Crystal structure of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase from the ESKAPE pathogenAcinetobacter baumannii

The enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the sixth step of the seven-step shikimate pathway. Chorismate, the product of the pathway, is a precursor for the biosynthesis of aromatic amino acids, siderophores and metabolites such as folate, ubiquinone and vitamin K. The shikimate pathway is present in bacteria, fungi, algae, plants and apicomplexan parasites, but is absent in humans. The EPSP synthase enzyme produces 5-enolpyruvylshikimate 3-phosphate and phosphate from phosphoenolpyruvate and shikimate 3-phosphateviaa transferase reaction, and is the target of the herbicide glyphosate. TheAcinetobacter baumanniigene encoding EPSP synthase,aroA, has previously been demonstrated to be essential during host infection for the growth and survival of this clinically important drug-resistant ESKAPE pathogen. Prephenate dehydrogenase is also encoded by the bifunctionalA. baumannii aroAgene, but its activity is dependent upon EPSP synthase since it operates downstream of the shikimate pathway. As part of an effort to evaluate new antimicrobial targets, recombinantA. baumanniiEPSP (AbEPSP) synthase, comprising residues Ala301–Gln756 of thearoAgene product, was overexpressed inEscherichia coli, purified and crystallized. The crystal structure, determined to 2.37 Å resolution, is described in the context of a potential antimicrobial target and in comparison to EPSP synthases that are resistant or sensitive to the herbicide glyphosate.

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Adaptation of bacteria to glyphosate: a microevolutionary perspective of the enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase

10.1101/2020.06.16.154005 ◽

2020 ◽

Author(s):

Miia J. Rainio ◽

Suvi Ruuskanen ◽

Marjo Helander ◽

Kari Saikkonen ◽

Irma Saloniemi ◽

...

Keyword(s):

Amino Acids ◽

Active Site ◽

Shikimate Pathway ◽

Aromatic Amino Acids ◽

Ecosystem Functions ◽

Epsp Synthase

ABSTRACTGlyphosate is the leading herbicide worldwide, but it also affects prokaryotes because it targets the central enzyme (EPSPS) of the shikimate pathway in the synthesis of the three essential aromatic amino acids in autotrophs. Our results reveal that bacteria easily become resistant to glyphosate through changes in the EPSPS active site. This indicates the importance of examining how glyphosate affects microbe-mediated ecosystem functions and human microbiomes.

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The Glyphosate Target Enzyme 5-Enolpyruvyl Shikimate 3-Phosphate Synthase (EPSPS) Contains Several EPSPS-Associated Domains in Fungi

Proceedings ◽

10.3390/iecge-07146 ◽

2020 ◽

Vol 76 (1) ◽

pp. 6

Author(s):

Tuomas Tall ◽

Pere Puigbò

Keyword(s):

Amino Acids ◽

Shikimate Pathway ◽

Aromatic Amino Acids ◽

Comprehensive Analysis ◽

Single Domain ◽

Differential Impact ◽

Herbicide Glyphosate ◽

Target Enzyme ◽

Phosphate Synthase

5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) is the central enzyme of the shikimate pathway to synthesize three aromatic amino acids in fungi, plants and prokaryotes. This enzyme is the target of the herbicide glyphosate. In most plants and prokaryotes, the EPSPS protein is constituted by a single domain, whereas in fungi, it contains several EPSPS-associated domains. Here, we perform a comprehensive analysis of 390 EPSPS proteins of fungi to determine the distribution and the evolution of the EPSPS-associated domains. The results of this study will be useful to determine the potential differential impact of glyphosate on alternative domain architectures in fungi.

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Evaluating aroA gene essentiality and EPSP synthase vulnerability in Mycobacterium smegmatis under different nutritional conditions

10.1101/2020.03.02.974360 ◽

2020 ◽

Author(s):

Mario Alejandro Duque-Villegas ◽

Bruno Lopes Abbadi ◽

Paulo Ricardo Romero ◽

Luiza Galina ◽

Pedro Ferrari Dalberto ◽

...

Keyword(s):

Amino Acids ◽

Mycobacterium Smegmatis ◽

Gene Knockout ◽

Shikimate Pathway ◽

Aromatic Amino Acids ◽

Gene Knockdown ◽

Epsp Synthase ◽

Gene Essentiality ◽

Nutritional Conditions ◽

Phosphate Synthase

AbstractThe epidemiological importance of bacteria from the genus Mycobacterium 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 metabolites in bacteria, represents a target 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 knockout, gene knockdown and kinetic assays to evaluate aroA gene essentiality and the vulnerability of its protein product, EPSPS synthase from Mycobacterium smegmatis (MsEPSPS), under different nutritional conditions. We demonstrate by an allelic exchange-based gene knockout approach the essentiality of MsEPSPS under rich and poor nutritional conditions. By performing gene complementation experiments with wild-type (WT) and point mutant versions of aroA gene, together with kinetic assays using WT and mutant recombinant proteins, we show that aroA gene essentiality depends on MsEPSPS activity. To evaluate MsEPSPS vulnerability, we performed gene knockdown experiments using the Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) system. The experiments were performed in both rich and defined (poor) media, using three different repression forces for aroA gene. We only observed growth impairment when bacteria were grown in defined medium without supplementation of aromatic amino acids, thereby indicating that MsEPSPS vulnerability depends on the environment conditions.ImportanceWe evaluated both gene essentiality and target vulnerability of the enzyme that catalyzes the sixth step of the shikimate pathway, the aroA-encoded 5-enolpyruvylshikimate-3-phosphate synthase from Mycobacterium smegmatis (MsEPSPS). Combining gene knockout experiments and kinetic assays, we established a causal link between aroA gene essentiality and the biological function of EPSPS protein, which we advocate is an indispensable step for target validation. Moreover, we characterized MsEPSPS vulnerability under different nutritional conditions and found it is a vulnerable target only when M. smegmatis is grown under poor nutritional conditions without supplementation with aromatic amino acids. Based on our findings, we suggest that gene essentiality information should be obtained from gene knockout experiments and not knockdown approaches, as even low levels of a protein after gene silencing can lead to a different growth phenotype when compared to that under its complete absence, as was the case with aroA and MsEPSPS in our study.

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

Microbiology Spectrum ◽

10.1128/spectrum.00009-21 ◽

2021 ◽

Vol 9 (3) ◽

Author(s):

Mario Alejandro Duque-Villegas ◽

Bruno Lopes Abbadi ◽

Paulo Ricardo Romero ◽

Letícia Beatriz Matter ◽

Luiza Galina ◽

...

Keyword(s):

Mycobacterium Smegmatis ◽

Model Organism ◽

Shikimate Pathway ◽

Aromatic Amino Acids ◽

Building Blocks ◽

Mycobacterial Species ◽

Epsp Synthase ◽

Content Type ◽

Viable Cells ◽

Starting Compound

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.

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Interaction of Plasmodium falciparum apicortin with α- and β-tubulin is critical for parasite growth and survival

Scientific Reports ◽

10.1038/s41598-021-83513-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Malabika Chakrabarti ◽

Nishant Joshi ◽

Geeta Kumari ◽

Preeti Singh ◽

Rumaisha Shoaib ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Life Cycle ◽

Specific Protein ◽

Parasite Growth ◽

Growth And Survival ◽

Apicomplexan Parasites ◽

Parasite Replication ◽

Main Components ◽

Β Tubulin

AbstractCytoskeletal structures of Apicomplexan parasites are important for parasite replication, motility, invasion to the host cell and survival. Apicortin, an Apicomplexan specific protein appears to be a crucial factor in maintaining stability of the parasite cytoskeletal assemblies. However, the function of apicortin, in terms of interaction with microtubules still remains elusive. Herein, we have attempted to elucidate the function of Plasmodium falciparum apicortin by monitoring its interaction with two main components of parasite microtubular structure, α-tubulin-I and β-tubulin through in silico and in vitro studies. Further, a p25 domain binding generic drug Tamoxifen (TMX), was used to disrupt PfApicortin-tubulin interactions which led to the inhibition in growth and progression of blood stage life cycle of P. falciparum.

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History of Herbicide-Tolerant Crops, Methods of Development and Current State of the Art – Emphasis on Glyphosate Tolerance

Weed Technology ◽

10.1017/s0890037x00035934 ◽

1992 ◽

Vol 6 (3) ◽

pp. 626-634 ◽

Cited By ~ 44

Author(s):

Ganesh M. Kishore ◽

Stephen R. Padgette ◽

Robert T. Fraley

Keyword(s):

Weed Management ◽

Effective Means ◽

Shikimate Pathway ◽

Management Program ◽

Crop Plants ◽

Epsp Synthase ◽

Glyphosate Tolerance ◽

Specific Expression ◽

Expression Of Genes ◽

Wide Range

Weed management is an integral part of agriculture; weeds lower both productivity and quality of agricultural products. A combination of mechanical, chemical, biological, and cultural methods is expected to deliver a sustainable weed management program for the next two decades. While chemical methods offer the most cost effective means of weed management, crop selectivity has hampered the use of the best chemicals for weed management. Recent progress in gene technology has facilitated the introduction and expression of genes to confer a wide range of traits to crop plants. Application of this technology has resulted in the development of crop plant genotypes that are resistant to a specific herbicide. This article describes the progress that has been made by our group toward the introduction of glyphosate tolerance to crop plants. Glyphosate [N-(phosphonomethyl)glycine] kills plants due to inhibition of the biosynthesis of aromatic compounds via the shikimate pathway. Our approach for introduction of glyphosate tolerance is based on insertion and expression in plants of a gene encoding a glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, a key enzyme of the shikimate pathway. The wild type enzyme present in plants is susceptible to inhibition glyphosate; variants of EPSP synthase have been produced that are less susceptible to inhibition by glyphosate. Expression of genes encoding these variants has been shown to confer glyphosate tolerance to plants. The degree of glyphosate tolerance is related to the tolerance characteristics of the EPSP synthase variant, its substrate activity, targeting to the plastid, and the level of expression of the variant gene. The tissue specificity of expression of the variant EPSP synthase has also been shown to be critical since glyphosate is a systemic herbicide and is translocated to many growing points within the plant. Our studies on glyphosate tolerance have substantially enhanced our understanding of the mode-of-action of glyphosate, the shikimate pathway, and protein sorting within plant cells, as well as developmental and tissue specific expression of genes in plants. Commercial use of glyphosate tolerance technology is expected to affect positively, the weed management arsenal available to the farmers, the sustainability of farm land and groundwater, and promote the use of a “soft” herbicide.

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PXO_RS20535, Encoding a Novel Response Regulator, Is Required for Chemotactic Motility, Biofilm Formation, and Tolerance to Oxidative Stress in Xanthomonas oryzae pv. oryzae

Pathogens ◽

10.3390/pathogens9110956 ◽

2020 ◽

Vol 9 (11) ◽

pp. 956

Author(s):

Abdulwahab Antar ◽

Mi-Ae Lee ◽

Youngchul Yoo ◽

Man-Ho Cho ◽

Sang-Won Lee

Keyword(s):

Oxidative Stress ◽

Mutant Strain ◽

Intracellular Signaling ◽

Biofilm Formation ◽

Response Regulator ◽

Xanthomonas Oryzae ◽

Knockout Mutant ◽

Gene Encoding ◽

Growth And Survival ◽

Intracellular Signaling Pathway

Xanthomonas oryzae pv. oryzae (Xoo), a causal agent of bacterial leaf blight of rice, possesses two-component regulatory systems (TCSs) as an intracellular signaling pathway. In this study, we observed changes in virulence, biofilm formation, motility, chemotaxis, and tolerance against oxidative stress of a knockout mutant strain for the PXO_RS20535 gene, encoding an orphan response regulator (RR). The mutant strain lost virulence, produced significantly less biofilm, and showed remarkably reduced motility in swimming, swarming, and twitching. Furthermore, the mutant strain lost glucose-guided movement and showed clear diminution of growth and survival in the presence of H2O2. These results indicate that the RR protein encoded in the PXO_RS20535 gene (or a TCS mediated by the protein) is closely involved in regulation of biofilm formation, all types of motility, chemotaxis, and tolerance against reactive oxygen species (ROS) in Xoo. Moreover we found that the expression of most genes required for a type six secretion system (T6SS) was decreased in the mutant, suggesting that lack of the RR gene most likely leads to defect of T6SS in Xoo.

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Shikimate pathway in apicomplexan parasites

Nature ◽

10.1038/16618 ◽

1999 ◽

Vol 397 (6716) ◽

pp. 219-220 ◽

Cited By ~ 54

Author(s):

Patrick J. Keeling ◽

Jeffrey D. Palmer ◽

Robert G. K. Donald ◽

David S. Roos ◽

Ross F. Waller ◽

...

Keyword(s):

Shikimate Pathway ◽

Apicomplexan Parasites

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Cloning, sequencing, expression, purification and preliminary characterization of a type II dehydroquinase from Helicobacter pylori

Biochemical Journal ◽

10.1042/bj3190559 ◽

1996 ◽

Vol 319 (2) ◽

pp. 559-565 ◽

Cited By ~ 21

Author(s):

Joanna R BOTTOMLEY ◽

Christopher L. CLAYTON ◽

Peter A. CHALK ◽

Colin KLEANTHOUS

Keyword(s):

Helicobacter Pylori ◽

Sequence Data ◽

Shikimate Pathway ◽

Cosmid Library ◽

Type Ii ◽

Gene Encoding ◽

Heat Stable ◽

Plasmid Construct ◽

H Pylori ◽

Type Ii Dehydroquinase

A heat-stable dehydroquinase was purified to near homogeneity from a plate-grown suspension of the Gram-negative stomach pathogen Helicobacter pylori, and shown from both its subunit and native molecular masses to be a member of the type II family of dehydroquinases. This was confirmed by N-terminal amino acid sequence data. The gene encoding this activity was isolated following initial identification, by random sequencing of the H. pylori genome, of a 96 bp fragment, the translated sequence of which showed strong identity to a C-terminal region of other type II enzymes. Southern blot analysis of a cosmid library identified several potential clones, one of which complemented an Escherichia coliaroD point mutant strain deficient in host dehydroquinase. The gene encoding the H. pylori type II dehydroquinase (designated aroQ) was sequenced. The translated sequence was identical to the N-terminal sequence obtained directly from the purified protein, and showed strong identity to other members of the type II family of dehydroquinases. The enzyme was readily expressed in E. coli from a plasmid construct from which several milligrams of protein could be isolated, and the molecular mass of the protein was confirmed by electrospray MS. The aroQ gene in H. pylori may function in the central biosynthetic shikimate pathway of this bacterium, thus opening the way for the construction of attenuated strains as potential vaccines as well as offering a new target for selective enzyme inhibition.

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Requirement for the Murine Zinc Finger Protein ZFR in Perigastrulation Growth and Survival

Molecular and Cellular Biology ◽

10.1128/mcb.21.8.2880-2890.2001 ◽

2001 ◽

Vol 21 (8) ◽

pp. 2880-2890 ◽

Cited By ~ 18

Author(s):

Madeleine J. Meagher ◽

Robert E. Braun

Keyword(s):

Cell Death ◽

Programmed Cell Death ◽

Zinc Finger ◽

Zinc Finger Protein ◽

Cellular Growth ◽

Gene Encoding ◽

Growth And Survival ◽

Finger Protein ◽

Development Delay ◽

Cellular Phenotypes

ABSTRACT The transition from preimplantation to postimplantation development leads to the initiation of complex cellular differentiation and morphogenetic movements, a dramatic decrease in cell cycle length, and a commensurate increase in the size of the embryo. Accompanying these changes is the need for the transfer of nutrients from the mother to the embryo and the elaboration of sophisticated genetic networks that monitor genomic integrity and the homeostatic control of cellular growth, differentiation, and programmed cell death. To determine the function of the murine zinc finger protein ZFR in these events, we generated mice carrying a null mutation in the gene encoding it. Homozygous mutant embryos form normal-appearing blastocysts that implant and initiate the process of gastrulation. Mutant embryos form mesoderm but they are delayed in their development and fail to form normal anterior embryonic structures. Loss of ZFR function leads to both an increase in programmed cell death and a decrease in mitotic index, especially in the region of the distal tip of the embryonic ectoderm. Mutant embryos also have an apparent reduction in apical vacuoles in the columnar visceral endoderm cells in the extraembryonic region. Together, these cellular phenotypes lead to a dramatic development delay and embryonic death by 8 to 9 days of gestation, which are independent of p53 function.

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