Effect of Mutagenesis of V111 and L112 on the Substrate Specificity of Zymomonas mobilis Pyruvate Decarboxylase

Duggleby, Ronald G .,Huang, Chang Yi,Nixon, Peter F . 생화학분자생물학회 1997 BMB Reports Vol.32 No.1

Pyruvate decarboxylase (PDC) catalyzes the conversion of pyruvate to acetaldehyde as the penultimate step in alcohol fermentation. The enzyme requires two cofactors, thiamin diphosphate (ThDP) and Mg^(2+), for activity. Zymomonas mobilis PDC shows a strong preference for pyruvate although it will use the higher homologues 2-ketobutyrate and 2-ketovalerate to some extent. We have investigated the effect of mutagenesis of valine 111 and leucine 112 on the substrate specificity. V111 was replaced by glycine, alanine, leucine, and isoleucine while L112 was replaced by alanine, valine, and isoleucine. With the exception of L1121, all mutants retain activity towards pyruvate with k_cat values ranging from 40% to 139% of wild-type. All mutants show changes from wild-type in the affinity for ThDP, and several (V111A, L112A, and L112V) show decreases in the affinity for Mg ^(2+). Two of the mutants, V111G and V111A, show an increase in the K_m for pyruvate. The activity of each mutant towards 2-ketobutyrate and 2-ketovalerate was investigated and some changes from wild-type were found. For the V111 mutants, the most notable of these is a 3.7-fold increase in the ability to use 2-ketovalerate. However, the largest effect is observed for the L112V mutation which increases the ability to use both 2-ketobutyrate (4.3-fold) and 2-ketovalerate (5.7-fold). The results suggest that L112 and, to a lesser extent, V111 are close to the active site and may interact with the alkyl side-chain of the substrate.

Acetohydroxyacid Synthase

Duggleby, Ronald G.,Pang, Siew Siew The Korea Science and Technology Center 2000 BMB Reports Vol.33 No.1

Acetohydroxyacid synthase (EC 4.1.3.18) catalyses the first reaction in the pathway for synthesis of the branched-chain amino acids. The enzyme is inhibited by several commercial herbicides and has been subjected to detailed study over the last 20 to 30 years. Here we review the progress that has been made in understanding its structure, regulation, mechanism, and inhibition.

Acetohydroxyacid Synthase

Duggleby, Ronald G.,Pang, Siew Siew Korean Society for Biochemistry and Molecular Biol 2000 Journal of biochemistry and molecular biology Vol.33 No.1

Acetohydroxyacid synthase (EC 4.1.3.18) catalyses the first reaction in the pathway for synthesis of the branched-chain amino acids. The enzyme is inhibited by several commercial herbicides and has been subjected to detailed study over the last 20 to 30 years. Here we review the progress that has been made in understanding its structure, regulation, mechanism, and inhibition.

Expression in Escherichia coli of a Putative Human Acetohydroxyacid Synthase

Duggleby, Ronald G.,Kartikasari, Apriliana E.R.,Wunsch, Rebecca M.,Lee, Yu-Ting,Kil, Mee-Wha,Shin, Ju-Young,Chang, Soo-Ik Korean Society for Biochemistry and Molecular Biol 2000 Journal of biochemistry and molecular biology Vol.33 No.3

A human gene has been reported that may encode the enzyme acetohydroxyacid synthase. Previously this enzyme was thought to be absent from animals although it is present in plants and many microorganisms. In plants, this enzyme is the target of a number of commercial herbicides and the use of these compounds may need to be reassessed if the human enzyme exists and proves to be susceptible to inhibition. Here we report the construction of several plasmid vectors containing the cDNA sequence for this protein, and their expression in Escherichia coli. High levels of expression were observed, but most of the protein proved to be insoluble. The small amounts of soluble protein contained little or no acetohydroxyacid synthase activity. Attempts to refold the insoluble protein were successful insofar as the protein became soluble. However, the refolded protein did not gain any acetohydroxyacid synthase activity. In vivo complementation tests of an E. coli mutant produced no evidence that the protein is active. Incorrect folding, or the lack of another subunit, may explain the data but we favor the interpretation that this gene does not encode an acetohydroxyacid synthase.

Acetohydroxyacid Synthase

Duggleby, Ronald G .,Pang, Siew Siew 생화학분자생물학회 2001 BMB Reports Vol.33 No.1

Acetohydroacyacid synthase (EC 4.13.18) catalyses the first reaction in the pathway for synthesis of the branched-chain amino acids. The enzyme is inhibited by several commercial herbicides and has been subjected to detailed study over the last 20 to 30 years. Here we review the progress that has been made in understanding its structure, regulation, mechanism, and inhibition.

Effect of Mutagenesis of V111 and L112 on the Substrate Specificity of Zymomonas mobilis Pyruvate Decarboxylase

Duggleby, Ronald G.,Huang, Chang-Yi,Nixon,Peter F. The Korea Science and Technology Center 1999 BMB Reports Vol.32 No.1

Pyruvate decarboxylase (PDC) catalyzes the conversion of pyruvate to acetaldehyde as the penultimate step in alcohol fermentation. The enzyme requires two cofactors, thiamin diphosphate (ThDP) and Mg²+ for activity. Zymomonas PDC shows a strong preference for pyruvate although it will use the higher homologues 2-ketobutyrate and 2-ketovalerate to some extent. We have investigated the effect of mutagenesis of valine 111 and leucine 112 on the substrate specificity. V111 was replaced by glycine, alanine, leucine, and isoleucine while L112 was replaced by alanine, valine, and isoleucine. With the exception of L112I, all mutants retains activity towards pyruvate with k cat values ranging from 40% to 139% of wild-type. All mutants show changes from wild-type in the affinity for ThDP, and several (V111A, L112A, and L112V) show decreases in the affinity for Mg²+. Two of the mutants, V111G and V111A, show an increase in the K m for pyruvate. The activity of each mutant towards 2-ketobutyrate and 2-ketovalerate was investigated and some changes from wild-type were found. For the V111 mutants, the most notable of these is a 3.7-fold increase in the ability to use 2-ketovalerate. However, the largest effect is observed for the L112V mutation which increases the ability to use both 2-ketobutyrate (4.3-fold) and 2-ketovalerate (5.7-fold). The results suggest that L112 and, to a lesser extent, V111 are close to the active site and may interact with the alkyl side-chain of the substrate.

Expression in Escherichia coli of a Putative Human Acetohydroxyacid Synthase

Chang, Soo Ik,Duggleby, Ronald G .,Kil, Mee Wha,Kartikasari, Apriliana E . R .,Wunsch, Rebecca M .,Lee, Yu Ting,Shin, Ju Young 생화학분자생물학회 2001 BMB Reports Vol.33 No.3

A human gene has been reported that may encode the enzyme acetohydroxyacid synthase. Previously this enzyme was thought to be absent from animals although it is present in plants and many microorganisms. In plants, this enzyme is the target of a number of commercial herbicides and the use of these compounds may need to be reassessed if the human enzyme exists and proves to be susceptible to inhibition. Here we report the construction of several plasmid vectors containing the cDNA sequence for this protein, and their expression in Escherichia coli. High levels of expression were observed, but most of the protein proved to be insoluble. The small amounts of soluble protein contained tittle or no acetohydroxyacid synthase activity. Attempts to refold the insoluble protein were successful insofar as the protein became soluble. However, the refolded protein did not gain any acetohydroxyacid synthase activity. In vivo complementation tests of an E. coli mutant produced no evidence that the protein is active. Incorrect folding, or the lack of another subunit, may explain the data but we favor the interpretation that this gene does not encode an acetohydroxyacid synthase.

Effect of Mutagenesis of V111 and L112 on the Substrate Specificity of Zymomonas mobilis Pyruvate Decarboxylase

Huang, Chang-Yi,Nixon, Peter F.,Duggleby, Ronald G. Korean Society for Biochemistry and Molecular Biol 1999 Journal of biochemistry and molecular biology Vol.32 No.1

Pyruvate decarboxylase (PDC) catalyzes the conversion of pyruvate to acetaldehyde as the penultimate step in alcohol fermentation. The enzyme requires two cofactors, thiamin diphosphate (ThDP) and $Mg^{2+}$, for activity. Zymomonas mobilis PDC shows a strong preference for pyruvate although it will use the higher homologues 2-ketobutyrate and 2-ketovalerate to some extent. We have investigated the effect of mutagenesis of valine 111 and leucine 112 on the substrate specificity. V111 was replaced by glycine, alanine, leucine, and isoleucine while L112 was replaced by alanine, valine, and isoleucine. With the exception of L112I, all mutants retain activity towards pyruvate with $k_{cat}$ values ranging from 40% to 139% of wild-type. All mutants show changes from wild-type in the affinity for ThDP, and several (V111A, L112A, and L112V) show decreases in the affinity for $Mg^{2+}$. Two of the mutants, V111G and V111A, show an increase in the $K_m$ for pyruvate. The activity of each mutant towards 2-ketobutyrate and 2-ketovalerate was investigated and some changes from wild-type were found. For the V111 mutants, the most notable of these is a 3.7-fold increase in the ability to use 2-ketovalerate. However, the largest effect is observed for the L112V mutation which increases the ability to use both 2-ketobutyrate (4.3-fold) and 2-ketovalerate (5.7-fold). The results suggest that L112 and, to a lesser extent, V111 are close to the active site and may interact with the alkyl side-chain of the substrate.

Expression in Escherichia coli of a Putative Human Acetohydroxyacid Synthase

Chang,Soo-Ik,Kartikasari,Apriliana E.R.,Wunsch,Rebecca M.,Lee,Yu-Ting,Kil,Mee-Wha,Shin,Ju-Young,Duggleby,Ronald G. The Korea Science and Technology Center 2000 BMB Reports Vol.33 No.3

A human gene has been reported that may encode the enzyme acetohydroxyacid synthase. Previously this enzyme was thought to be absent from animals although it is present in plants and many microorganisms. In plants, this enzyme is the target of a number of commercial herbicides and the use of these compounds may need to be reassessed if the human enzyme exists and proves to be susceptible to inhibition. Here we report the construction of several plasmid vectors containing the cDNA sequence for this protein, and their expression in Escherichia coli. High levels of expression were observed, but most of the protein proved to be insoluble. The small amounts of soluble protein contained little or no acetohydroxyacid synthase activity. Attempt to refold the insoluble protein were successful insofar as the protein became soluble. However, the refolded protein did not gain any acetohydroxyacid synthase activity. In vivo complementation tests of an E. coli mutant produced no evidence that the protein is active. Incorrect folding, or the lack of another subunit, may explain the data but we favor the interpretation that this gene does not encode an acetohydroxyacid synthase.