Rapid and Specific Determination of Threonine

Flavin, Martin; Slaughter, Clarence

doi:10.1021/ac60156a028

Cited by 32 publications

(21 citation statements)

References 7 publications

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“…Threonine synthase has been purified from Neurospora crassa [5] and from Escherichia coli [6]. The corresponding gene has been isolated from a number of bacteria [6][7][8][9][10][11] and from Saccharomyces cerevisiae [12].…”

Section: Introductionmentioning

confidence: 99%

Characterization of an Arabidopsis thaliana cDNA encoding an S‐adenosylmethionine‐sensitive threonine synthase Threonine synthase from higher plants

et al. 1996

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An Arabidopsis thaliana cDNA encoding an Sadenosylmethionine-sensitive threonine synthase (EC 4.2.99.2) has been isolated by functional complementation of an Escherichia coli mutant devoid of threonine synthase activity. Threonine synthase from A. thaliana was shown to be synthesized with a transit peptide. The recombinant protein is activated by Sadenosylmethionine in the same range as the plant threonine synthase and evidence is presented for an involvement of the Nterminal part of the mature enzyme in the sensitivity to Sadenosylmethionine.

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Section: Introductionmentioning

confidence: 99%

Characterization of an Arabidopsis thaliana cDNA encoding an S‐adenosylmethionine‐sensitive threonine synthase Threonine synthase from higher plants

et al. 1996

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“…Instead, homoserine kinase is feedback-inhibited by threonine (17,18). TS from the fungal class are unique among PLP-dependent enzymes, because they fulfill their physiological function in monomeric state (8,10). Recently, the crystal structure of the aTS apoprotein structure has been solved (19) indicating its domain organization and its quaternary assembly.…”

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confidence: 99%

Structure and Function of Threonine Synthase from Yeast

Garrido-Franco

Ehlert

Messerschmidt

et al. 2002

Journal of Biological Chemistry

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Threonine synthase catalyzes the final step of threonine biosynthesis, the pyridoxal 5-phosphate (PLP)-dependent conversion of O-phosphohomoserine into threonine and inorganic phosphate. Threonine is an essential nutrient for mammals, and its biosynthetic machinery is restricted to bacteria, plants, and fungi; therefore, threonine synthase represents an interesting pharmaceutical target. The crystal structure of threonine synthase from Saccharomyces cerevisiae has been solved at 2.7 Å resolution using multiwavelength anomalous diffraction. The structure reveals a monomer as active unit, which is subdivided into three distinct domains: a small N-terminal domain, a PLP-binding domain that covalently anchors the cofactor and a so-called large domain, which contains the main of the protein body. All three domains show the typical open ␣/␤ architecture. The cofactor is bound at the interface of all three domains, buried deeply within a wide canyon that penetrates the whole molecule. Based on structural alignments with related enzymes, an enzyme-substrate complex was modeled into the active site of yeast threonine synthase, which revealed essentials for substrate binding and catalysis. Furthermore, the comparison with related enzymes of the ␤-family of PLP-dependent enzymes indicated structural determinants of the oligomeric state and thus rationalized for the first time how a PLP enzyme acts in monomeric form.Threonine synthase (TS, EC 4.2.99.2) 1 is a pyridoxal 5Ј-phosphate (PLP)-dependent enzyme that catalyzes the ultimate step in threonine biosynthesis, the PLP-dependent ␤,␥-replacement reaction (Reaction 1) of O-phosphohomoserine (OPHS) yielding threonine and inorganic phosphate (1-3).Together with tryptophan synthase (TRPS), threonine deaminase (TDA), O-acetylserine sulfhydrylase (OASS), cystathione ␤-synthase (CBS), and 1-aminocyclopropane-1-carboxylate deaminase (ACCD), TS constitutes the core of the fold-type II family of PLP enzymes (4, 5) (also referred to as ␤-family (6)). Detailed amino acid sequence alignments revealed that TS can be grouped into a plant and a fungal subfamily (7). The former one (class I subfamily) comprises TS from higher plants, cyanobacteria, archaebacteria, and the eubacterial groups of Mycobacteria, Aquificaceae, and Bacillus species. The second subfamily (class II subfamily) contains the enzymes from fungi and from the eubacterial groups of Proteobacteria and corneyform bacteria. Only 5 from ϳ500 residues are invariant between both subfamilies, including the PLP-binding lysine as part of a phenylalanine-lysine-aspartate consensus sequence.During the last decades, TS was purified and characterized from several bacteria and fungi (8 -12) and from Arabidopsis thaliana (7, 13). In plants, the substrate of TS, OPHS, is the branching point for threonine and methionine biosynthesis. Flux coordination between both synthetic pathways is accomplished by allosteric activation of plant threonine synthase. The allosteric effector is S-adenosyl methionine (SAM) (14, 15), a product of methioni...

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“…In all cases, with or without maleimide, a slightly lower yield of the keto acids as compared to the amount of threonine disappeared was due to a small amount of radioactivity distributed throughout the paper and not included in the calculations. From the cumulative results presented thus far, we conclude that N-ethylmaleimide was indeed trapping 2-aminocrotonate to form 2-oxo-3[3'-(N'-ethyl-2',5'-dioxypyrrolidyl)]butyric acid as shown by Flavin and Slaughter [7,8] and that, in the absence of Nethylmaleimide, 2-aminocrotonate was the reducing equivalent for the reduction of ferricyanide.…”

Section: Trapping Of the Intermediate By N-ethylmaleimidementioning

confidence: 63%

“…Addition of N-ethylmaleimide traps this material as a keto acid positive compound (as revealed by reactions with semicarbazide [9] and dinitrophenyl hydrazine) which fails to reduce ferricyanide. From several lines of evidence obtained with various enzymes catalyzing ct, p elimination reactions (including threonine dehydratase), Falvin and Slaughter [8] have shown conclusively that 2-aminocrotonate, and not 2-iminobutyrate, reacts with maleimide to form an adduct…”

Section: Discussionmentioning

confidence: 99%

“…The time of incubation with N-ethylmaleimide, shown in the abscissa, represents the time, in seconds, the reaction mixture was in contact with maleimide prior to ferricyanide addition: Control experiments revealed that in 120 s only a 20% decrease of ferricyanide reduction was observed in the absence of maleimide aminocrotonate which then tautomerizes to form 2-iminobutyrate; the latter compound, free in solution, undergoes non-enzymatic hydrolysis to liberate ammonia and 2-oxobutyrate. Using the same enzyme from E. coli, Flavin and Slaughter [7,8] Fig. 3).…”

Section: Trapping Of the Intermediate By N-ethylmaleimidementioning

confidence: 99%

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Biodegradative Threonine Dehydratase. Reduction of Ferricyanide by an Intermediate of the Enzyme-Catalyzed Reaction

Datta

Bhadra

1978

Eur J Biochem

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The threonine-dependent reduction of ferricyanide catalyzed by the purified biodegradative threonine dehydratase of Escherichiu coli has been studied. The rate of production of 2-oxobutyrate in the presence of ferricyanide was lower than that found in the absence of ferricyanide. The concentrations of threonine required for half-maximal effects for the reduction of ferricyanide and, in the presence of the dye, for 2-oxobutyrate production, were 3 mM and 9 mM, respectively. Reduction of ferricyanide was accompanied by evolution of C02, and even within a very short incubation time with the enzyme, the ratio of ferricyanide reduced over C02 evolved was approximately 7. Stopping the enzyme activity after a brief exposure to threonine at pH 9.7 resulted in the accumulation of an intermediate (with a half-life of 4 min at 25 "C) which formed an adduct with N-ethylmaleimide; the accumulated intermediate, in the absence of N-ethylmaleimide, reduced ferricyanide with concomitant evolution of C02. We conclude from these results that 2-aminocrotonate is the intermediate which serves as a source of reducing equivalent for ferricyanide, and nonstoichiometric amount of ferricyanide reduction may be attributed to some secondary reactions of ferricyanide with compounds derived from the oxidation product of 2-aminocrotonate.With phenazine methosulfate and nitroblue tetrazolium, Feldberg and Datta [l] developed an activity stain for threonine dehydratase of Rhodospirillum rubrum on polyacrylamide gels. To explore the chemical basis for this activity stain, the reaction was studied spectrophotometrically using ferricyanide as an artificial electron acceptor. The results showed that in potassium phosphate buffer, pH 8.0, the threonine-dependent reduction of ferricyanide was linear for approximately one minute and then decreased rapidly to a near-zero rate after the reduction of about 0.5 mM ferricyanide. Neither the products of the dehydration reaction, 2-oxobutyrate and ammonia, nor the combination of products plus ferrocyanide affected the total amount of ferricyanide reduced (0.5 mM) in the presence of enzyme and threonine.

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Rapid and Specific Determination of Threonine

Cited by 32 publications

References 7 publications

Characterization of an Arabidopsis thaliana cDNA encoding an S‐adenosylmethionine‐sensitive threonine synthase Threonine synthase from higher plants

Characterization of an Arabidopsis thaliana cDNA encoding an S‐adenosylmethionine‐sensitive threonine synthase Threonine synthase from higher plants

Structure and Function of Threonine Synthase from Yeast

Biodegradative Threonine Dehydratase. Reduction of Ferricyanide by an Intermediate of the Enzyme-Catalyzed Reaction

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