Divergent properties and phylogeny of cyanobacterial 5‐ enol ‐pyruvyl‐shikimate‐3‐phosphate synthases: evidence for horizontal gene transfer in the Nostocales (original) (raw)

Classification of the glyphosate target enzyme (5-enolpyruvylshikimate-3-phosphate synthase)

2020

ABSTRACTGlyphosate is the most common broad-spectrum herbicide. It targets the key enzyme of the shikimate pathway, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), which synthesizes three essential aromatic amino acids (phenylalanine, tyrosine and tryptophan) in plants. Because the shikimate pathway is also found in many prokaryotes and fungi, the widespread use of glyphosate may have unsuspected impacts on the diversity and composition of microbial communities, including the human gut microbiome. Here, we introduce the first bioinformatics method to assess the potential sensitivity of organisms to glyphosate based on the type of EPSPS enzyme. We have precomputed a dataset of EPSPS sequences from thousands of species that will be an invaluable resource to advancing the research field. This novel methodology can classify sequences from >90% of eukaryotes and >80% of prokaryotes. A conservative estimate from our results shows that 54% of species in the core human gut microb...

Reevaluating Glyphosate as a Transition-State Inhibitor of EPSP Synthase: Identification of an EPSP Synthase.cntdot.EPSP.cntdot.Glyphosate Ternary Complex

Biochemistry, 1995

Numerous studies have confirmed that glyphosate forms a tight ternary complex with EPSP synthase and shikimate 3-phosphate. It has been proposed 351-3571 that in this complex glyphosate functions as a transition-state analog of the putative phosphoenolpyruvoyl oxonium ion. For this to be true, glyphosate must occupy the space in the enzyme active site that is normally associated with PEP and, through turnover, the carboxyvinyl group of the product EPSP. According to this model, one would predict that, in the reverse EPSP synthase reaction with EPSP and phosphate as substrates, there should be little if any interaction of glyphosate with enzyme or enzymewbstrate complexes. In contrast to this expectation, rapid gel filtration experiments provided direct evidence that glyphosate could be trapped on the enzyme in the presence of EPSP to form a ternary complex of EPSPS-EPSP-glyphosate. The experimentally determined stoichiometry for this complex, 0.62 equiv of glyphosate/mole of EPSPS, is similar to that found for the EPSPSS3P-glyphosate ternary complex (0.66). This direct binding result was corroborated and quantitated by fluorescence titration experiments which demonstrated that glyphosate forms a reasonably tight (& = 56 f 1 pM) ternary complex with enzyme and EPSP. This finding was further verified, and its impact on substrate turnover analyzed, by steady-state kinetics. Glyphosate was found to be an uncompetitive inhibitor versus EPSP with Kii(app) = 54 f 2 pM. Taking these results together, it is apparent that the carboxyvinyl group in EPSP does not prevent glyphosate binding, and in fact it strongly facilitates the binding of this inhibitor to the enzyme. It has been previously demonstrated that glyphosate has little (& = 12 mM) interaction with free enzyme [Ream, J. E., Yuen, H. K., Frazier, R. B., & Sikorski, J. A. (1992) Biochemistry 31, 5528-55341. Interestingly, glyphosate was a mixed inhibitor (Kis(app1 = 18.2 f 0.4 pM, Kii(app) = 23.9 f 0.3 pM)

Divergent properties and phylogeny of cyanobacterial 5‐enol‐pyruvyl‐shikimate‐3‐phosphate synthases: evidence for horizontal gene transfer in the Nostocales

New Phytologist, 2014

Summary As it represents the target of the successful herbicide glyphosate, great attention has been paid to the shikimate pathway enzyme 5‐enol‐pyruvyl‐shikimate‐3‐phosphate (EPSP) synthase. However, inconsistent results have been reported concerning the sensitivity of the enzyme from cyanobacteria, and consequent inhibitory effects on cyanobacterial growth. The properties of EPSP synthase were investigated in a set of 42 strains representative of the large morphological diversity of these prokaryotes. Publicly available protein sequences were analyzed, and related to enzymatic features. In most cases, the native protein showed an unusual homodimeric composition and a general sensitivity to micromolar doses of glyphosate. By contrast, eight out of 15 Nostocales strains were found to possess a monomeric EPSP synthase, whose activity was inhibited only at concentrations exceeding 1 mM. Sequence analysis showed that these two forms are only distantly related, the latter clustering sep...

Characterization and plant expression of glyphosate-tolerant enolpyruvylshikimate phosphate synthase

2008

BACKGROUND: Glyphosate tolerance is a dominant trait in modern biotech crops. RESULTS: A gene encoding a glyphosate-tolerant EPSP synthase (aroA 1398 ) from bacterial strain ATX1398 was cloned and characterized. The protein is initiated at a GTG translational start codon to produce a protein that provides robust glyphosate resistance in Escherichia coli (Mig) Cast & Chalm. The aroA 1398 protein was expressed and purified from E. coli, and key kinetic values were determined (K i = 161 µM; K m (PEP) = 11.3 µM; k cat = 28.3 s −1 ). The full-length enzyme is 800-fold more resistant to glyphosate than the maize EPSP synthase while retaining high affinity for the substrate phosphoenol pyruvate. To evaluate further the potential of aroA 1398 , transgenic maize events expressing the aroA 1398 protein were generated. T 0 plants were screened for tolerance to glyphosate sprays at 1.3× commercial spray rates, and T 1 plants were selected that completely resisted glyphosate sprays at 1×, 2× and...

Glyphosate Resistant 5-ENOLPYRUVYLSHIKIMATE-3-PHOSPHATE (Epsp) Synthase

2013

A plant comprising SEQ. ID. NO. 2 or a functional portion thereof, Wherein SEQ ID NO. 2 is not native to said plant. A glyphosate resistant grass of economic value comprises a nucleic acid molecule that encodes a EPSPS enzyme. In some embodiments, the nucleic acid molecule comprises a sequence of SEQ. ID. NO. 1, or a functional portion thereof. In some embodiments, the EPSPS enzyme can be a polypep tide molecule comprising an amino acid sequence that is essentially of SEQ. ID. NO. 2, or portion thereof. Embodi ments include a DNA construct comprising a SEQ. ID. NO. 1 or a functional portion thereof and transgenic methods for inserting the DNA construct into a plant. Some embodiments include non-transgenic glyphosate resistant turfgrasses.

Glyphosate-Resistant Goosegrass. Identification of a Mutation in the Target Enzyme 5-Enolpyruvylshikimate-3Phosphate Synthase

Plant …, 2002

The spontaneous occurrence of resistance to the herbicide glyphosate in weed species has been an extremely infrequent event, despite over 20 years of extensive use. Recently, a glyphosate-resistant biotype of goosegrass (Eleusine indica) was identified in Malaysia exhibiting an LD 50 value approximately 2-to 4-fold greater than the sensitive biotype collected from the same region. A comparison of the inhibition of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) activity by glyphosate in extracts prepared from the resistant (R) and sensitive (S) biotypes revealed an approximately 5-fold higher IC 50 (glyphosate) for the (R) biotype. Sequence comparisons of the predicted EPSPS mature protein coding regions from both biotypes revealed four single-nucleotide differences, two of which result in amino acid changes. One of these changes, a proline to serine substitution at position 106 in the (R) biotype, corresponds to a substitution previously identified in a glyphosate-insensitive EPSPS enzyme from Salmonella typhimurium. Kinetic data generated for the recombinant enzymes suggests that the second substitution identified in the (R) EPSPS does not contribute significantly to its reduced glyphosate sensitivity. Escherichia coli aroAϪ (EPSPS deficient) strains expressing the mature EPSPS enzyme from the (R) biotype exhibited an approximately 3-fold increase in glyphosate tolerance relative to strains expressing the mature EPSPS from the (S) biotype. These results provide the first evidence for an altered EPSPS enzyme as an underlying component of evolved glyphosate resistance in any plant species. ; fax 662-915-1035.

Variation in 5-Enolpyruvylshikimate-3-Phosphate Synthase (EPSPS) Coding Sequences and Glyphosate Response among Cyperus rotundus L. Populations

American Journal of Plant Sciences, 2019

The gene sequence encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), the enzymatic target site of the herbicide glyphosate, was determined for several purple nutsedge (Cyperus rotundus L.) accessions from geographically distant locations and these were aligned to generate a consensus sequence. The EPSPS sequences each had single nucleotide polymor-How to cite this paper: Molin, W.T. and Bryson, C.T. (2019) Variation in 5-Enolpyruvylshikimate-3-Phosphate Synthase (EPSPS) Coding Sequences and Glyphosate Response among Cyperus rotundus L. Populations.

Enzymological Basis for Herbicidal Action of Glyphosate

Plant Physiology, 1982

The effects of 1 milliolar glyphosate (N-Iphosphonomethyllglycine) upon the activities ofenzymes of aromatic amino acid biosynthesis, partially purified by ion-exchange chromatography from mung bean seedings (Vigna radiata IL.I Wilczek), were examined. Multiple isozyme species of shikimate dehydrogenase, chorismate mutase, and aromatic aminotransferase were separated, and these were all insensitive to inhibition by glyphosate. The activities of prephenate dehydrogenase and arogenate dehydrogenase were also not sensitive to inhibition. Two molecular species of 3-deoxy-Darabino-heptulosonate 7-phosphate (DAHP) synthase were resolved, one stimulated several-fold by MnI2+ (DAHP synthase-Mn), and the other absolutely dependent upon the presence of Co2, for activity (DAHP synthase-Co). Whereas DAHP synthase-Mn was invulnerable to glyphosate, greater than 95% inhibition of DAHP synthase-Co was found in the presence of glyphosate. Since Co02 is a V33.. activator with respect to both substrates, glyphosate cannot act simply by Co2' chelation because inhibition is competitive with respect to erythrose4-phosphate. The accumulation of shikimate found in glyphosate-treated seedlings is consistent with in vivo inhibition of both 5-enolpyruvylshikimc acid 3-phosphate synthase and one of the two DAHP synthase isozymes. Aromatic amino acids, singly or in combination, only showed a trend towards reversal ofgrowth inhibition in 7-day seedlfings ofmung bean. The possibilities are raised that glyphosate may act at multiple enzyme targets in a given organism or that different plants may vary in the identity of the prime enzyme target. Glyphosate (N-[phosphonomethyl]glycine) is an exceedingly effective broad-spectrum herbicide which is readily translocated between plant tissues (29). Residual glyphosate also biodegrades very satisfactorily in soil environments (32). Beyond its herbicidal properties, glyphosate is often a potent inhibitor of microbial growth. The general finding that glyphosate inhibition is antagonized by one or more of the aromatic amino acids extends to a wide range of organisms, including prokaryotes, eukaryotic algae, and plants (15). In buckwheat and cultured cells of Galium mollugo L. (Rubiaceae), physiological experiments have shown that glyphosate causes shikimate accumulation and blocks the entry of radiolabeled shikimate into aromatic amino acids (1). Evidently, the accumulation of shikimate in response to glyphosate treatment generally occurs in plants, inasmuch as such data have now been obtained from dozens of diverse species (2, 3). These data are consistent with the possibility that EPSP2 synthase might be the 'Supported by Department of Energy Contract EP-78-024967