Flavin adenine dinucleotide causes oligomerization of acetohydroxyacid synthase from black Mexican sweet corn cells

Singh, Bijay; Schmitt, Gail K.

doi:10.1016/0014-5793(89)81628-x

Cited by 20 publications

(13 citation statements)

References 16 publications

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“…Consequently, these values are not strictly comparable with the K i of 11n3 µM that we have measured. Nevertheless, they are all of a similar magnitude : maize (imazapyr 5n0-12n3 µM [54] ; imazaquin 12 µM [49]), wheat (imazaquin 2n5 µM, imazethapyr 5 µM, imazaquin 10 µM [18]), barley (imazaquin 3n8-6 µM [49]) and Arabidopsis (imazethapyr, 2 µM [20]). Some workers have recognized that the inhibition is time-dependent and have attempted to determine an initial K i .…”

Section: Discussionmentioning

confidence: 99%

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%

Expression, purification and characterization of Arabidopsis thaliana acetohydroxyacid synthase

Chang

Duggleby

1997

Biochemical Journal

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“…Our earlier results with AHAS from Black Mexican Sweet corn cells have demonstrated that a monomeric form of the enzyme is insensitive to valine and leucine, whereas the t 234 dimeric and the tetrameric forms of the enzyme are inhibited by these inhibitors (16,19). Therefore, insensitivity of the Arabidopsis AHAS extracted from PA10 cells to the feedback inhibitors could be due to inability of the enzyme to aggregate to the dimeric or the tetrameric form.…”

Section: Inhibition Of Ahas Activitymentioning

confidence: 95%

“…tivity of the enzyme (3). AHAS from plants also exists as high molecular mass aggregate (11,16); however, it is not known whether AHAS from plants is composed of homologous or heterologous subunits. Because AHAS is highly conserved across bacteria, yeast, and plants (1,8), it is possible that plants also contain a small subunit of AHAS.…”

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Arabidopsis Acetohydroxyacid Synthase Expressed in Escherichia coli Is Insensitive to the Feedback Inhibitors

et al. 1992

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Acetohydroxyacid synthase (AHAS), the first enzyme unique to the biosynthesis of isoleucine, leucine, and valine, is the target enzyme for several classes of herbicides. The AHAS gene from Arabidopsis thaliana, including the chloroplast transit peptide, was cloned into the bacterial expression plasmid pKK233-2. The resulting plasmid was used to transform an AHAS-deficient Escherichia coli strain MF2000. The growth of the MF2000 strain of E. coli was complemented by the functional expression of the Arabidopsis AHAS. The AHAS protein was processed to a molecular mass of 65 kilodaltons that was similar to the mature protein isolated from Arabidopsis seedlings. The AHAS activity extracted from the transformed E. coli cells was inhibited by imidazolinone and sulfonylurea herbicides. AHAS activity extracted from Arabidopsis is inhibited by valine and leucine; however, this activity was insensitive to these feedback inhibitors when extracted from the transformed E. coli.AHAS' (also known as acetolactate synthase; EC 4.1.3.18) catalyzes the first enzymic reaction leading to the biosynthesis of the branched chain amino acids valine, leucine, and isoleucine. The enzyme catalyzes two parallel reactions: condensation of 2 mol pyruvate to give rise to acetolactate, and condensation of pyruvate and a-ketobutyrate to yield acetohydroxybutyrate. Biochemical and genetic studies have shown that AHAS is the target site of several classes of structurally unrelated herbicides, which include the imidazolinones, the sulfonylureas, and the triazolopyrimidines (1,11,15).

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“…AHAS from plants has been isolated as high mol wt aggregate (18,19,27). However, it is not known whether this aggregate is composed of homologous or heterologous subunits of the enzyme.…”

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confidence: 99%

Overexpression of Acetohydroxyacid Synthase from Arabidopsis as an Inducible Fusion Protein in Escherichia coli

et al. 1991

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Acetohydroxyacid synthase (AHAS, EC 4.1.3.18) is the first enzyme unique to the biosynthesis of valine, leucine, and isoleucine. This enzyme is the target site of several classes of structurally unrelated herbicides. The conventional method of antibody production using purified protein has not been successful with this enzyme. Two separate fragments of a gene encoding a portion of the mature region of AHAS from Arabidopsis were fused with the trpE gene from Escherichia coli using the pATH1 vector. E. coli cells transformed with each respective plasmid expressed a fusion protein at levels greater than 10% of the total cell protein. The fusion protein was purified and used to immunize rabbits. Antisera obtained from the immunized rabbits immunoprecipitated AHAS activity from Arabidopsis cell free extracts. The anti-AHAS antisera reacted with a 65 kilodalton protein band in electrophoretically resolved extracts of Arabidopsis. In crossreactivity tests, this antibody was able to immunoprecipitate AHAS activity from various plant species. Furthermore, a protein band with a molecular mass of 65 kilodaltons was detected in the crude extracts of all plant species tested on a Westem blot. These results indicate that the 65 kilodalton protein represents AHAS in various plant species. The wide spectrum of crossreactivity for the antisera supports the view that the AHAS enzyme is highly conserved across all plant species.

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Flavin adenine dinucleotide causes oligomerization of acetohydroxyacid synthase from black Mexican sweet corn cells

Cited by 20 publications

References 16 publications

Expression, purification and characterization of Arabidopsis thaliana acetohydroxyacid synthase

Expression, purification and characterization of Arabidopsis thaliana acetohydroxyacid synthase

Arabidopsis Acetohydroxyacid Synthase Expressed in Escherichia coli Is Insensitive to the Feedback Inhibitors

Overexpression of Acetohydroxyacid Synthase from Arabidopsis as an Inducible Fusion Protein in Escherichia coli

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