N-(Phosphonomethyl)glycine (glyphosate) tolerance in Euglena gracilis acquired by either overproduced or resistant 5-enolpyruvylshikimate-3-phosphate synthase

The recently characterized amino acid L-arogenate (Zamir et al., J. Am. Chem. Soc. 102:4499-4504, 1980) may be a precursor of either L-phenylalanine or L-tyrosine in nature. Euglena gracilis is the first example of an organism that uses L-arogenate as the sole precursor of both L-tyrosine and L-phenylalanine, thereby creating a pathway in which L-arogenate rather than prephenate becomes the metabolic branch point. E. gracilis ATCC 12796 was cultured in the light under myxotrophic conditions and harvested in late exponential phase before extract preparation for enzymological assays. Arogenate dehydrogenase was dependent upon nicotinamide adenine dinucleotide phosphate for activity. L-Tyrosine inhibited activity effectively with kinetics that were competitive with respect to L-arogenate and noncompetitive with respect to nicotinamide adenine dinucleotide phosphate. The possible inhibition of arogenate dehydratase by L-phenylalanine has not yet been determined. Beyond the latter uncertainty, the overall regulation of aromatic biosynthesis was studied through the characterization of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase and chorismate mutase. 3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase was subject to noncompetitive inhibition by L-tyrosine with respect to either of the two substrates. Chorismate mutase was feedback inhibited with equal effectiveness by either L-tyrosine or L-phenylalanine. L-Tryptophan activated activity of chorismate mutase, a pH-dependent effect in which increased activation was dramatic above pH 7.8 L-Arogenate did not affect activity of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase or of chorismate mutase. Four species of prephenate aminotransferase activity were separated after ion-exchange chromatography. One aminotransferase exhibited a narrow range of substrate specificity, recognizing only the combination of L-glutamate with prephenate, phenylpyruvate, or 4-hydroxyphenylpyruvate. Possible natural relationships between Euglena spp. and fungi previously considered in the literature are discussed in terms of data currently available to define enzymological variation in the shikimate pathway.

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The aromatic amino acid pathway branches at L-arogenate in Euglena gracilis.

Molecular and Cellular Biology ◽

10.1128/mcb.1.5.426 ◽

1981 ◽

Vol 1 (5) ◽

pp. 426-438 ◽

Cited By ~ 35

Author(s):

G S Byng ◽

R J Whitaker ◽

C L Shapiro ◽

R A Jensen

Keyword(s):

Amino Acid ◽

Euglena Gracilis ◽

Nicotinamide Adenine Dinucleotide ◽

Nicotinamide Adenine Dinucleotide Phosphate ◽

Shikimate Pathway ◽

Exchange Chromatography ◽

Chorismate Mutase ◽

Nicotinamide Adenine ◽

Acid Pathway ◽

Phosphate Synthase

The recently characterized amino acid L-arogenate (Zamir et al., J. Am. Chem. Soc. 102:4499-4504, 1980) may be a precursor of either L-phenylalanine or L-tyrosine in nature. Euglena gracilis is the first example of an organism that uses L-arogenate as the sole precursor of both L-tyrosine and L-phenylalanine, thereby creating a pathway in which L-arogenate rather than prephenate becomes the metabolic branch point. E. gracilis ATCC 12796 was cultured in the light under myxotrophic conditions and harvested in late exponential phase before extract preparation for enzymological assays. Arogenate dehydrogenase was dependent upon nicotinamide adenine dinucleotide phosphate for activity. L-Tyrosine inhibited activity effectively with kinetics that were competitive with respect to L-arogenate and noncompetitive with respect to nicotinamide adenine dinucleotide phosphate. The possible inhibition of arogenate dehydratase by L-phenylalanine has not yet been determined. Beyond the latter uncertainty, the overall regulation of aromatic biosynthesis was studied through the characterization of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase and chorismate mutase. 3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase was subject to noncompetitive inhibition by L-tyrosine with respect to either of the two substrates. Chorismate mutase was feedback inhibited with equal effectiveness by either L-tyrosine or L-phenylalanine. L-Tryptophan activated activity of chorismate mutase, a pH-dependent effect in which increased activation was dramatic above pH 7.8 L-Arogenate did not affect activity of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase or of chorismate mutase. Four species of prephenate aminotransferase activity were separated after ion-exchange chromatography. One aminotransferase exhibited a narrow range of substrate specificity, recognizing only the combination of L-glutamate with prephenate, phenylpyruvate, or 4-hydroxyphenylpyruvate. Possible natural relationships between Euglena spp. and fungi previously considered in the literature are discussed in terms of data currently available to define enzymological variation in the shikimate pathway.

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Enhancement of glyphosate tolerance in rice (Oryza sativa L.) through mutation induction in EPSPS (5-enolpyruvylshikimate-3-phosphate synthase)

Plant Gene ◽

10.1016/j.plgene.2020.100225 ◽

2020 ◽

Vol 22 ◽

pp. 100225 ◽

Cited By ~ 2

Author(s):

Sedigheh Nasr Ramzi ◽

Mohammad Mehdi Sohani ◽

Reza Shirzadian-Khorramabad ◽

Jafar Asghari ◽

Mehriar Amininasab

Keyword(s):

Oryza Sativa ◽

Oryza Sativa L ◽

Mutation Induction ◽

Glyphosate Tolerance ◽

Phosphate Synthase

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Improvement of Glyphosate Resistance through Concurrent Mutations in Three Amino Acids of the Ochrobactrum 5-Enopyruvylshikimate-3-Phosphate Synthase

Applied and Environmental Microbiology ◽

10.1128/aem.05271-11 ◽

2011 ◽

Vol 77 (23) ◽

pp. 8409-8414 ◽

Cited By ~ 27

Author(s):

Yong-Sheng Tian ◽

Jing Xu ◽

Ai-Sheng Xiong ◽

Wei Zhao ◽

Xiao-Yan Fu ◽

...

Keyword(s):

Amino Acids ◽

Three Dimensional ◽

Glyphosate Resistance ◽

Site Directed Mutagenesis ◽

Dimensional Structure ◽

Ochrobactrum Anthropi ◽

Glyphosate Tolerance ◽

Content Type ◽

Important Region ◽

Phosphate Synthase

ABSTRACTA mutant of 5-enopyruvylshikimate-3-phosphate synthase fromOchrobactrum anthropiwas identified after four rounds of DNA shuffling and screening. Its ability to restore the growth of the mutant ER2799 cell on an M9 minimal medium containing 300 mM glyphosate led to its identification. The mutant had mutations in seven amino acids: E145G, N163H, N267S, P318R, M377V, M425T, and P438L. Among these mutations, N267S, P318R, and M425T have never been previously reported as important residues for glyphosate resistance. However, in the present study they were found by site-directed mutagenesis to collectively contribute to the improvement of glyphosate tolerance. Kinetic analyses of these three mutants demonstrated that the effectiveness of these three individual amino acid alterations on glyphosate tolerance was in the order P318R > M425T > N267S. The results of the kinetic analyses combined with a three-dimensional structure modeling of the location of P318R and M425T demonstrate that the lower hemisphere's upper surface is possibly another important region for glyphosate resistance. Furthermore, the transgenicArabidopsiswas obtained to confirm the potential of the mutant in developing glyphosate-resistant crops.

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