPSP synthase as a function of the pincer angles.

Confirmation and mechanism of glyphosate resistance in tall windmill grass (Chloris elata) from Brazil.

BACKGROUND: Overreliance on glyphosate as a single tool for weed management in agricultural systems in Brazil has selected glyphosate-resistant populations of tall windmill grass (Chloris elata Desv.). RESULTS: Two C. elata populations, one glyphosate resistant (GR) and one glyphosate susceptible (GS), were studied in detail for a dose-response experiment and for resistance mechanism. The dose causing 50% reduction in dry weight was 620 g a.e. ha(-1) for GR and 114 g ha(-1) for GS, resulting in an R/S ratio of 5.4. GS had significantly higher maximum (14) C-glyphosate absorption into the treated leaf (51.3%) than GR (39.5%), a difference of 11.8% in maximum absorption. GR also retained more (14) C-glyphosate in the treated leaf (74%) than GS (51%), and GR translocated less glyphosate (27%) to other plant parts (stems, roots and root exudation) than GS (36%). There were no mutations at the Pro106 codon in the gene encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). There was no difference in EPSPS genomic copy number or EPSPS transcription between GS and GR populations. CONCLUSION: Based on these data, reduced glyphosate absorption and increased glyphosate retention in the treated leaf contribute to glyphosate resistance in this C. elata population from Brazil. © 2015 Society of Chemical Industry.

Construction and assessment of reaction models of Class I EPSP synthase. Part II: investigation of the EPSP ketal.

Although the proposed mechanisms are reasonable, there are still many questions about the 5-enolpyruvyl shikimate-3-phosphate (EPSP) synthase mechanism that are difficult to answer by experimental means alone. EPSP synthase is a key enzyme in the shikimic acid pathway, which is found only in plants and some micro-organisms and is also molecular target of glyphosate, active component of one of the top-selling herbicides. In the study of reaction mechanism of EPSP synthase, in addition to inorganic phosphate and EPSP products, after long time at equilibrium, it was shown that a side product is formed, the EPSP ketal. In this line, studies using density functional theory (DFT) techniques were performed to investigate the reaction mechanism of formation of EPSP and the corresponding ketal. Our findings indicate some key amino acid residues in the EPSP synthase mechanism and a possible route for the formation of the EPSP ketal.

Bio-inspired algorithms applied to molecular docking simulations.

Nature as a source of inspiration has been shown to have a great beneficial impact on the development of new computational methodologies. In this scenario, analyses of the interactions between a protein target and a ligand can be simulated by biologically inspired algorithms (BIAs). These algorithms mimic biological systems to create new paradigms for computation, such as neural networks, evolutionary computing, and swarm intelligence. This review provides a description of the main concepts behind BIAs applied to molecular docking simulations. Special attention is devoted to evolutionary algorithms, guided-directed evolutionary algorithms, and Lamarckian genetic algorithms. Recent applications of these methodologies to protein targets identified in the Mycobacterium tuberculosis genome are described.

Construction and assessment of reaction models of class I EPSP synthase: molecular docking and density functional theoretical calculations.

The high frequency of contamination by herbicides suggests the need for more active and selective herbicides. Glyphosate is the active component of one of the top-selling herbicides, which is also a potent EPSP synthase inhibitor. That is a key enzyme in the shikimic acid pathway, which is found only in plants and some microorganisms. Thus, EPSP synthase is regarded as a prime target for herbicides. In this line, molecular modeling studies using molecular dynamics simulations and DFT techniques were performed to understand the interaction of glyphosate and its analogs with the wild type enzyme and Gly96Ala mutant EPSP synthase. In addition, we investigated the reaction mechanism of the natural substrate. Our findings indicate some key points to the design of new selective glyphosate derivates.

Dynamics of glyphosate-induced conformational changes of Mycobacterium tuberculosis 5-enolpyruvylshikimate-3-phosphate synthase (EC 2.5.1.19) determined by hydrogen-deuterium exchange and electrospray mass spectrometry.

The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) catalyzes the reaction between shikimate 3-phosphate and phosphoenolpyruvate to form 5-enolpyruvylshikimate 3-phosphate, an intermediate in the shikimate pathway, which leads to the biosynthesis of aromatic amino acids. EPSPS exists in an open conformation in the absence of substrates and/or inhibitors and in a closed conformation when bound to the substrate and/or inhibitor. In the present report, the H/D exchange properties of EPSPS from Mycobacterium tuberculosis ( Mt) were investigated for both enzyme conformations using ESI mass spectrometry and circular dichroism (CD). When the conformational changes identified by H/D exchanges were mapped on the 3-D structure, it was observed that the apoenzyme underwent extensive conformational changes due to glyphosate complexation, characterized by an increase in the content of alpha-helices from 40% to 57%, while the beta-sheet content decreased from 30% to 23%. These results indicate that the enzyme underwent a series of rearrangements of its secondary structure that were accompanied by a large decrease in solvent access to many different regions of the protein. This was attributed to the compaction of 71% of alpha-helices and 57% of beta-sheets as a consequence of glyphosate binding to the enzyme. Apparently, MtEPSPS undergoes a series of inhibitor-induced conformational changes, which seem to have caused synergistic effects in preventing solvent access to the core of molecule, especially in the cleft region. This may be part of the mechanism of inhibition of the enzyme, which is required to prevent the hydration of the substrate binding site and also to induce the cleft closure to avoid entrance of the substrates.

5-Enolpyruvylshikimate-3-phosphate synthase: determination of the protonation state of active site residues by the semiempirical method.

EPSP synthase (EPSPS) catalyzes the addition of shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP) to form a tetrahedral intermediate (TI) that is converted to 5-enolpyruvylshikimate-3-phosphate (EPSP) and inorganic phosphate. A semiempirical molecular modeling study of the EPSPS active site containing the TI was implemented for the assignment of the protonation states of four basic residues, Lys22, Lys340, His385, and Lys411, based on the evaluation of 16 different protonation states and comparison of the resulting energy minimized heavy atoms coordinates with available X-ray crystallographic data of the D313A mutant of EPSPS. The results, employing both gas phase and continuum solvent models, are indicative that after the TI formation the histidine residue is most probably in neutral form (N(epsilon)-protonated) and the lysine residues are in protonated form, which suggests that none of the presently proposed assignments of aminoacid residues involved in the reaction mechanism could be completely correct. The protonated state of Lys22 in the presence of the TI supports the proposal that this residue is a general acid catalyst for TI breakdown. Modeling of the native enzyme active site suggests that Asp313 residue has only minor effects on the definition of the TI position inside the active site. Hydrogen-bonds distances suggest that, in order to act as a base, Asp313 needs the intermediacy of a hydroxyl group of the TI for effecting the attack on the TI methyl group in the elimination step leading to EPSP, as suggested previously in the literature.

GMOs: building the future on the basis of past experience.

Biosafety of genetically modified organisms (GMOs) and their derivatives is still a major topic in the agenda of government and societies worldwide. The aim of this review is to bring into light that data that supported the decision taken back in 1998 as an exercise to stimulate criticism from the scientific community for upcoming discussions and to avoid emotional and senseless arguments that could jeopardize future development in the field. It must be emphasized that Roundup Ready soybean is just one example of how biotechnology can bring in significant advances for society, not only through increased productivity, but also with beneficial environmental impact, thereby allowing more rational use of agricultural pesticides for improvement of the soil conditions. The adoption of agricultural practices with higher yield will also allow better distribution of income among small farmers. New species of genetically modified plants will soon be available and society should be capable of making decisions in an objective and well-informed manner, through collegiate bodies that are qualified in all aspects of biosafety and environmental impact.

Phosphate closes the solution structure of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) from Mycobacterium tuberculosis.

The 5-enolpyruvylshikimate-3-phosphate synthase catalyses the sixth step of the shikimate pathway that is responsible for synthesizing aromatic compounds and is absent in mammals, which makes it a potential target for drugs development against microbial diseases. Here, we report the phosphate binding effects at the structure of the 5-enolpyruvylshikimate-3-phosphate synthase from Mycobacterium tuberculosis. This enzyme is formed by two similar domains that close on each other induced by ligand binding, showing the occurrence of a large conformation change. We have monitored the phosphate binding effects using analytical ultracentrifugation, small angle X-ray scattering and, circular dichroism techniques. The low resolution results showed that the enzyme in the presence of phosphate clearly presented a more compact structure. Thermal-induced unfolding experiments followed by circular dichroism suggested that phosphate rigidified the enzyme. Summarizing, these data suggested that the phosphate itself is able to induce conformational change resulting in the closure movement in the M. tuberculosis 5-enolpyruvylshikimate-3-phosphate synthase.