Purification and properties of a glyphosate-tolerant 5-enolpyruvylshikimate 3-phosphate synthase from the cyanobacterium Anabaena variabilis

Powell, Hilary A.; Kerby, Nigel W.; Rowell, P.; Mousdale, David M.; Coggins, John R.

doi:10.1007/bf00197039

Cited by 20 publications

(28 citation statements)

References 49 publications

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“…The deduced molecular mass of the subunit being c . 46 kDa (Powell et al ., ; Table in this work), the data suggest that either monomeric or homodimeric enzymes may occur among the representatives of Subsection IV .…”

Section: Methodssupporting

confidence: 56%

“…Interestingly, while the enzymes from Nostoc sp. PCC 7937 (‘ A. variabilis ’ ATCC 29413) and all other bacterial and plant species characterized to date show a monomeric structure, with a subunit relative molecular mass ranging from 40 to 60 kDa (Powell et al ., ; Forlani, ), the ‘ S. platensis ’ C1 EPSP synthase appeared to be homodimeric (Forlani & Campani, ).…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2014

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SummaryAs 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 separately in a clade composed of diverse bacterial phyla.The results are consistent with the occurrence of a horizontal gene transfer event involving an evolutionarily distant organism. Moreover, data suggest that the existence of class I (glyphosate-sensitive) and class II (glyphosate-tolerant) EPSP synthases representing two distinct phylogenetic clades is an oversimplification because of the limited number of analyzed samples.

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Section: Methodssupporting

confidence: 56%

Section: Introductionmentioning

confidence: 99%

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

et al. 2014

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“…In contrast to AroL, the activity of AroK in the cell is independent of both the amount of extracellular aromatic amino acids and the level of tyrR gene product67. 5-Enolpyruvyl shikimate 3-phosphate synthase (AroA) catalyzes the transfer of the enolpyruvoyl moiety from phosphoenolpyruvate (PEP) to the hydroxyl group of carbon 5 of S3P with the elimination of phosphate to produce EPSP891011. This is an addition-elimination reaction, which introduces the three-carbon fragment destined to become the side chain of phenylalanine.…”

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confidence: 99%

Improving the Production of L-Phenylalanine by Identifying Key Enzymes Through Multi-Enzyme Reaction System in Vitro

Ding

Liu

et al. 2016

Sci Rep

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L-Phenylalanine (L-Phe) is an important amino acid used in both food and medicinal applications. We developed an in vitro system that allowed a direct, quantitative investigation of phenylalanine biosynthesis in E. coli. Here, the absolute concentrations of six enzymes (AroK, AroL, AroA, AroC, PheA and TyrB) involved in the shikimate (SHIK) pathway were determined by a quantitative proteomics approach and in vitro enzyme titration experiments. The reconstitution of an in vitro reaction system for these six enzymes was established and their effects on the phenylalanine production were tested. The results showed that the yield of phenylalanine increased 3.0 and 2.1 times when the concentrations of shikimate kinase (AroL) and 5-enolpyruvoyl shikimate 3-phosphate (EPSP) synthase (AroA) were increased 2.5 times. Consistent results were obtained from in vivo via the overexpression of AroA in a phenylalanine-producing strain, and the titer of phenylalanine reached 62.47 g/l after 48 h cultivation in a 5-liter jar fermentor. Our quantitative findings provide a practical method to detect the potential bottleneck in a specific metabolic pathway to determine which gene products should be targeted to improve the yield of the desired product.

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“…Except for the glyphosate‐resistant enzyme isolated from A. variabilis (Powell et al. , 1992), this is the first report describing the purification and characterization of EPSP synthase from a cyanobacterium.…”

Section: Discussionmentioning

confidence: 99%

A dimeric 5‐enol‐pyruvyl‐shikimate‐3‐phosphate synthase from the cyanobacterium Spirulina platensis

Forlani

Campani

2001

New Phytologist

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Summary• Isolation and biochemical characterization is reported here of 5-enol -pyruvylshikimate-3-phosphate (EPSP) synthase, the enzyme that catalyses the sixth step in the common prechorismate pathway of aromatic amino acid biosynthesis and the target of the widely used herbicide glyphosate, from the cyanobacterium Spirulina platensis .• Homogeneous enzyme preparations were obtained by ammonium sulphate fractionation, anion-exchange and substrate-elution chromatography, and chromatofocusing. Protein characterization was carried out by conventional kinetic analysis, PAGE and gel permeation.• A 2800-fold purification was achieved, with a recovery of 20% of initial activity. Unusually low apparent affinities for both substrates, phospho enol pyruvate and shikimate-3-phosphate, did not correspond to decreased glyphosate sensitivity. During SDS-PAGE, the protein migrated as a single band corresponding to a molecular mass of c. 49 kDa. The behaviour of the protein upon gel permeation chromatography under nondenaturing conditions was, however, consistent with a mass of c. 91 kDa.• The native enzyme appears to be homodimeric, a remarkable feature that has not been previously reported for EPSP synthases from either cyanobacteria or higher plants. The presence of mono-and dimeric EPSP synthases could represent an important tool for cyanobacterial classification.

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Purification and properties of a glyphosate-tolerant 5-enolpyruvylshikimate 3-phosphate synthase from the cyanobacterium Anabaena variabilis

Cited by 20 publications

References 49 publications

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

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

Improving the Production of L-Phenylalanine by Identifying Key Enzymes Through Multi-Enzyme Reaction System in Vitro

A dimeric 5‐enol‐pyruvyl‐shikimate‐3‐phosphate synthase from the cyanobacterium Spirulina platensis

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