• Title, Summary, Keyword: Threonine Dehydrogenase

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Producyion of Threonine Using Methanol Dehydrogenase and Serine Hydroxyltransferase in a New Methylotrophic Bacterium KJ29 (New Methylotrophic Bacterium KJ29의 Methanol Dehydrogenase와 Serine Hydroxymethyltransferase를 이용한 Threonine의 생산에 관한 연구)

  • 김경자
    • Microbiology and Biotechnology Letters
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    • v.21 no.6
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    • pp.577-581
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    • 1993
  • The amino acid threonine was produced from glycine and ethanol in a reaction mixture using cell free extract of the methylotrophic bacterium isolated from soil and identified as mellthylo-bacterium sp. KJ29. Although the isolate could grow on carbon source other than methanol, only the cell free extract from the cells grown on methanol produced threonine. Methanol dehydrogenase (MDH) activity was present only in the cells grown on methanol when compared to the cells grown on heterotrophic substrates.

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Effect of Protein and Threonine Level in the Diet on Liver Threonine Dehydrogenase Activity in Growing Rat (성장하는 랫트에 있어서 단백질과 Threonine의 급여 수준이 간 Threonine Dehydrogenase 활성에 미치는 영향)

  • 이철원
    • The Korean Journal of Food And Nutrition
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    • v.15 no.3
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    • pp.235-240
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    • 2002
  • The aim of this investigation was to determine the influence of different protein and threonine (Thr) levels on the liver threonine dehydroaenase (TDG) activity b\ulcorner rats. In rats fed on CP (crude Protein) - diets, TDG activity was increased during an CP rise to 12.0% CP, decreased slightly down to 18.0% CP and showed .a trend to increase from 18.0 to 24.0% CP. In rats the feeding with graded Protein supply gavee no indication for additional stimulation of threonine-oxidation by TDG over a wide range of CP-content in the diets. The increase in threonine content from 0.28 to 0.72% in the presence of 12.0% CP caused a gradual increase in TDG activity in rat liver. This similarly applied to the feed admixed with 18.0% CP, but at a higher level.

Biotechnology for the Production of Threonine Production (Threonine의 생물공학적 생산)

  • Kim, Kyoung-Ja
    • YAKHAK HOEJI
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    • v.34 no.6
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    • pp.447-456
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    • 1990
  • Various methods are available for the production of L-threonine. The microbial production of L-threonine has been achieved by breeding L-threonine analog-resistant auxotrophic mutants of various bacteria. The enzymatic production of L-threonine has been demonstrated by use of threonine metabolic enzymes such as threonine deaminase, threonine aldolase, or threonine dehydrogenase complex. Threonine synthesis from glycine and ethanol seems to be catalyzed by the enzymes Methanol dehydrogenase(MDH) and Serine hydroxymethyltransferase(SHMT), which was also found to catalyze the aldol condensation of glycine with acetaldehyde. The improved production of L-threonine has been achieved by amplifying the genes for the L-threonine biosynthetic enzymes using recombinant DNA techniques.

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Effects of Dietary Protein and Threonine Supply on In vitro Liver Threonine Dehydrogenase Activity and Threonine Efficiency in Rat and Chicken

  • Lee, C.W.;Oh, Y.J.;Son, Y.S.;An, W.G.
    • Asian-Australasian Journal of Animal Sciences
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    • v.24 no.10
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    • pp.1417-1424
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    • 2011
  • This study was conducted to assess the relation between threonine (Thr) oxidation rate and threonine efficiency on rat and chicken fed with graded levels of protein and threonine. The increase in threonine content from 0.28 to 0.72% in a diet containing 12.0% crude protein (CP) caused a gradual increase in threonine dehydrogenase (TDG) activity in rat liver. Similar, but more pronounced results were observed after 18.0% CP in the diet. Both protein levels in combination with the highest level of threonine supplementation increased liver TDG activity significantly, indicating enhanced threonine catabolism. Parameters of efficiency of threonine utilization calculated from parallel nitrogen balance studies decreased significantly and indicated threonine oversupply after a maximum of threonine supplementation. At the lower levels of threonine addition the efficiency of threonine utilization was not significantly changed. In the chicken liver up to 0.60% true digestible threonine (dThr) in the 18.5% CP diet produced no effect on the TDG activity. However, TDG activity in the liver was elevated by the diet containing 22.5% CP (0.60% dThr) and the efficiency of threonine utilization decreased, indicating the end of threonine limiting range. In conclusion, the in vitro TDG activity in the liver of rat and growing chicken has an indicator function for the dietary supply of threonine.

Identification and Characterization of Protein Encoded by orf382 as $\small{L}$-Threonine Dehydrogenase

  • Ma, Fei;Wang, Tianwen;Ma, Xingyuan;Wang, Ping
    • Journal of Microbiology and Biotechnology
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    • v.24 no.6
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    • pp.748-755
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    • 2014
  • In the genome annotation of Escherichia coli MG1655, the orf382 (1,149 bp) is designated as a gene encoding an alcohol dehydrogenase that may be Fe-dependent. In this study, the gene was amplified from the genome by PCR and overexpressed in Escherichia coli BL21(DE3). The recombinant $6{\times}$His-tag protein was then purified and characterized. In an enzymatic assay using different hydroxyl-containing substrates (n-butanol, $\small{L}$-threonine, ethanol, isopropanol, glucose, glycerol, $\small{L}$-serine, lactic acid, citric acid, methanol, or $\small{D}$-threonine), the enzyme showed the highest activity on $\small{L}$-threonine. Characterization of the mutant constructed using gene knockout of the orf382 also implied the function of the enzyme in the metabolism of $\small{L}$-threonine into glycine. Considering the presence of tested substrates in living E. coli cel ls and previous literature, we believed that the suitable nomenclature for the enzyme should be an $\small{L}$-threonine dehydrogenase (LTDH). When using $\small{L}$-threonine as the substrate, the enzyme exhibited the best catalytic performance at $39^{\circ}C$ and pH 9.8 with $NAD^+$ as the cofactor. The determination of the Km values towards $\small{L}$-threonine (Km = $11.29{\mu}M$), ethanol ($222.5{\mu}M$), and n-butanol ($8.02{\mu}M$) also confirmed the enzyme as an LTDH. Furthermore, the LTDH was shown to be an ion-containing protein based on inductively coupled plasma-atomic emission spectrometry with an isoelectronic point of pH 5.4. Moreover, a circular dichroism analysis revealed that the metal ion was structurally and enzymatically essential, as its deprivation remarkably changed the ${\alpha}$-helix percentage (from 12.6% to 6.3%).

Development of L-Threonine Producing Recombinant Escherichia coli using Metabolic Control Analysis (대사 조절 분석 기법을 이용한 L-Threonine 생산 재조합 대장균 개발)

  • Choi, Jong-Il;Park, Young-Hoon;Yang, Young-Lyeol
    • KSBB Journal
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    • v.22 no.1
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    • pp.62-65
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    • 2007
  • New strain development strategy using kinetic models and metabolic control analysis was investigated. In this study, previously reported mathematical models describing the enzyme kinetics of intracellular threonine synthesis were modified for mutant threonine producer Escherichia coli TF5015. Using the modified models, metabolic control analysis was carried out to identify the rate limiting step by evaluating the flux control coefficient on the overall threonine synthesis flux exerted by individual enzymatic reactions. The result suggested the production of threonine could be enhanced most efficiently by increasing aspartate semialdehyde dehydrogenase (asd) activity of this strain. Amplification of asd gene in recombinant strain TF5015 (pCL-$P_{aroF}$-asd) increased the threonine production up to 23%, which is much higher than 14% obtained by amplifying aspartate kinse (thrA), other gene in threonine biosynthesis pathway.

Purification and Properties of Homoseine Dehydrogenases in Canavalia Iineata Cotyledons (해녀콩(Canavalia lineata) 자엽에서 Homoserine Dehydrogenase의 정제 및 특성)

  • 최영명
    • Journal of Plant Biology
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    • v.39 no.1
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    • pp.41-48
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    • 1996
  • Two forms of homo serine dehydrogenase have been isolated from 8-day-old cotyledons of Canavalin lineata by a heat denaturation, ammonium sulfate fractionation, DEAE-8ephacel ion exchange and Sephacryl 8-300 gel filtration chromatographies, and Pro cion red dye, Cibacron blue dye and Resource Q column chromatographies. The molecular weights of T -form (threonine-sensitive) and K-form(threonine- insensitive) were estimated to 230 kD and 135 kD, respectively. In the presence of 10 mM threonine, the activity of T-form was inhibited with almost 70%, but that of K-form was not at all. The Km values tor homo serine of T- and Kform were 1.6 mM and 0.3 mM, respectively. The Km values for NAD of T- and K-form were 2.34 mM and 0.03 mM, respectively. And Km values for NADP of two isozymes were the same as 0.01 mM. The activities of T- and K-form were markedly stimulated up to 4.9and 2.8-fold, respectively, by 400 mM KCI. The partial purified(gel filtration) enzymes(Tform and K-form) can be reversibly converted.verted.

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Effects of Dietary Levels of Glycine, Threonine and Protein on Threonine Efficiency and Threonine Dehydrogenase Activity in Hepatic Mitochondria of Chicks

  • Lee, C.W.;Cho, I.J.;Lee, Y.J.;Son, Y.S.;Kwak, I.;Ahn, Y.T.;Kim, S.C.;An, W.G.
    • Asian-Australasian Journal of Animal Sciences
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    • v.27 no.1
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    • pp.69-76
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    • 2014
  • This study was carried out to evaluate the relationship between threonine (Thr) efficiency and Thr dehydrogenase (TDG) activity as an indicator of Thr oxidation on chicks fed with levels of diets (CP [17.5% and 21.5%] and Thr [3.8 and 4.7 g/100 g CP]; glycine [Gly][0.64% and 0.98%] and true digestible Thr [dThr] [0.45% and 0.60%]). Calculation of the Thr efficiency was based on N-balance data and an exponential N-utilization model, and TDG activity was determined as accumulation of aminoacetone and Gly during incubation of hepatic mitochondria. This study found that in the liver of chicks who received a diet containing up to 0.79% Thr (4.7 g Thr/100 g of CP) in the 17.5% CP diet, no significant (p>0.05) effect on TDG activity was observed. However, significantly (p = 0.014) increased TDG activity was observed with a diet containing 21.5% CP (4.7 g Thr/100 g of CP) and the efficiency of Thr utilization showed a significant (p = 0.001) decrease, indicating the end of the Thr limiting range. No significant (p>0.05) effect on the total TDG activity and accumulation of Gly was observed with addition of Gly to a diet containing 0.45% dThr. In addition, addition of Gly to a diet containing 0.60% dThr also did not result in a change in accumulation of Gly. Due to an increase in accumulation of aminoacetone, an elevated effect on total TDG activity was also observed. No significant (p>0.05) reduction in the efficiency of Thr utilization was observed after addition of Gly at the level of 0.45% dThr. However, significantly (p<0.001) reduced efficiency of Thr utilization was observed after addition of Gly at the level of 0.60% dThr. Collectively, we found that TDG was stimulated not only by addition of Thr and protein to the diet, but also by addition of Gly, and efficiency of Thr utilization was favorably affected by addition of Gly at the level near to the optimal Thr concentration. In addition, no metabolic requirement of Gly through the TDG pathway was observed with almost the same accumulation of Gly and a slight increase in TDG activity by addition of Gly. Thus, our findings suggest that determination of TDG activity and parameter of efficiency of Thr utilization may be useful for evaluation of dietary Thr level.

Activity of Human Dihydrolipoamide Dehydrogenase Is Reduced by Mutation at Threonine-44 of FAD-binding Region to Valine

  • Kim, Hak-Jung
    • BMB Reports
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    • v.35 no.4
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    • pp.437-441
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    • 2002
  • Dihydrolipoamide dehydrogenase (E3) is a member of the pyridine nucleotide-disulfide oxidoreductase family. Thr residues are highly conserved. They are at the active site disulfide-bond regions of most E3s and other oxidoreductases,. The crystal structure of Azotobacter vinelandii E3 suggests that the hydroxyl group of Thr that are involved in the FAD binding interact with the adenosine phosphate of FAD. However, several prokaryotic E3s have Val instead of Thr. To investigate the meaning and importance of the Thr conservation in many E3s, the corresponding residue, Thr-44, in human E3 was substituted to Val by site-directed mutagenesis. The mutant’s E3 activity showed about a 2.2-fold decrease. Its UV-visible and fluorescence spectra indicated that the mutant might have a slightly different microenvironment at the FAD-binding region.

Purification and Crystallization of the Recombinant Catalytic Subunit of Pyruvate Dehydrogenase Phosphatase (Pyruvate Dehydrogenase Phosphatase의 Catalytic Subunit의 분리정제 및 결정화)

  • Kim, Young-Mi
    • Journal of Food Hygiene and Safety
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    • v.18 no.3
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    • pp.146-152
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    • 2003
  • Pyruvate dehydrogenase phosphatase (PDP) is a mitochondrial protein serine/threonine phosphatase that catalyzes the dephosphorylation and concomitant reactivation of the pyruvate dehydrogenase component of the pyruvate dehydrogenase complex (PDC). PDP consists of a catalytic subunit (PDPc, Mr 52,600) and regulatory subunit (PDPr, Mr 95,600). In the presence of $Ca^{2+}$, PDPc binds to the dihydrolipoamide acetyltransferase (E2) component of the pyruvate dehydrogenase complex in proximity to its substrate, the phosphorylated E1 component, thereby increasing the rate of dephosphorylation. PDPc possesses and intrinsic $Ca^{2+}$ binding site and a second $Ca^{2+}$ site is generated in the presence of E2. Using the unique interaction, highly pure PDPc was produced by the GSH-Sepharose-GST-L2 matrix with a specific activity of approx. 1000 U/mg and a yield of about 80%.