• Journal of the Korean Chemical Society
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• v.39 no.6
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• pp.459-465
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• 1995

Acetolactate Synthase (ALS) was partially purified from the yeast and its basic biochemical studies were carried out. Yeast was grown in the minimum media containing 0.5% glucose, 51 mM $K_2HPO_4$, 22 mM $KH_2PO_4$, 8 mM $(NH_4)2SO_4,\;0.4\;m M\;MgSO_4$ for 18 hours at 37 $^{\circ}C$. The cell was ruptured in the buffer (20 mM phosphate buffer pH 7.0, 0.1 mM TPP, 0.5 mM DTT, 1 ${\mu}M$ FAD, and 1 mM MgCl_2$) following an overnight suspension. The supernatant fraction was collected from $10,000{\times}g$ and the enzyme was further purified by ammonium sulfate fractionation, DEAE-Sephacel chromatography and leucine-agarose chromatography. The enzyme activity was measured under the various conditions by the function of protein concentration, time, temperature, pH, and substrate. The optimum temperature was found to be 50$^{\circ}C$, optimum pH 8.0∼8.5. The kinetic parameters, $K_m\;and\;V_{max}$ were 8.4 mM and 17.9 nmol/mg/min respectively. Stability of the enzyme was studied with ethylene glycol and glycerol added to the enzyme solution. Both ethylene glycol and glycerol improved the enzyme stability up to 50%. The study of feedback inhibition showed that valine was a strong inhibitor while leucine was a weak inhibitor.

• The Korean Journal of Pesticide Science
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• v.2 no.3
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• pp.1-20
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• 1998

Chlorsulfuron, one of sulfonylurea herbicides acts through inhibition of acetolactate syuthase (EC 4.1.3.18; ALS, also known as acetohydroxyacid synthase) in the branched-chain amino acid biosynthesis process. After chlorsulfuron-ALS interaction, many physiological and metabolic disruptions occur in plants. However, it is not clear how this chlorsulfuron-ALS interaction affects those physiological and metabolic processes and how this interaction leads subsequently to plant death. Several researchers suggested that the death of chlorsulfuron-treated plants might be due to a shortage of the branched-chain amino acids, an accumulation of toxic metabolites, and/or a depletion of photoassimilates. It remains as a mystery presently, however, if such changes result in the plant death. In this review, we discussed how the chlorsulfuran-ALS interaction leads to physiological and metabolic disruptions in plants.

• BMB Reports
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• v.34 no.3
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• pp.244-249
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• 2001

The anabolic acetolactate synthase III was purified to homogeneity from Serratia marcescens using DEAE-Sepharose, Phenyl-Sepharose, and hydroxylapatite column chromatography The native molecular weight of the enzyme was approximately 165 kDa. The enzyme is composed of two large and two small subunits with molecular weights of 64 and 15 kDa, respectively. The N-terminal sequence of the large and small subunit of the enzyme was Ser-Ala-Thr-Pro-Gln-Pro-Ser-Thr-Arg-Phe-Thr-Cys-Ala-Gln-Leu-Ile-Ala-His-Leu and Met-Leu-Gln-Pro-Gln-Asp-Lys-Pro-Gln-Val-Ile-Leu-Glu-Leu-Ala-Val-Arg-Asn-His-Pro-Gly-Val-Met-Ser-His-Val, respectively. The optimum pH and pI value were 7.5 and 5.5, respectively The $IC_{50}$ values were $20\;{\mu}M$ and $14\;{\mu}M$ for valine and herbicide SU7, respectively. The substrate specificity ratio, R value, was determined to be approximately 40, which suggests that this enzyme prefers the formation of $\alpha$-aceto-$\alpha$-hydroxybutyrate leading to the synthesis of isoleucine.

• Proceedings of the Korean Journal of Food and Nutrition Conference
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• 2001.12a
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• pp.121-121
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• 2001

최소 배지에 여러 아미노산과 대사 산물을 첨가하여 배양시킨 Serratia marcescens ATCC 25419 세포추출물에서여 biodegradative threonine dehydratase (BDTD), biosynthetic threonine dehydratase (BSTD)와 acetolactate syntase (ALS)의 비활성도를 조사하였다. S. marcescens BDTD와 ALS는 낮은 농도 (0.5-2 mM)의 cAMP에 의해 촉진적 조절을 받으며, 비교적 낮은 농도의 isoleucine (1-4 mM)에 의해서는 S. marcescens BSTD의 생합성이 증가되고 높은 농도의 isoleucine (10-30 mM)에서는 감소되고 비교적 낮은 농도의 valine (2-4 mM)에 의해서 S. marcescens ALS의 생합성이 증가되는 것으로 보아 S. marcescens ATCC 25419에서 branched chain 아미노산 생합성 과정의 조절 양상은 Escherichia coli K-12와는 달리, isoleucine의 생합성 과정은 BSTD에 의해 조절되고, valine의 생합성 과정은 ALS에 의해 조절되는 것으로 사료된다.

• Proceedings of the Korean Society for Bioinformatics Conference
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• 2003.10a
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• pp.277-287
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• 2003

Acetolactate synthase (ALS, EC 4.1.3.18; also referred to as acetohydroxy acid synthase) catalyzes the first common step in the biosynthesis of valine, leucine, and isoleucine in microorganisms and plants. Recently X-ray structure of yeast ALS was available. Pair-wise alignment of yeast and tobacco ALS sequences revealed 63% sequence similarity. Using Deep View and automatic modeling on Swiss model server, we have generated reliable models of tobacco ALS based on yeast ALS template with a calculated pair-wise RMSD of 0.86 Angstrom. Functional roles of four residues located on the subunit interface (H142, El43, M350, and R376) were examined by site-directed mutagenesis. Seven mutants were generated and purified, of which three mutants (H142T, M350V, and R376F) were found to be inactivated under various assay conditions. The H142k mutant showed moderately altered kinetic properties. The E143A mutant increased 10-fold in K$_m$ value while other parameters remained unchanged. The M350C mutant was strongly resistant to three tested herbicides, while the R376k mutant can bind with herbicide carder at similar affinity to that of wild type enzyme, as determined by tryptophan quenching study. Except M350V mutant, all other mutants were ate to bind with cofactor FAD. Taken together, it is likely that residues H142 and E143 are located at the active site, while residues M350 and R376 are possibly located at the overlapping region of active site and herbicide binding site of the enzyme. Our data also allows us to hypothesize that the interaction between side chains of residues M350 and R376 are probably essential for the correct conformation of the active site. It remains to be elucidated that, whether the herbicide, upon binding with enzyme, inactivates the enzyme by causing change in the active site allosterically, which is unfavorable for catalytic activity.

• The Korean Journal of Pesticide Science
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• v.11 no.4
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• pp.269-275
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• 2007

Four sulfonylurea(SU)-resistant Monochoria vaginalis(M. vaginalis) accessions were tested for levels of resistance to four SU herbicides which have been widely using in paddy fields of Korea, based on whole plant response and sensitivity of the target enzyme, acetolactate synthase(ALS). The resistant Naju, Nonsan and Gimje accessions were not affected to the survival by treatment with recommended dose of all SU herbicides tested. The $GR_{50}$ values for the Naju, Nonsan and Gimje accessions were 8- to 33-fold, 8- to 30-fold and 7- to 32-fold higher to recommended doses of all SU herbicides tested than the susceptible Cheongdo accession, respectively. However, the $GR_{50}$ values for Kimhae accession displayed an intermediate response and was only 4-to 13-fold more resistant than the susceptible accession. The ALS $I_{50}$ values for the Naju, Nonsan and Gimje accessions were 25- to 66-fold, 9- to 26-fold and 10- to 24-fold higher to recommended doses of all SU herbicides tested than the susceptible Cheongdo accession, respectively. However, the $I_{50}$ value for Kimhae accession was 4- to 9-fold more resistant than the susceptible accession, as determined by $I_{50}$ values of ALS.

• Korean Journal of Weed Science
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• v.18 no.4
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• pp.333-340
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• 1998

The combined effects of primisulfuron and imazethapyr treated with piperonyl butoxide(PBO) on growth inhibition and acetolactate synthase(ALS) activity in corn were studied to identify the tolerance mechanism among corn cultivars. Pioneer 3751 IR showed resistance to primisulfuron and imazethapyr, but Pioneer 3751 and Chalok 2 were susceptible to them. Pioneer 3751 IR was tolerant to primisulfuron regardless of PBO treatment, but Pioneer 3751, Suwon 118 and Chalok 2 were more greatly inhibited by combined treatment of primisulfuron with PBO. Synergistic effect on growth inhibition in Pioneer 3751 IR, Pioneer 3751, Suwon 118 and Chalok 2 was not occurred when imazethapyr was treated with PBO, but Pioneer 3751, Suwon 118 and Chalok 2 tended to inhibit by combined treatment of imazethapyr with PBO. Pioneer 3751 IR showed higher ALS activity than Pioneer 3751 when primisulfuron or imazethapyr was treated. ALS activity was inhibited by primisulfuron at $0.01{\mu}M$ or above and imazethapyr at $1{\mu}M$ or above, and inhibitory effect on ALS activity was observed by primisufuron and imazethapyr treatments with PBO.

• Weed & Turfgrass Science
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• v.3 no.2
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• pp.110-113
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• 2014

The continuous use of acetolactate synthase (ALS) and acetyl-CoA carboxylase (ACCase) inhibitors has led to the selection of herbicide resistant barnyardgrass populations in direct-seeded rice fields of Korea. This study was conducted to identify herbicide resistant barnyardgrass biotypes and to determine the cross- and multiple-resistance of them. 25% of the population collected from Taeahn was partially resistant to ACCase inhibitors and 22% collected from Kimjae were partially resistant to ALS inhibitors. However, 8.2% of the population from both sites was resistant to ALS and ACCase inhibitors. Resistance to sulfonylurea herbicide, flazasulfuron was identified from two barnyardgrass accessions collected from both Taeahn and Kimjae. One barnyardgrass accession from both sites was resistant to ACCase inhibitor, sethoxydim. The cross-resistance to ALS inhibitors was identified at one barnyardgrass accession from Taeahn and at two accessions from Kimjae. Further, crossresistance to ACCase inhibitors was also identified at barnyardgrass accessions from Taeahn and Kimjae. Multiple-resistance to flazasulfuron and sethoxydim was determined at four barnyardgrass accessions from Taeahn and at six accessions from Kimjae. Therefore, the herbicide mixture and sequences within a growing season or the herbicide rotation with different modes of actions across growing seasons are recommended to control herbicide-resistant barnyardgrass in infested fields.

• Korean Journal of Weed Science
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• v.18 no.2
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• pp.171-178
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• 1998

Growth response and acetolactate synthase(ALS) activity were examined to determine the resistance of corn cultivars to primisulfuron when primisulfuron and/ or insecticide Terbufos were applied. Pioneer 3571 IR showed resistance to primisulfuron regardless of Terbufos treatment, but Pioneer 3571 was greatly injured with primisulfuron plus Terbufos treatment. Suwon 118 was relatively tolerant to primisulfuron compared to Chalok 2, but crop injury was occurred at both cultivars by primisulfuron plus Terbufos treatment. ALS activity at Pioneer 3751 IR was very high in primisulfuron and/ or Terbufos treatment. Suwon 118 also showed higher ALS activity compared to Pioneer 3751 and Chalok 2, but ALS activities were greatly decreased by primisulfuron plus Terbufos treatment at Suwon 118 and Chalok 2. The $I_{50}$ concentration for 50% inhibition of the ALS enzyme was 10.0, 0.06, 7.75, and 0.04${\mu}M$ for Pioneer 3751 IR, Pioneer 3751, Suwon 118, and Chalok 2, respectively. Consequently, resistance of corn cultivars to primisulfuron was significantly related to ALS activity. Crop injury and lower ALS activity were recognized in susceptible corn cultivars by primisulfuron plus Terbufos treatment.

• Bulletin of the Korean Chemical Society
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• v.24 no.12
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• pp.1799-1804
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• 2003

Acetolatate synthase(ALS) catalyzes the first common step in the biosynthesis of valine, leucine, isoleucine in plants and microorganisms. ALS is the target of several classes of herbicides, including the sulfonylureas, the imidazolinones, and the triazolopyrimidines. To elucidate the roles of arginine residues in tobacco ALS, chemical modification and site-directed mutagenesis were performed. Recombinant tobacco ALS was expressed in E. coli and purified to homogeneity. The ALS was inactivated by arginine specific reagents, phenylglyoxal and 2,3-butanedione. The rate of inactivation was a function of the concentration of modifier. The inactivation by butanedione was enhanced by borate, and the inactivation was reversible on removal of excess butanedione and borate. The substrate pyruvate and competitive inhibitors fluoropyruvate and phenylpyruvate protected the enzyme against inactivation by both modifiers. The mutation of well-conserved Arg198 of the ALS by Gln abolished the enzymatic activity as well as the binding affinity for cofactor FAD. However, the mutation of R198K did not affect significantly the binding of FAD to the enzyme. Taken together, the results imply that Arg198 is essential for the catalytic activity of the ALS and involved in the binding of FAD, and that the positive charge of the Arg is crucial for the interaction with negatively charged FAD.