• BMB Reports
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• 제30권1호
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• pp.13-17
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• 1997

The inhibitory effect of herbicides such as sulfonylurea derivatives, imidazolinones and pyrimidylsalicylate has been examined on the purified valine sensitive acetolactate synthase (ALS) from Serratia marcescens. The concentration of sulfometuron methyl which inhibits 50% of the ALS activity was 2.5 mM. The required concentrations of triasulfuron, primisulfuron methyl and imazaquin for the 50% inhibition of the ALS activity were 1 mM. The resistance of Serratia ALS to sulfometuron methyl, imazapyr and imazaquin is similar to that of E. coli ALS 1. However, pyrimidylsalicylate showed a potent inhibitory effect on the Serratia ALS almost 13 times more potent than on E. coli ALS II, which is known as herbicide-sensitive isozyme. The inhibitory mode was competitive against pyruvate. 150 value was determined to be $17{\mu}M$ in an assay mixture containing 20 mM pyruvate, and the $K_1$, value was calculated to be $0.4{\mu}m$ from the modified double reciprocal plot of 1/V versus $1/S^2$.

• BMB Reports
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• 제29권5호
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• pp.462-467
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• 1996

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 mazolopyrimidines, the pyrimidyl-oxy-benzoates, the pyrimidyl-thio-benzens, and the 4,6-dimethoxypyrimidines. An amino-terminal fragment of the sulfonylurea-resistant ALS gene (SurB) from Nicotiana tabaccum was cloned into the bacterial expression vector pGEX-2T. The resulting recombinant plasmid pGEX-ALS1 was used to transform Escherichia coli strain BL21, and the tobacco ALS was expressed in the bacteria as a protein fused with glutathione S-transferase (GST). Polyclonal antibodies against the fusion product (GST-ALS) were produced, and the sensitivity of GST-ALS with the rabbit anti-GST-ALS IgG was up to 50 ng. This antibody was used for Western blot analysis of the partially purified ALS from barley shoots. The results suggest that the polyclonal antibody produced in this study can be used to detect plant ALS.

• 농약과학회지
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• 제5권3호
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• pp.41-46
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• 2001

분지아미노산 생합성 과정에 관여하는 첫 번째 효소인 acetolactate synthase (ALS)를 대상으로 수행할 수 있도록 고효율 검색방법(High Throughput Screening, HTS)을 개발하였고, 이를 이용하여 식물특이적 효소 저해제로 알려진 107개의 기존 화합물 중에서 새로운 ALS 저해 화합물을 선발하였다. 기존의 방법과 비교할 경우 한사람이 1회 수행한다고 하면 8 배 효율이지만 연속적으로 수행한다고 할 경우 1/10 이하의 양, 동일한 재료의 적용, 측정 결과의 계산, enzyme kinetics 등을 감안하면 최소 100 배 이상의 효과를 얻을 수 있다. 새로운 ALS 저해제로 탐색된 화학물질은, ammooxyacetic acid, azelaic acid, citric acid, cyanuric fluoride, glyoxylic acid, itaconic acid, malonic acid, niclosamid, oxalic acid, 2-oxoglutaric acid, suramin 등이었다. 앞으로 이들을 기본 구조로 하여 신규 ALS 저해 제초제의 개발을 위한 유도체의 합성에 이용되었으면 한다.

• BMB Reports
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• 제31권1호
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• pp.37-43
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• 1998

The catabolic ${\alpha}$-acetolactate synthase was purified to homogeneity from Serratia marcescens ATCC 25419 using ammonium sulfate fractionation, DEAE-Sepharose, Phenyl-Sepharose, and Hydroxylapatite column chromatography. The native molecular weight of the enzyme was approximately 150 kDa and composed of two identical subunits with molecular weights of 64 kDa each. The N-terminal amino acid sequence of the enzyme was determined to be Ala-Gln-Glu-Lys-Thr-Gly-Asn-Asp-Trp-Gln-His-Gly-Ala-Asp-Leu-Val-Val-Lys-Asn-Leu. It was not inhibited by the branched chain amino acids and sulfometuron methyl herbicide. The optimum pH of the enzyme was around pH 5.5 and the pI value was 6.1. The catabolic ${\alpha}$-acetolactate synthase showed weak immunological relationships with recombinant tobacco ALS, barley ALS, and the valine-sensitive ALS isozyme from Serratia marcescens.

• BMB Reports
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• 제32권4호
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• pp.393-398
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• 1999

Acetolactate synthase (ALS) catalyzes the first reaction common to the biosynthesis of L-valine, L-leucine, and L-isoleucine. ALS is the target site of several classes of herbicides, including the sulfonylureas, the imidazolinones, and the triazolopyrimidines. Two forms of ALS (ALS I and ALS II) which have different affinity for Heparin have been separated from etiolated pea seedlings. The substrate saturation curves of both ALS I and ALS II were hyperbolic in contrast to previous reports. The two forms of ALS showed significant differences in their physical and kinetic properties. The values of $K_m$ for ALS I and ALS II were 9.0 mM and 4.8 mM, respectively. The pI values for ALS I and ALS II were determined to be 5.3 and 5.75 by isoelectric focusing, respectively. The native molecular weights for ALS I and ALS II obtained by nondenaturing gel electrophoresis and activity staining were 124 and 244 kDa, respectively. They also exhibited different sensitivity to feedback inhibition by end-product amino acids and inhibition by Cadre, an imidazolinone herbicide.

• 한국미생물·생명공학회지
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• 제20권2호
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• pp.115-121
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• 1992

최소 배지에 여러가지 아미노산들을 첨가하여 배양한 Serratia marcescens ATCC 25419 세포 추출물에서 acetolactate syhthase(ALS)의 비활성도를 0.5mM에서 40 증가시킨 반면 8mM에서 60, 20mM에서 90 감소시켰다. Valine은 효소의 비활성도를 2-4 mM에서 20-40 정도 증가시켰고 20mM의 높은 농도에서 10 정도 감소시켰다.

• BMB Reports
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• 제31권3호
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• pp.287-295
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• 1998

Acetolactate synthase (ALS) is the first common enzyme in the biosynthesis of L-Ieucine, L-isoleucine, and L-valine. The sulfonylurea-resistant ALS gene from Nicotiana tabacum was cloned into the bacterial expression vector pGEX-2T. The resulting recombinant plasmid pGEX-ALS3 was used to transform Escherichia coli strain XL1-Blue, and the mutant tobacco ALS (mALS) was expressed in the bacteria as a protein fused with glutathione S-transferase (GST). The fusion product GST-mALS was purified in a single step on a glutathione-Sepharose column. ALS activities of 0.9-2.5 ${\mu}mol/min/mg$ protein were observed in the GST-mALS, and the Km values for pyruvate, FAD, and TPP were 10.8-24.1, $(1.9-8.9){\times}10^{-3}$, and 0.14-0.38 mM, respectively. The purified GST-mALS was resistant to both the sulfonylurea and the triazolopyrimidine herbicides, and lost its sensitivity to end products, L-valine and L-leucine. For comparision, the tobacco wild-type recombinant ALS fused with GST, GST-wALS, was also characterized with respect to its pyruvate and cofactor bindings. These results suggest that the purified mutant recombinant tobacco ALS was functionally active, that the mutations resulting in herbicide resistance has affected pyruvate and cofactor bindings," and that the two classes of herbicides interact at a common site on the plant ALS.

• BMB Reports
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• 제31권3호
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• pp.258-263
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• 1998

Acetolactate synthase (ALS) is the common enzyme in the biosynthetic pathways leading to leucine, valine, and isoleucine. Tobacco ALS was expressed in E. coli and purified to homogeneity. The recombinant tobacco ALS was inactivated by thiol-specific reagents, N-ethylmaleimide (NEM) and 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB). Inactivation of the ALS by NEM followed pseudo-first order kinetics and was first order with respect to the modifier. The substrate pyruvate protected the enzyme against the inactivation by NEM and DTNB. Extrapolation to complete inactivation of the enzyme by DTNB showed modification of approximately 2 out of 4 total cysteinyl residues (or 2 cysteinyl and 1 cysteinyl residues), with approximately 1 residue protected by pyruvate. The tobacco ALS was also inactivated by the tryptophanspecific reagent, N-bromosuccinimide (NBS), and was similarly protected by pyruvate. The kinetics of the inactivation was first-order with respect to NBS. The present data suggest that cysteinyl and tryptophanyl residues play a key role in the catalytic function of the enzyme.

• BMB Reports
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• 제36권5호
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• pp.456-461
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• 2003

Acetolactate synthase (ALS) catalyzes the first common step in the biosynthesis of valine, leucine, and isoleucine. ALS is the target site for several classes of herbicides, including sulfonylureas, imidazolinones, and triazolopyrimidines. Two forms of ALS (designated ALS I and ALS II) were separated from barley shoots by heparin affinity column chromatography. The molecular masses of native ALS I and ALS II were determined to be 248 kDa and 238 kDa by nondenaturing gel electrophoresis and activity staining. Similar molecular masses of two forms of ALS were confirmed by a Western blot analysis. SDS-PAGE and Western blot analysis showed that the molecular masses of the ALS I and ALS II subunits were identical - 65 kDa. The two ALS forms exhibited different properties with respect to the values of $K_m$, pI and optimum pH, and sensitivity to inhibition by herbicides sulfonylurea and imidazolinone as well as to the feedback regulation by the end-product amino acids Val, Leu, and Ile. These results, therefore, suggest that the two ALS forms are not different polymeric forms of the same enzyme, but isozymes.

• BMB Reports
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• 제32권2호
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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.