• Bulletin of the Korean Chemical Society
• /
• v.27 no.4
• /
• pp.549-555
• /
• 2006

Acetohydroxyacid synthase (AHAS, EC 2.2.1.6 also referred to as acetolactate synthase) catalyzes the first common step in the metabolic pathway leading to biosynthesis of the branched-chain amino acids in plants and microorganisms. Due to its presence in plants, AHAS is a target for the herbicides (sulfonylurea and imidazolinone), which act as potent inhibitors of the enzyme. Recently, we have shown [J. Kim, D.G. Baek, Y.T. Kim, J.D. Choi, M.Y. Yoon, Biochem. J. (2004) 384, 59-68] that the residues in the “mobile loop” 567-582 on the C-termini are involved in the binding/stabilization of the active dimer and ThDP (thiamin diphosphate) binding. In this study, we have demonstrated the role of the W573 in the mobile loop of the C-termini of tobacco AHAS. The substitution of this W573 residue caused significant perturbations in the activation process and in the binding site of ThDP. Position W573 plays a structurally important role in the binding of FAD, maintaining the enzyme active site in the required geometry for catalysis to occur. In here we propose that the tryptophan at position 573 is important for the catalytic process.

• Proceedings of the Plant Resources Society of Korea Conference
• /
• 2023.04a
• /
• pp.62-62
• /
• 2023

Zanthoxylum piperitum ("chopi" in Korean) has been used as traditional medicinal plants with high anti-inflammatory, antioxidant, and antifungal activities. The aims of the study were to identify marker compounds and to investigate metabolites variation of chopi according to different parts and harvest times. Every month from June to September, chopi were harvested with three different parts: leaves, leaf-twig mixtures, twigs. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), two main marker compounds (quercitrin and quercetin-3-O-glucoside) were characterized in 70% ethanol extracts of chopi. Quantification of the two marker compounds were subsequently conducted by high performance liquid chromatography (HPLC), representing that contents of these compounds were higher in leaves and leaf-twig mixtures rather than twigs. For the comprehensive analysis of metabolites associated with production of marker compounds, 35 primary metabolites were identified using gas chromatography-mass spectrometry (GC-MS). Multivariate analysis results represented that plant parts were main contributors to the separation of chopi. However, significant differences were not observed between leaves and leaf-twig mixtures samples. The partial least square (PLS) predictive model revealed that monosaccharides (fructose, galactose, glucose, mannose, xylose) and branched-chain amino acids (isoleucine, valine, leucine) were important determinants for the production of marker compounds together with alanine, inositol, GABA, and theronic acid. This study could be extended to stabilize and utilize chopi as an industrial material, as well as to find good candidates with various nutritional traits.

• Korean Journal of Food Science and Technology
• /
• v.51 no.6
• /
• pp.537-542
• /
• 2019

Proteins were isolated from four species of edible insects, Bombyx mori, B; Tenebrio molitor, T; Protaetia brevitarsis, P; and Gryllus bimaculatus, G, and their properties were analyzed and compared with those of whey proteins. The yield of the protein isolated from raw materials was 65.0-75.0%. Among three branched chain amino acids (BCAA), the amounts of valine in all the insects were similar to that of the whey protein. The BCAA content was 16.8 and 16.4% in G and T, respectively. The total polyphenolic content and the antioxidant activity of the insect proteins were significantly higher than those of the whey proteins. Based on the sensory test of the protein powder, the whey protein could be replaced by 25% of the isolated protein from the insects, but the taste and flavor needs to be improved. Although the BCAA content of insect proteins was lower than that of the whey protein (by 73-76%), their application could be useful because of high polyphenolic content and the anti-oxidative activity.

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

• Korean Journal of Microbiology
• /
• v.43 no.1
• /
• pp.19-22
• /
• 2007

Acetohydroxyacid synthase (E.C.2.2.1.6., AHAS) is the enzyme that catalyses the first step in the synthesis of the branched-chain amino acids valine, leucine and isoleucine. The AHAS gene (TIGR access code HI2585) from Heamophilus influenzae was cloned into the bacterial expression vector pET-28a and expressed in the Escherichia coli strain BL21(DE3). The expressed enzyme was purified by $Ni^{2+}-charged$ HiTrap chelating HP column. The purified enzyme appears as a single band on SDS-PAGE with a molecular mass of about 63.9 kDa. The enzyme exhibits absolute dependence on the three cofactors FAD, $MgCl_{2}$ and thiamine diphosphate for activity. Specific activity of purified enzyme has 3.22 unit/mg and optimum activity in the pH 7.5 at $37^{\circ}C$. This enzyme activity has an effect on the buffer. When comparing the enzyme activity against the organic solvent, it followed in type and the difference it is but even from the aqueous solution where the organic solvent is included with the fact that the enzyme activity is maintained.

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

• Clinical and Experimental Pediatrics
• /
• v.51 no.9
• /
• pp.964-970
• /
• 2008

Purpose : Methylmalonic aciduria (MMA) and propionic aciduria (PA) are inborn errors in the catabolism of branched-chain amino acids. The study was undertaken to investigate the genotypes and clinical features of Korean patients with MMA and PA. Methods : This study examined 12 patients with MMA and eight with PA. We analyzed various clinical features, laboratory findings, treatments, and neuro-developmental outcomes. Diagnoses were based on the presence of characteristic compounds detected by amino acid analysis in serum and organic acid analysis in urine. Mutation analysis was performed in the genes of MUT, MMAA, MMAB, and MMACHC for MMA and PCCA and PCCB for PA. Results : Among the 20 patients, six patients were diagnosed before one month of age and nine patients were diagnosed after the newborn period. Five patients were diagnosed via a neonatal screening test. Patients with early-onset forms had more severe illness at presentation and generally poor outcomes. A favorable outcome was obtained in 55% patients; most of them were of a late-onset type or diagnosed by neonatal mass screening test without symptoms. Genotypes were confirmed in all patients with MMA. We detected 11 different mutations by MUT gene analysis in 10 patients, and three different mutations in MMACHC genes in two patients. PCCA and PCCB gene mutations were identified in 14 of the 16 alleles, in eight patients with PA. Conclusion : Organic aciduria is a fatal disease; however, better outcomes are expected whenever early diagnosis and prompt management are made possible. Mutation analysis is useful for confirming diagnoses and planning management strategies.