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Bacterial expression and isolation of Petunia hybrida 5-enol-pyruvylshikimate-3-phosphate synthase. 1987

S R Padgette, and Q K Huynh, and J Borgmeyer, and D M Shah, and L A Brand, and D Biest Re, and B F Bishop, and S G Rogers, and R T Fraley, and G M Kishore
Plant Molecular Biology, Corporate Research and Development Staff, Chesterfield, Missouri 63198.

5-enol-Pyruvylshikimate-3-phosphate synthase (EPSP synthase, EPSPS), an in vivo enzyme target of the herbicide glyphosate (N-phosphonomethyl glycine), was purified from a Petunia hybrida suspension culture line, MP4-G, by a small-scale high-performance chromatographic purification procedure. The cDNA encoding the mature petunia EPSPS (lacking the chloroplast transit sequence) was cloned into a plasmid, pMON342, for expression in Escherichia coli. This clone complemented the EPSPS deficiency of an E. coli aroA- mutant, and the plant enzyme constituted approximately 1% of the total extractable protein. Large-scale purification of the enzyme from E. coli cells resulted in a highly active protein which was homogeneous as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino terminal sequencing. Antibodies raised against the purified enzyme also reacted with the E. coli EPSPS in Western analyses. The availability of large quantities of the plant enzyme will significantly facilitate mechanistic investigations as well as a comparative study with EPSPS from bacteria and fungi.

UI MeSH Term Description Entries
D007700 Kinetics The rate dynamics in chemical or physical systems.
D010944 Plants Multicellular, eukaryotic life forms of kingdom Plantae. Plants acquired chloroplasts by direct endosymbiosis of CYANOBACTERIA. They are characterized by a mainly photosynthetic mode of nutrition; essentially unlimited growth at localized regions of cell divisions (MERISTEMS); cellulose within cells providing rigidity; the absence of organs of locomotion; absence of nervous and sensory systems; and an alternation of haploid and diploid generations. It is a non-taxonomical term most often referring to LAND PLANTS. In broad sense it includes RHODOPHYTA and GLAUCOPHYTA along with VIRIDIPLANTAE. Plant
D010957 Plasmids Extrachromosomal, usually CIRCULAR DNA molecules that are self-replicating and transferable from one organism to another. They are found in a variety of bacterial, archaeal, fungal, algal, and plant species. They are used in GENETIC ENGINEERING as CLONING VECTORS. Episomes,Episome,Plasmid
D002460 Cell Line Established cell cultures that have the potential to propagate indefinitely. Cell Lines,Line, Cell,Lines, Cell
D003001 Cloning, Molecular The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells. Molecular Cloning
D004247 DNA A deoxyribonucleotide polymer that is the primary genetic material of all cells. Eukaryotic and prokaryotic organisms normally contain DNA in a double-stranded state, yet several important biological processes transiently involve single-stranded regions. DNA, which consists of a polysugar-phosphate backbone possessing projections of purines (adenine and guanine) and pyrimidines (thymine and cytosine), forms a double helix that is held together by hydrogen bonds between these purines and pyrimidines (adenine to thymine and guanine to cytosine). DNA, Double-Stranded,Deoxyribonucleic Acid,ds-DNA,DNA, Double Stranded,Double-Stranded DNA,ds DNA
D004926 Escherichia coli A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc. Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D001483 Base Sequence The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence. DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA
D014166 Transferases Transferases are enzymes transferring a group, for example, the methyl group or a glycosyl group, from one compound (generally regarded as donor) to another compound (generally regarded as acceptor). The classification is based on the scheme "donor:acceptor group transferase". (Enzyme Nomenclature, 1992) EC 2. Transferase
D051229 3-Phosphoshikimate 1-Carboxyvinyltransferase An enzyme of the shikimate pathway of AROMATIC AMINO ACID biosynthesis, it generates 5-enolpyruvylshikimate 3-phosphate and ORTHOPHOSPHATE from PHOSPHOENOLPYRUVATE and shikimate-3-phosphate. The shikimate pathway is present in BACTERIA and PLANTS but not in MAMMALS. 3-Enol-Pyruvoylshikimate-5-Phosphate Synthase,3-Enolpyruvylshikimate 5-Phosphate Synthase,5-Enol-Pyruvyl Shikimate-3-Phosphate Synthase,5-Enolpyruvyl Shikimic Acid 3-Phosphate Synthetase,5-Enolpyruvylshikimate-3-Phosphate Synthase,5-Enolpyruvylshikimic Acid-3-Phosphate Synthase,EPSP Synthase,aroA 3-Phosphoshikimate 1-Carboxyvinyltransferase,1-Carboxyvinyltransferase, 3-Phosphoshikimate,1-Carboxyvinyltransferase, aroA 3-Phosphoshikimate,3 Enol Pyruvoylshikimate 5 Phosphate Synthase,3 Enolpyruvylshikimate 5 Phosphate Synthase,3 Phosphoshikimate 1 Carboxyvinyltransferase,3-Phosphoshikimate 1-Carboxyvinyltransferase, aroA,5 Enol Pyruvyl Shikimate 3 Phosphate Synthase,5 Enolpyruvyl Shikimic Acid 3 Phosphate Synthetase,5 Enolpyruvylshikimate 3 Phosphate Synthase,5 Enolpyruvylshikimic Acid 3 Phosphate Synthase,5-Phosphate Synthase, 3-Enolpyruvylshikimate,Acid-3-Phosphate Synthase, 5-Enolpyruvylshikimic,Shikimate-3-Phosphate Synthase, 5-Enol-Pyruvyl,Synthase, 3-Enol-Pyruvoylshikimate-5-Phosphate,Synthase, 3-Enolpyruvylshikimate 5-Phosphate,Synthase, 5-Enol-Pyruvyl Shikimate-3-Phosphate,Synthase, 5-Enolpyruvylshikimate-3-Phosphate,Synthase, 5-Enolpyruvylshikimic Acid-3-Phosphate,Synthase, EPSP,aroA 3 Phosphoshikimate 1 Carboxyvinyltransferase

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