• BMB Reports
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• v.32 no.2
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• pp.210-213
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• 1999

In Serratia marcescens, acetolactate produced by the catabolic acetolactate synthase (ALS) is converted into acetoin, its physiological role of which is to maintain intracellular pH homeostasis. In this study, the expression mode of catabolic ALS by aeration and branched-chain amino acids was examined by the ELISA method. The amount of catabolic ALS decreased approximately 93% under aerobic conditions. We also showed that the expression of catabolic ALS decreased approximately 34 % and 65 % in the presence of 2.5 mM and 10 mM leucine, respectively. The repression of catabolic ALS by leucine has not been reported previously. In contrast to leucine, catabolic ALS levels increased approximately 13% and 38% by treatment with 2.5 mM and 10 mM isoleucine, respectively, while valine alone did not have any significant effect on the synthesis of catabolic ALS. The amount of catabolic ALS was also reduced to approximately 32% and 45% in the presence of 10 mM Leu+Ile and Leu+Ile+Val, respectively. The regulatory mode of the Serratia catabolic ALS suggests that catabolic ALS may also have a role in supplying acetolactate as an intermediate of valine and leucine biosynthesis in addition to the maintenance of internal pH.

• Korean Journal of Weed Science
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• v.17 no.1
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• pp.66-70
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• 1997

LGC-40863(proposed common name ; pyribenzoxim), (benzophenone O-[2,6-bis[(4,6-dimethoxy-2-pyrimidinyl)oxy]benzoyl]oxime) is a new rice herbicide being developed by LG Chemical Ltd. The herbicide is highly selective between rice(Oryza sativa L.) and weeds including barnyardgrass (Echinochloa crus-galli(L.) P. Beauv.), and assumed to inhibit acetolactate synthase(ALS ; EC 4.1.3.18) because other structurally related herbicides inhibit the enzyme. To know inhibitory activity and the mode of inhibition of LGC-40863, $I_{50}$(concentration inhibiting ALS activity by 50%) and inhibition kinetics were investigated using ALS extracted from rice and barnyardgrass. $I_{50}$ values of LGC-40863 were 14 and 16mM in rice and barnyardgrass, respectively. In contrast to imazapyr(2-[4,5-dihydro-4-mythyl-4-(1-methylethyl)-5-oxo-1H-imidazol-2-yl]-3-pyridine-carboxylic acid) which showed an uncompetitive inhibition pattern, LGC-40863 was a noncompetitive inhibitor to ALS with respect to pyruvate similar to chlorsulfuron(2-chloro-N-((4-methoxy-6-methyl-l,3,5-triazin-2-yl) aminocarbonyl)benz-enesulfonamide) in both plants.

• Korean Journal of Weed Science
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• v.16 no.2
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• pp.122-131
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• 1996

The inhibition characteristics of chlorsulfuron [CHL, 2-chloro-N-[{ (4-methoxy-6-methyl-1,3,5-triazin-2-yl)amino}carbonyl]benzenesulfonamide] and imazaquin [IMA, 2-{4,5-dihydro-4-methyl-4-(1-methy-lethyl)-5-oxo-1H-imidazol-2-yl}-3-quinolinecarboxylic acid] on acetolactate synthase(ALS) activity of corn plants were investigated. CHL and IMA rapidly inhibited ALS activity of corn plants in vitro. Their $I_{50}$ values for ALS activity were 100nM and $5{\mu}M$, respectively, indicating that CHL had 50 times more inhibitory effect on ALS activity than IMA. The first applied herbicide had a dominant inhibitory effect on ALS activity when the two herbicides were applied sequentially. Branched-chain amino acids, valine(Val), leucine(Leu), and isoleucine(Ile) showed a feedback inhibition on ALS activity ; Val or Leu had a more inhibitory effect on ALS activity than Ile. Branchedchain amino acids and CHL or IMA exhibited an additive effect on inhibiting ALS activity. This suggests that branched-chain amino acids inhibit ALS activity by a different mechanisms) from that of CHL or IMA. Apparent ALS activity, which was measured on the basis of the conversion of pyruvate to acetolactate, was decreased by the addition of 2-ketobutyrate into the ALS reaction mixture in a concentration-dependent manner. In addition, kinetic studies revealed that CHL acts as a noncompetitive inhibitor, while IMA acts as an uncompetitive inhibitor to ALS with respect to pyruvate.

• Journal of Microbiology and Biotechnology
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• v.22 no.9
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• pp.1258-1263
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• 2012

2,3-Butanediol (2,3-BD) is a major metabolite produced by Klebsiella pneumoniae KCTC2242, which is a important chemical with wide applications. Three genes important for 2,3-BD biosynthesis acetolactate decarboxylase (budA), acetolactate synthase (budB), and alcohol dehydrogenase (budC) were identified in K. pneumoniae genomic DNA. With the goal of enhancing 2,3-BD production, these genes were cloned into pUC18K expression vectors containing the lacZ promoter and the kanamycin resistance gene to generate plasmids pSB1-7. The plasmids were then introduced into K. pneumoniae using electroporation. All strains were incubated in flask experiments and 2,3-BD production was increased by 60% in recombinant bacteria harboring pSB04 (budA and budB genes), compared with the parental strain K. pneumoniae KCTC2242. The maximum 2,3-BD production level achieved through fed-batch fermentation with K. pneumoniae SGJSB04 was 101.53 g/l over 40 h with a productivity of 2.54 g/l.h. These results suggest that overexpression of 2,3-BD synthesis-related genes can enhance 2,3-BD production in K. pneumoniae by fermentation.

• BMB Reports
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• v.28 no.3
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• pp.265-270
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• 1995

Acetolactate synthase (ALS, EC 4.1.3.18) is the first common enzyme in the biosynthesis of leucine, isoleucine, and valine. It is the target enzyme for several classes of herbicides, including the sulfonylureas, the imidazolinones, the triazolopyrimidines, the pyrimidyl-oxy-benzoates and the pyrimidyl-thio-benzens. The sulfonylurea-resistant ALS gene (SurB) from Nicotiana tabaccum [Lee et al. (1988) The EMBO J. 7, 1241-1248] was cloned into the bacterial expression plasmid pT7-7. The resulting recombinant plasmid pT7-ALS was used to transform an ALS-deficient Escherichia coli strain MF2000. MF2000 cells transformed with pT7-ALS grew in the absence of valine and isoleucine. ALS activities of 0.042 and 0.0002 ${\mu}mol/min/mg$ protein were observed in the crude extracts prepared from MF2000 cells transformed with plasmids pT7-ALS and pT7-7, respectively. In addition, the former crude extract containing mutant ALS was insensitive to inhibition by K11570, a new chemical class of herbicides. $IC_{50}$ values for K11570 were $0.13{\pm}0.01$ mM. For comparison, a plasmid pTATX containing the wild-type Arabidopsis thaliana ALS coding sequences was also expressed in MF2000. ALS activities of 0.037 ${\mu}mol/min/mg$ protein were observed, and the wild type ALS was sensitive to two different classes of herbicides, K11570 and ALLY, a sulfonylurea. $IC_{50}$ values for K11570 and ALLY were $0.63{\pm}0.07$ and $80{\pm}5.6$ nM, respectively. Thus, the results suggest that the sulfonylurea-resistant tobacco ALS was functionally expressed in the bacteria, and that K11570 herbicides bind to the regulatoty site of ALS enzymes.

• Applied Biological Chemistry
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• v.41 no.1
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• pp.99-103
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• 1998

Triazolopyrimidine sulfonanilide (TP) derivative is one of excellent herbicide compounds. We have synthesized three classes of a new sulfonamide derivative (TPP) as Acetolactate synthase (ALS) inhibitors, in which the benzene ring in TP skeleton was converted to substituted pyrimidyl ring and examined their inhibitory activities on barley for ALS. $I_{50}$ values of the inhibitors ranged from 0.005 to 2 mM. Comparing the $I_{50}$ value of each class of TPP derivatives, the substituents in pyrimidine and triazolopyrimidine ring were found to affect the degree of ALS inhibition. TPP with substituted methyl group in pyrimidine ring showed higher inhibitory activity than that with methoxy group, while the substitution of the cyclopentano group in triazolopyrimidine ring gave very large inhibitory activity than that of methyl group. The present study established that variation of the electron density by substitution at heterocyclic ring is a very important factor for ALS inhibition, but showed no dependence on steric effect by substituents.

• Korean Journal of Weed Science
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• v.15 no.1
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• pp.54-62
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• 1995

Acetolactate synthase activity inhibition and herbicidal activities were investigated with 2 sulfonylureas [chlorsulfuron{2-chloro-N-{{(4-methoxy-6-methyl-1,3,5-triazin-2-yl) amino} carboxyl} benzenesulfonamide}, metsulfuron-methyl{methyl-2{{{{(4-methoxy-6-methyl-1,3,5-triazin-2-yl)amino}carbonyl}amino}sulfonyl}benzoic acid}, and 2 imidazoli-nones [imazethapyr{2-{4,5-dihydro-4-methyl-4-(1-methyl)-5-oxo-1H-imidazol-2-yl}-5-ethyl-3-pyridinecarboxylicacid}, imazaquin{2-{4,5-dihydro-4-methyl-4-(1-methyl)-5-oxo-1H-imidazol-2-yl}-3-quinoline carboxylic acid} herbicides. A broad weeding spectrum was observed with the treated herbicides at low application rates. Both corn(Zea mays L.) and sorghum(Sorghum bicolor Moench) were very sensitive to the two herbicide groups. Although legumes, such as soybean(Glycine max Merr.), clover(Trifolium repense L.), and indian jointvetch(Aeschnomene indica L.) were sensitive to the sulfonylureas, they were tolerant to the imidazolinones. On the contrary, wheat(Triticum aestivum L.) and barley(Hoderum sativum Jess.) showed the reverse responses of the legumes to the two herbicide groups. Quackgrass(Agropyron repens(L.) P. Beauv.). however, was commonly tolerant to the two herbicide groups. Degrees of crop injury and acetolactate synthase inhibition also varied with the crops examined. The 50% inhibition concentrations of sulfonylureas on acetolactate synthase in vitro activity($IC_{50}$) from corn, wheat, and soybean did not relate to the greenhouse herbicidal activities ($GI_{50}$). With chlorsulfuron, for example, wheat had more than 100 times higher $GI_{50}$ than corn and soybean, but the $IC_{50}$ was 4 to 10 times lower. Similar observation was made with metsulfuron-methyl. However, closer relationships between $IC_{50}$ and $GI_{50}$ were found with the imidazolinones. When imazethapyr was applied, the order of $GI_{50}$ values against com, wheat, and soybean was the same as that of $IC_{50}$.

• Journal of the Korean Chemical Society
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• v.42 no.3
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• pp.306-314
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• 1998

Acetolactate synthase (ALS) is the common enzyme in the biosynthesis of branched chain amino acids, Val, Leu, and Ile in bacteria, yeast, and higher plants. The enzyme is target site of several classes of structually diverse herbicides, including the sulfonylureas, the imidazolinones, the triazolopyrimidines, and the primidyl-oxy-benzoates. We have synthesized new triazolopyrimidine (TP) derivatives, and determined their inhibitory activities on barley ALS. $lC_{50}$ values for the active compounds were 3.2 nM-0.62 mM, and some of them appeared to be potent inhibitors. The progress curves for inhibition of ALS by TP4, a representative derivative, indicated that the extent of inhibition increased with incubation time. The inhibition of ALS by TP4 showed mixed-type inhibition with respect to pyruvate. Dual inhibition analyses of TP4 versus imidazolinone Cadre and feedback inhibitor Leu suggested that three different classes of inhibitors bind to ALS in a mutually exclusive manner. Chemical modification of tyrosyl residues of ALS decreased sensitivity of ALS to TP4, while modification of tryptophan and cysteine did not affect the sensitivity.

• The Korean Journal of Pesticide Science
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• v.13 no.4
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• pp.290-297
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• 2009

This research aims to contribute the characterization of acetolactate synthase (Ec 4.1.3.18; ALS) and the resistance mechanism by sequence analysis of ALS gene of the sulfonylurea-resistant and -susceptible Monochoria vaginalis. The ALS gene was obtained from susceptible (S) and resistant (R) M. vaginalis to sulfonylurea herbicides (SUs). The 815 bp the fragment and the genomic DNA sequence coding for acetolactate synthase (ALS) of S and R biotypes of M. vaginalis were cloned and sequenced. Nineteen clones were divided greatly into 4 groups as result of sequencing. The first group was not difference to S type, the second group was amino acid of P197S which found point mutations causing substitution of serine for proline at amino acid 197, the third group was observed greatly other part of 6 places than group 1, and the fourth group appeared the intergrade of group 1 and 3. Therefore, it could be assumed what ALS gene of various types can be one plant. The peptide of the 13 amino acid Domain A region for ALS genes from R biotype of M. vaginalis differed from that of the S biotype by one base substitution at proline codon of Domain A. It could also be confirmed that point mutation of serine for proline at amino acid 197.

• Journal of the Korean Chemical Society
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• v.43 no.4
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• pp.461-468
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• 1999

Acetolactate synthase (ALS) catalyzes the first common reaction in the biosynthesis of branched-chain amino acids, valine, leucine, and isoleucine. ALS is the common target of several classes of structurally diverse herbicides, the triazolopyrimidines, the imidazolinones, the sulfonylureas, and pyrimidyl-oxy-benzoates. We examined ihibitory activities of newly synthesized triazolopyrimidine sulfonamide derivatives using partially purified ALS from barley. $IC_{50}$ values for the active derivatives are 0.5nM∼8$\mu$M. Among them TP1 and TP2 are the most potent ALS inhibitors with $IC_{50}$ values of 0.5nM and 1.6nM, respectively. These inhibitors are more potent in the inhibition of barley ALS than commercial herbicides, Metosulam ($IC_{50}$;3.6 nM), Flumetsulam ($IC_{50}$;126 nM), and Cadre ($IC_{50};4 {\mu}M$). The progress curves for inhibition of ALS by TP2 showed that the amount of inhibition increases with time. The inhibition of ALS by TP2 was mixed-type inhibition with respect to pyruvate. Dual inhibition analyses of TP2 versus an imidazolinone, Cadre, and Leu showed parallel and intercepting kinetic pattern, respectively. The results suggest that TP2 binds to ALS competively with Cadre but not with Leu. Chemical modification of cysteinly residues in ALS decreased the sensitivity of ALS to Leu, while the modification did not affect the sensitivity of ALS to TP2 and Cadre.