• Korean Journal of Weed Science
• /
• v.17 no.2
• /
• pp.199-206
• /
• 1997

Chlorsulfuron, an acetolactate-synthase-inhibiting sulfonylurea herbicide, induces many metabolic and physiological changes in susceptible plants. The objective of this study was to determine to what extent chlorsulfuton-induced phytotoxicity was due to a shortage of final products(the branched-chain amino acids valine, leucine, and isoleucine) or to an accumulation of a toxic metabolite(2-ketobutyrate), or both, in a susceptible species. Chlorsulfuron-treated canola seedlings showed growth inhibition and injury symptoms that included chlorosis, downward leaf rolling, and accumulation of anthocyanins. Supplementation with valine, leucine, and isoleucine prevented the chlorsulfuron-induced growth inhibition and injury symptoms only partially, suggesting that factor(s) other than a shortage of the branched-chain amino acids also are involved in the phytotoxicity. Canola seedlings treated with 2-ketobutyrate showed reduced growth, but they showed different changes in metabolites than seedlings treated with chlorsulfuron. The results suggest that 2-ketobutyrate is not involved in chlorsulfuron-induced phytotoxicity. We conclude that chlorsulfuron-induced phytotoxicity is due at least in part to a shortage of branched-chain amino acids.

• BMB Reports
• /
• v.28 no.1
• /
• pp.40-45
• /
• 1995

Acetolactate synthase purified from Serratia marcescens ATCC 25419 was rapidly inactivated by the thiol specific reagent p-chloromercuribenzoate (PCMB), the tryptophan specific reagent N-bromosuccinimide (NBS), and the arginine modifying reagent phenylglyoxal (PGO). Inactivation by PCMB was prevented by both ${\alpha}$-ketobutyrate and pyruvate, and the second order rate constant for the inactivation was $2480\;M^{-1}{\cdot}min^{-1}$. The reaction order with respect to PCMB was 0.94. The inactivation of the enzyme by NBS was also substantially reduced by both ${\alpha}$-ketobutyrate and pyruvate. The second order rate constant for inactivation by NBS was $15,000\;M^{-1}{\cdot}min^{-1}$, and the reaction order was 2.0. On the other hand, inactivation by PGO was partially prevented by ${\alpha}$-ketobutyrate, but not by pyruvate. The second order rate constant for the inactivation was $1480\;M^{-1}{\cdot}min^{-1}$ and the order of reaction with respect to PGO was 0.75. These results suggest that essential cysteine, tryptophan and arginine are located at or near the substrate binding site.

• BMB Reports
• /
• v.32 no.1
• /
• pp.39-44
• /
• 1999

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.

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

• The Korean Journal of Food And Nutrition
• /
• v.10 no.4
• /
• pp.475-479
• /
• 1997

The effects of amino acids and metabolites in growth media on the biosynthesis of prodigiosin from Serratia marcescens ATCC 25419 were examined. The prodigiosin synthesis was decreased approximately by 50 to 80% by several amino acids and metabolites tested. The prodigiosin synthesis was increased approximately by 20 to 40% by a low concentration of glyoxylate(1 to 3mM) and outstandingly increased by 122% at 5mM concentration under anaerobic condition. However, the prodigiosin synthesis was decreased approximately by 50 to 90% at a high concentration(20 to 30mM) under anaerobic condition. The prodigiosin was not synthesized by pyruvate and $\alpha$-ketobutyrate under aerobic and anaerobic condition, with addition to glyoxylate under aerobic condition, among the range from 0.5 to 30mM, while the cell growth under anaerobic condition was decreased distinctly by a high concentration(20mM above) of glyoxylate. These data suggest that the growth and prodigiosin of S. marcescens is positively regulated by a low concentration of glyoxylate (1-5mM), but repressed by a high concentration of glyoxylate(20mM above) unlike pyruvate and $\alpha$-ketobutyrate.

• Korean Journal of Microbiology
• /
• v.49 no.1
• /
• pp.46-50
• /
• 2013

Some rhizobacteria producing 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase can make plant to continue growth under the stress conditions through lowering the level of phytohormone, ethylene which inhibits the plant growth and accelerates plant aging. In this study, some rhizobacteria producing ACC deaminase have been isolated from the rhizosphere of plants grown at sand beaches, and identified as Escherichia hermannii m-2, Enterobacter asburiae m-4, Pseudomonas thivervalensis BD2-26 and Pseudomonas brassicacearum subsp. neoaurantiaca BD3-35 through sequencing of 16S rRNA genes. Strain BD3-35 showed the highest activity of ACC deaminase among the isolates, 20.26 ${\alpha}$-ketobutyrate ${\mu}M/mg$ protein/h. Strains BD3-35 and BD2-26 secreted a phytohormone cytokinin, and strains m-4 and m-2 could produce auxin and abscisic acid, respectively. When these bacteria were applied to the 7-day old tomato plant under drought stress for 7 days, strains BD3-35, m-2, and m-4 increased the length of tomato root by 14, 15, and 35%, respectively, and strains m-2, BD2-26 and BD3-35 increased the dry weight of tomato plant by 22, 33, and 68%, respectively compared to the uninoculated control tomatoes. Therefore, these rhizobacteria may be utilized as a microbial fertilizer for the plants under drought stress.

• Korean Journal of Microbiology
• /
• v.53 no.4
• /
• pp.251-256
• /
• 2017

Some rhizobacteria were isolated, that have copper resistance and can confer copper resistance to plants allowing growth under copper stress. Isolated strains Pseudomonas veronii MS1 and P. migulae MS2 produced 0.13 and 0.26 mmol/ml of siderophore, that is a metal-chelating agent, and also showed 64.6 and 77.9% of biosorption ability for Cu in 20 mg/L Cu solution, respectively. Copper can catalyze a formation of harmful free radicals, which may cause oxidative stress in organisms. Removal activity of 1,1-diphenyl-2-picryl hydrazyl radical and antioxidant capacity of strains MS1 and MS2 increased up to 82.6 and 78.1%, respectively compared to those of control at 24 h of incubation. They exhibited 7.10 and $6.42{\mu}mol$ ${\alpha}$-ketobutyrate mg/h of 1-aminocyclopropane-1-carboxylic acid deaminase activity, respectively, which reduced levels of stress hormone, ethylene in plants, and also produced indole-3-acetic acid and salicyclic acid that can help plant growth under abiotic stress. All these results indicated that these copper-resistant rhizobacteria could confer copper resistance and growth promotion to plants.

• The Korean Journal of Food And Nutrition
• /
• v.23 no.2
• /
• pp.171-177
• /
• 2010

The catabolic threonine dehydratase from Serratia marcescens ATCC 25419 was purified to homogeniety using Sephadex G-200 gel filtration and AMP-Sepharose 4B affinity chromatography. The molecular weight of the native enzyme was 120,000 by native pore gradient PAGE. The enzyme was composed of four identical subunits with subunit molecular weights of 30,000 by SDS-PAGE. The Km values of the enzyme for L-threonine with and without AMP were 7.3 and 92 mM, respectively. There were 2 moles of pyridoxal phosphate and 16 moles of free -SH groups per 1 mole of enzyme. The enzyme was inhibited by $\alpha$-ketobutyrate, pyruvate, glyoxylate, and phosphoenol pyruvate(PEP) in the presence of AMP, yet stimulated by cAMP and ADP. For enzyme properties in comparison with S. marcescens, E. coli, and S. typhimurium enzyme, such as the PLP content, number of free sulfhydryl groups, and existence of ADP binding site, the S. marcescens enzyme was more similar to the S. typhimurium enzyme than the E. coli enzyme. Of the three enteric bacteria, the E. coli and S. typhimurium enzyme was increased the activity by ADP and cAMP, respectively, but only the S. marcescens enzyme was increased the activity by both ADP and cAMP. Therefore, the subtle differences in the properties between enzymes from the three enteric bacteria may represent minor structural differences among these enzymes and warrants further study.

• BMB Reports
• /
• v.28 no.2
• /
• pp.118-123
• /
• 1995

Biodegradative threonine dehydratase was purified to homogeneity from Serratia marcescens ATCC 25419 by streptomycin sulfate treatment, Sephadex G-200 gel filtration chromatography followed by AMP-Sepharose 4B affinity chromatography. The molecular weight of the purified enzyme was 118,000 by fast protein liquid chromatography using superose 6-HR. The enzyme was determined to be a homotetrameric protein with subunit molecular weights of 30,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was inhibited by ${\alpha}-Keto$ acids and their derivatives such as ${\alpha}-ketobutyrate$, pyruvate, glyoxlyate, and phosphoenol pyruvate, but not by ${\alpha}-aminobutyrate$ and ${\alpha}-hydroxybutyrate$. The inhibition of the enzyme by pyruvate and glyoxylate was observed in the presence of AMP. The inhibitory effect of glyoxylate was decreased at high enzyme concentration, whereas the inhibition by pyruvate was independent of the enzyme concentration. The kinetics of inhibition of the enzyme by pyruvate and glyoxylate revealed a noncompetitive and mixed-type inhibition by the two inhibitors with respect to L-threonine and AMP, respectively.

• Bulletin of the Korean Chemical Society
• /
• v.24 no.5
• /
• pp.627-632
• /
• 2003

The pH dependence of the kinetic parameters of recombinant acetohydroxy acid synthase catalyzed reaction was determined in order to obtain information about the chemical mechanism, particularly acid-base chemistry. The maximum velocity and V/K for pyruvate were bell-shaped with estimated pK values of 6.5-6.7 and 8.6-8.9, respectively. The maximum velocity and V/K for 2-ketobutyrate were also bell-shaped with estimated pK values of 6.6-7.0 and 8.4-8.6. The pH dependence of 1/Ki for 3-bromopyruvate, a competitive inhibitor of pyruvate, was also bell-shaped, giving pK values almost identical with those obtained for pyruvate. Since the same pK values were observed in the $pK_{i 3-bromopyruvate}$, V/K pH profiles and $V_{max}$ profiles, both enzyme groups must be in their optimum protonation state for efficient binding of reactants. These results reflect that two enzyme groups are necessary for binding of substrate and/or catalysis.