HORT640 - Metabolic Plant Physiology
Aromatic amino acid biosynthesis
The shikimate pathway - synthesis of chorismate
Glyphosate (N-phosphonomethylglycine) is a non-selective, broad spectrum herbicide that is symplastically translocated to the meristems of growing plants. It causes shikimate accumulation through inhibition of the chloroplast localized EPSP synthase (5-enolpyruvylshikimate-3-phosphate synthase; EPSPs) [EC 220.127.116.11] (Amrhein et al, 1980; Hollander and Amrhein, 1980; Jaworski, 1972; Klee et al, 1987; Rubin et al, 1982; Smart et al, 1985; Sost et al, 1984; Stalker et al, 1985; Steinrucken and Amrhein, 1980).
Genes encoding EPSP synthase have been cloned from Arabidopsis (Klee et al, 1987), tomato (Gasser et al, 1988), tobacco (Wang et al, 1991), and petunia (Gasser et al, 1988). Two distinct cDNAs encoding EPSPs have been identified in tobacco cell cultures; one of these genes (EPSPS-1) was amplified in glyphosate-tolerant cultures (Wang et al, 1991). Likewise selection for glyphosate-tolerant carrot cell cultures results in EPSPs gene amplification (Shyr et al, 1992). Expression of the Arabidopsis EPSPs gene under the direction of the cauliflower mosaic virus 35 S promoter results in overproduction of EPSPs and leads to glyphosate tolerance in transformed callus and Arabidopsis plants (Klee et al, 1987). Either amplification of (or a single amino acid substitution in) the aroA gene encoding EPSPs is sufficient to confer glyphosate resistance in Escherichia coli, Salmonella typhimurium and Klebsiella pneumoniae (Rogers et al, 1983; Stalker et al, 1985; Sost and Amrhein, 1990). Consistent with this, two distinct mechanisms of glyphosate resistance in Euglena gracilis have been identified (Reinbothe et al, 1991). One is characterized by the overproduction and 40-fold accumulation of EPSPs; the second is connected with a herbicide-insensitive enzyme (Reinbothe et al, 1991). Glyphosate tolerance of cultured Corydalis sempervirens cells is acquired by an increased rate of transcription of EPSPs as well as by a reduced turnover of the enzyme (Hollander-Czytko et al, 1992).
Chorismate synthase [EC 18.104.22.168], the seventh enzyme in the shikimate pathway, catalyzes the transformation of 5-enolpyruvylshikimate 3-phosphate (EPSP) to chorismate, which is the last common precursor in the biosynthesis of numerous aromatic compounds in bacteria, fungi and plants. Like other enzymes of the shikimate pathway, chorismate synthase is a plastid localized enzyme (Henstrand et al, 1995). The chorismate synthase reaction involves a 1,4-trans-elimination of phosphoric acid from EPSP and requires a reduced flavin cofactor (Bornemann et al, 1996); chorismate synthase has an absolute requirement for reduced FMN as a cofactor (Macheroux et al, 1999). The role of the reduced FMN in catalysis is obscure, but may involve an initial electron transfer from tightly bound reduced flavin to the substrate, resulting in C-O bond cleavage (Macheroux et al, 1999).
Amrhein N, Deus B, Gehrke P, Steinrucken HC 1980 The site of the inhibition of the shikimate pathway by glyphosate. II. Interference of glyphosate with chorismate formation in vivo and in vitro. Plant Physiol. 66: 830-834.
Bornemann S, Lowe DJ, Thorneley RN 1996 The transient kinetics of Escherichia coli chorismate synthase: substrate consumption, product formation, phosphate dissociation, and characterization of a flavin intermediate. Biochemistry 35: 9907-9916.
Gasser CS, Winter JA, Hironaka CM, Shah DM 1988 Structure, expression, and evolution of the 5-enolpyruvylshikimate-3-phosphate synthase genes of petunia and tomato. J. Biol. Chem. 263: 4280-4287.
Henstrand JM, Schmid J, Amrhein N 1995 Only the mature form of the plastidic chorismate synthase is enzymatically active. Plant Physiol. 108: 1127-1132.
Hollander H, Amrhein N 1980 The site of action of the inhibition of the shikimate pathway by glyphosate. I. Inhibition by glyphosate of phenylpropanoid synthesis in buckwheat (Fagopyrum esculentum Moench). Plant Physiol. 66: 823-829.
Hollander-Czytko H, Sommer I, Amrhein N 1992 Glyphosate tolerance of cultured Corydalis sempervirens cells is acquired by an increased rate of transcription of 5-enolpyruvylshikimate 3-phosphate synthase as well as by a reduced turnover of the enzyme. Plant Mol. Biol. 20: 1029-1036.
Jaworski EG 1972 Mode of action of N-phosphonomethylglycine: inhibition of aromatic amino acid biosynthesis. J. Agr. Food Chem. 20: 1195-1198.
Klee Hj, Muskopf YM, Gasser CS 1987 Cloning of an Arabidopsis thaliana gene encoding 5-enolpyruvyl-shikimic acid-3-phosphate synthase: sequence analysis and manipulation to obtain glyphosate-tolerant plants. Mol. Gen. Genet. 210: 437-442.
Macheroux P, Schmid J, Amrhein N, Schaller A 1999 A unique reaction in a common pathway: mechanism and function of chorismate synthase in the shikimate pathway. Planta 207: 325-334.
Reinbothe S, Nelles A, Parthier B 1991 N-(phosphonomethyl)glycine (glyphosate) tolerance in Euglena gracilis acquired by either overproduced or resistant 5-enolpyruvylshikimate-3-phosphate synthase. Eur. J. Biochem. 198: 365-373.
Rogers SG, Brand LA, Holder SB, Sharps ES, Brackin MJ 1983 Amplification of the aroA gene from Escherichia coli results in tolerance to the herbicide glyphosate. Appl. Environ. Microbiol. 46: 37-43.
Rubin JL, Gaines CG, Jensen RA 1982 Enzymological basis for herbicidal action of glyphosate. Plant Physiol. 70: 833-839.
Smart CC, Johanning D, Muller G, Amrhein N 1985 Selective overproduction of 5-enolpyruvyl-shikimic acid 3-phosphate synthase in a plant cell culture which tolerates high doses of the herbicide glyphosate. J. Biol. Chem. 260: 4724-4728.
Sost D, Amrhein N 1990 Substitution of Gly-96 to Ala in the 5-enolpyruvylshikimate-3-phosphate synthase of Klebsiella pneumoniae results in a greatly reduced affinity for the herbicide glyphosate. Arch. Biochem. Biophys. 282: 433-436.
Sost D, Schulz A, Amrhein N 1984 Characterization of a glyphosate-insensitive 5-enolpyruvyl-shikimic acid-3-phosphate synthase. FEBS Lett. 173: 238-242.
Stalker DM, Hiatt WR, Comai L 1985 A single amino acid substitution in the enzyme 5-enolpyruvyl-shikimic acid-3-phosphate synthase confers resistance to the herbicide glyphosate. J. Biol. Chem. 260: 4724-4728.
Steinrucken HC, Amrhein N 1980 The herbicide glyphosate is a potent inhibitor of 5-enolpyruvyl-shikimic acid-3-phosphate synthase. Biochem. Biophys. Res. Commun. 94: 1207-1212.
Wang YX, Jones JD, Weller SC, Goldsbrough PB 1991 Expression and stability of amplified genes encoding 5-enolpyruvylshikimate-3-phosphate synthase in glyphosate-tolerant tobacco cells. Plant Mol. Biol. 17: 1127-1138.
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