Purification and Some Properties of Threonine Dehydratase from Yeast

Katsunuma, Tsunehiko; Elsässer, Sigrid; Holzer, Helmut

doi:10.1111/j.1432-1033.1971.tb19657.x

Cited by 25 publications

(6 citation statements)

References 14 publications

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“…This would be a prerequisite for the catabolism of threonine via 2-oxobutyrate by the bacterium. The properties of the dehydratase from the Corynebacterium were similar to those of the enzyme in Rhodopseudomonas spheroides (Datta, 1966;Barritt, 1971), a yeast (Katsunuma et al, 1971) and a species of Brevibacterium (Miyajima & Shiio, 1972) rather than in the enteric bacteria. In the latter case, valine only normalizes the substrate-response curve in the presence of isoleucine (Umbarger, 1973).…”

Section: Discussionmentioning

confidence: 67%

Bacterial catabolism of threonine. Threonine degradation initiated by l-threonine hydro-lyase (deaminating) in a species of Corynebacterium

Bell

Turner

1977

Biochemical Journal

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1. Three bacterial isolates capable of growth on L-threonine medium only when supplemented with branched-chain amino acids, and possessing high L-threonine dehydratase activity, were examined to elucidate the catabolic route for the amino acid. 2. Growth, manometric, radiotracer and enzymic experiments indicated that L-threonine was catabolized by initial deamination to 2-oxobutyrate and thence to propionate. No evidence was obtained for the involvement of L-threonine 3-dehydrogenase or L-threonine aldolase in threonine catabolism. 3. L-Threonine dehydratase of Corynebacterium sp. F5 (N.C.I.B. 11102) was partially purified and its kinetic properties were examined. The enzyme exhibited a sigmoid kinetic response to substrate concentration. The concentration of substrate giving half the maximum velocity, [SO.5], was 40mM and the Hill coefficient (h) was 2.0. L-Isoleucine inhibited enzyme activity markedly, causing 50% inhibition at 60,UM, but did not affect the Hill constant. At the fixed L-threonine concentration of 10mM, the effect of L-valine was biphasic, progressive activation occurring at concentrations up to 2mM-L-valine, but was abolished by higher concentrations. Substrate-saturation plots for the L-valine-activated enzyme exhibited normal MichaelisMenten kinetics with a Hill coefficient (h) of 1.0. The kinetic properties of the enzyme were thus similar to those of the 'biosynthetic' isoenzyme from Rhodopseudomonas spheroides rather than those of the enteric bacteria. 4. The synthesis of L-threonine dehydratase was constitutive and was not subject to multivalent repression by L-isoleucine or other branched-chain amino acids either singly or in combination. 5. The catabolism of L-threonine, apparently initiated by a 'biosynthetic' L-threonine dehydratase in the isolates studied, depended on the concomitant catabolism of branched-chain amino acids. The biochemical basis of this dependence appeared to lie in the further catabolism of 2-oxobutyrate by enzymes which required branched-chain 2-oxo acids for their induction.

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Section: Discussionmentioning

confidence: 67%

Bacterial catabolism of threonine. Threonine degradation initiated by l-threonine hydro-lyase (deaminating) in a species of Corynebacterium

Bell

Turner

1977

Biochemical Journal

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“…Our results demonstrated that phosphate changes both the ligand binding and the temperature effects including temperature op- timum, activation energy, and thermal stability. The enzyme from C. malfosa is activated by valine, inhibited by isoleucine, and a sigmoid substrate saturation curve is obtained in the absence of isoleucine, comparable to the enzyme from S. cerevisiae [3,11]. In the presence of phosphate, the K, for threonine, the Ki for isoleucine, and the K, for valine increase, while the co-operativity between substrate molecules and the C. maltosa enzyme decreases.…”

Section: Cphosphatel (M)mentioning

confidence: 79%

Threonine dehydratase activity from several yeast species is activated and affected by phosphate

Bode

Birnbaum

1986

FEMS Microbiology Letters

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The threonine dehydratase activity of several yeast species was examined. Of 14 species tested, Candida maltosa, Pichia guilliermondii, Pichia pinus and Yarrowia lipolytica were found to produce an enzyme whose activity was increased about 20‐fold in the presence of phosphate ions. It is suggested that phosphate affects the allosteric properties of the enzyme and leads, thereby, to an alteration of the binding of threonine (substrate), isoleucine (negative effector), and valine (positive effector) as well as to an alteration of the effects of temperature on the enzyme including changes in temperature optimum, energy of activation, and heat inactivation.

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“…It was reported that the L Ile concentrations measured in the cells of C. glutamicum were always higher than the extracellu lar ones at all fermentation process [16], so the TDH of the strains was exposed in a high concentration of L Ile. A comparison of amino acid sequences between the tdcB polypeptide and the biosynthetic TDH of yeast (encoded by ILV1) and E. coli (encoded by ilvA) and other strains revealed various extents of homology [17][18][19]. The surprising difference from those homol ogies is in the carboxy terminal region [14,20], and analysis of the structure of TDH by site directed mutation in this site may be a strategy for further increasing L Ile production.…”

Section: Discussionmentioning

confidence: 99%

Expression of the Escherichia Coli TdcB gene encoding threonine dehydratase in L-isoleucine-overproducing Corynebacterium Glutamicum Yilw

Xie

Shi

et al. 2013

Appl Biochem Microbiol

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125 1 As an essential branched chain amino acid, L Ile holds an important part of amino acid market share. It could not be directly synthesized by mammals and its main application is in the field of synthetic nutrition for infusions or special diets. In microorganisms, it is synthesized via several steps, starting from the central precursor metabolite, L Asp, with branches to L Lys, L Met, additionally with L Thr being produced as intermediates. Enzymes involved in this biosynthesis include: aspartate kinase, homoserine dehydrogenase, homoserine kinase, threonine dehydratase (TDH), and acetohydroxyacid synthase. The last enzyme is the first enzyme in a series of enzymes that catalyze paral lel reactions to Ile and Val, both TDH and acetohy droxyacid synthase are feedback inhibited by L Ile [1]. Because of this tight regulation, fermentative pro cesses for L Ile are in general not as well developed as those for L Lys, for which there seems to be rather simple types of flux control [2][3][4][5].Although earlier work with direct manipulations of genes and enzymes in the L Ile biosynthetic pathway have proven very useful for improving its biosynthesis, in fact, one enzyme activity was found to affect the L Ile yield markedly, such as TDH [6]. TDH (EC 4.3.1.19), which catalyzes the degradation of Thr to α ketobutyrate, is a rate limiting enzyme in the L Ile pathway. The Escherichia. coli ilvA and tdcB genes encode two apparent TDH isozymes and both gene products catalyze the conversion of Thr to 2 oxobu tanoate in vivo [7]. However, the genes are not physio logically redundant and E. coli ilvA mutants are Ile auxotrophs even though a wild type copy of tdcB is 1 The article is published in the original. still present in the genome. In vitro, the tdcB enzyme is insensitive to feedback regulation by L Ile [7]. Homoserine, homoserine kinase and threonine dehy dratase diverted much carbon flow from L Thr to L Ile, we found that only expression of tdcB is sufficient to sup port enough intermediates into L Ile pathway [8].The aim of the work was to study enzymatic prop ertities and application of the E. coli TdcB. We first cloned and expressed the tdcB gene in the E. coli BL21 (DE3), and determined several characteristics of TdcB. Finally, we expressed the tdcB gene in the L iso leucine producing strain Corynebacterium glutamicum YILW. The analysis of biomass, residual glucose, L Ile and byproducts, helps to investigate the effect of expression tdcB on L Ile production with the indus trial production strain. MATERIALS AND METHODS Strains, Plasmids, and Growth Conditions.The strains and plasmids used in this study are listed in the Table. E. coli were grown in LB medium at 37°C [10], C. glutamicum were grown in LBG medium (LB with 0.5% glucose) at 32°C. The following media were used for L Ile production with C. glutamicum. The seed medium (g/L): glucose-30, yeast extract-5, (NH 4 ) 2 SO 4 -3, KH 2 PO 4 ⋅ 3H 2 O-1.5, MgSO 4 ⋅ 7H 2 O-0.6, FeSO 4 ⋅ 7H 2 O-0.01, MnSO 4 ⋅ H 2 O-0.01, containing 30 ml/L of corn steep liquor and 30...

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Purification and Some Properties of Threonine Dehydratase from Yeast

Cited by 25 publications

References 14 publications

Bacterial catabolism of threonine. Threonine degradation initiated by l-threonine hydro-lyase (deaminating) in a species of Corynebacterium

Bacterial catabolism of threonine. Threonine degradation initiated by l-threonine hydro-lyase (deaminating) in a species of Corynebacterium

Threonine dehydratase activity from several yeast species is activated and affected by phosphate

Expression of the Escherichia Coli TdcB gene encoding threonine dehydratase in L-isoleucine-overproducing Corynebacterium Glutamicum Yilw

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