Rational Molecular Design and Genetic Engineering of Herbicide Resistant Crops by Structure Modeling and Site-directed Mutagenesis of Acetohydroxyacid Synthase

Ott, Karl‐Heinz; Kwagh, Jae-Gyu; Stockton, Gerald W.; Sidorov, V. A.; Kakefuda, Genichi

doi:10.1006/jmbi.1996.0580

Cited by 85 publications

(73 citation statements)

References 18 publications

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“…The purified enzyme is moderately stable and could be kept at 4 mC for several days with only minor losses (10 % in three weeks) ; for long-term storage, the enzymic activity could be maintained at k70 mC (10 % loss in 6 months) provided intervening freeze-thaw cycles were avoided. Recently, Ott et al [23] described the purification of Arabidopsis AHAS, expressed as a GST fusion protein in E. coli. Although no mention was made of the stability of their preparation, their final product, after thrombin digestion to excise the GST portion, had a specific activity that is less than one-quarter of that which we have obtained.…”

Section: Discussionmentioning

confidence: 99%

“…More recently, Arabidopsis AHAS has been expressed in E. coli and purified as a glutathione S-transferase (GST) fusion protein. After cleavage of the GST portion, a functional enzyme was obtained without the first 59 amino acids but with an additional glycine at the N-terminus [23]. Unfortunately, few data were presented on the purity of the final product or its enzymic properties.…”

Section: Introductionmentioning

confidence: 99%

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Expression, purification and characterization of Arabidopsis thaliana acetohydroxyacid synthase

Chang

Duggleby

1997

Biochemical Journal

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Acetohydroxyacid synthase (EC 4;1;3;18) is the enzyme that catalyses the first step in the synthesis of the branched-chain amino acids valine, leucine and isoleucine. The AHAS gene from Arabidopsis thaliana with part of the chloroplast transit sequence removed was cloned into the bacterial expression vector pT7-7 and expressed in the Escherichia coli strain BL21(DE3). The expressed enzyme was purified by an extensive procedure involving (NH % ) # SO % fractionation followed by hydrophobic and anion-exchange chromatography. The purified enzyme appears as a single band on SDS\PAGE with a molecular mass of about 61 kDa. On gel filtration the enzyme is a dimer, migrating as a single peak with molecular masses of 109 and 113 kDa in the absence and presence of FAD respectively. Ion spray MS analysis yielded a mass of 63 864 Da. The enzyme has optimum activity in

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Expression, purification and characterization of Arabidopsis thaliana acetohydroxyacid synthase

Chang

Duggleby

1997

Biochemical Journal

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“…The fundamental requirement of amino acids for plant survival, as well as the absence of essential amino acid biosynthesis in humans and other animals, makes the aspartate-derived amino acid pathway an attractive target for herbicide development. For instance, acetolactate synthase, an enzyme in the biosynthetic pathway leading from threonine to isoleucine, is the target of several classes of economically important herbicides, including sulfonylureas, imidazolinones, triazolopyrimidines, and pyrimidinyl oxybenzoates (Mourad and King, 1992;Ott et al, 1996).…”

Section: Introductionmentioning

confidence: 99%

Aspartate-Derived Amino Acid Biosynthesis inArabidopsis thaliana

Jander

Joshi

2009

The Arabidopsis Book

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The aspartate-derived amino acid pathway in plants leads to the biosynthesis of lysine, methionine, threonine, and isoleucine. These four amino acids are essential in the diets of humans and other animals, but are present in growthlimiting quantities in some of the world's major food crops. Genetic and biochemical approaches have been used for the functional analysis of almost all Arabidopsis thaliana enzymes involved in aspartate-derived amino acid biosynthesis. The branch-point enzymes aspartate kinase, dihydrodipicolinate synthase, homoserine dehydrogenase, cystathionine gamma synthase, threonine synthase, and threonine deaminase contain well-studied sites for allosteric regulation by pathway products and other plant metabolites. In contrast, relatively little is known about the transcriptional regulation of amino acid biosynthesis and the mechanisms that are used to balance aspartatederived amino acid biosynthesis with other plant metabolic needs. The aspartate-derived amino acid pathway provides excellent examples of basic research conducted with A. thaliana that has been used to improve the nutritional quality of crop plants, in particular to increase the accumulation of lysine in maize and methionine in potatoes.

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“…Thus, the recent characterization of the x-ray structure of the glyphosate-insensitive form of the enzyme 5-enolpyruvylshikimate 3-P (ESP) synthase and its interaction with glyphosate (Funke et al, 2006), provides an explanation for the molecular basis of herbicide resistance of Roundup Ready crops. Whereas the ESP synthase was an introduced heterologous protein, acetohydroxyacid synthase (also known as acetolactate synthase) was the first example of a rationally engineered enzyme designed to confer resistance to the sulfonylurea and imidazolinone class of herbicides (Ott et al, 1996). Currently, transgenic soybean (Glycine max) resistant to these herbicides is commercially available.…”

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confidence: 99%

The Outlook for Protein Engineering in Crop Improvement

Rao

2008

Plant Physiology

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Rational Molecular Design and Genetic Engineering of Herbicide Resistant Crops by Structure Modeling and Site-directed Mutagenesis of Acetohydroxyacid Synthase

Cited by 85 publications

References 18 publications

Expression, purification and characterization of Arabidopsis thaliana acetohydroxyacid synthase

Expression, purification and characterization of Arabidopsis thaliana acetohydroxyacid synthase

Aspartate-Derived Amino Acid Biosynthesis inArabidopsis thaliana

The Outlook for Protein Engineering in Crop Improvement

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