Purification of biosynthetic threonine deaminase from Escherichia coli

Koerner, K.; Rahimi-Laridjani, I.; Grimminger, H.

doi:10.1016/0005-2744(75)90195-3

Cited by 20 publications

(7 citation statements)

References 25 publications

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“…Early studies on the regulation of threonine deaminase also suggested an intricate mechanism of control, despite complications from questionable preparations of wild-type enzyme that exhibited hyperbolic steady-state kinetics, substoichiometric cofactor levels, and noncooperative binding of isoleucine and valine (Bums & Zarlengo, 1968;Harding, 1969;Hatfield & Umbarger, 1970;Calhoun et al, 1973;Decedue et al, 1975;Koemer et al, 1975). Of particular relevance are the findings by Decedue et al (1975) that threonine was an effective competitor of valine for binding to a modified enzyme that was inactivated by borohydride reduction of the internal aldimine Schiff base.…”

Section: Discussionmentioning

confidence: 99%

An Expanded Two-State Model Accounts for Homotropic Cooperativity in Biosynthetic Threonine Deaminase from Escherichia coli

Eisenstein

Yu²,

Fisher³

et al. 1995

Biochemistry

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The linkage between substrate and regulatory effector binding to separate sites on allosteric enzymes results in shifts in their sigmoidal kinetics to regulate metabolism. Control of branched chain amino acid biosynthesis in Escherichia coli occurs in part through shifts in the sigmoidal dependence of alpha-ketobutyrate production promoted by isoleucine and valine binding to biosynthetic threonine deaminase. The structural similarity of threonine, valine, and isoleucine have given rise to suggestions that there may be competition among different ligands for the same sites on this tetrameric enzyme, resulting in a complex pattern of regulation. In an effort to provide a coherent interpretation of the cooperative association of ligands to the active sites and to the effector sites of threonine deaminase, binding studies using single amino acid variants were undertaken. A previously-isolated, feedback-resistant mutant identified in Salmonella typhimurium, ilvA219, has been cloned and sequenced. The phenotype is attributable to a single amino acid substitution in the regulatory domain of the enzyme in which leucine at position 447 is substituted with phenylalanine. The mutant exhibits hyperbolic saturation curves in both ligand binding and steady-state kinetics. These results, in addition to calorimetric and spectroscopic measurements of isoleucine and valine binding, indicate that the low affinity (T) state is destabilized in the mutant and that it exists predominantly in the high affinity (R) conformation in the absence of ligands, providing an explanation for its resistance to isoleucine. Chemical and spectroscopic analyses of another mutant, in which alanine has replaced an essential lysine at position 62 that forms a Schiff base with pyridoxal phosphate, indicate that the cofactor is complexed to exogenous threonine and is therefore unable to bind additional amino acids at the active sites. Isoleucine and valine binding to this inactive, active site-saturated enzyme revealed that it too was stabilized in the R state, yielding binding constants in excellent agreement with the leucine to phenylalanine mutant. The lysine to alanine mutant was further utilized to demonstrate that both threonine and 2-aminobutyrate bind with stronger affinity to the regulatory sites than to the active sites. A direct consequence of these results is that substrates and analogs have a synergistic effect on the allosteric transition since, in effect, they act as both homotropic and heterotropic effectors.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Discussionmentioning

confidence: 99%

An Expanded Two-State Model Accounts for Homotropic Cooperativity in Biosynthetic Threonine Deaminase from Escherichia coli

Eisenstein

Yu²,

Fisher³

et al. 1995

Biochemistry

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“…Native enzyme in 0.1 M potassium phosphate (pH 7.4) containing 1 mM mercaptoethanol and 1 mM ethylenediaminetetraacetate. threonine deaminase of strain IP7 contains 1/2 molecule of coenzyme per subunit; we have shown previously that the wild-type enzyme has the same ratio of bound pyridoxal phosphate and protein subunits (15).…”

Section: Resultsmentioning

confidence: 94%

“…Biosynthetic threonine deaminase ofE. coli BL3 (15) and of mutant IP7 (8) was purified as described previously. The assay of biosynthetic threonine deaminase has been described (8).…”

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confidence: 99%

Threonine deaminase from a nonsense mutant of Escherichia coli requiring isoleucine or pyridoxine: evidence for half-of-the-sites reactivity

Feldner¹,

Grimminger²

1976

J Bacteriol

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The mutant IP7 of Escherichia coli B requires isoleucine or pyridoxine for growth as a consequence of a mutation in the gene coding for biosynthetic threonine deaminase. The mutation of IP7 was shown to be of the nonsense type by the following data: (1) reversion to isoleucine prototrophy involves the formation of external suppression at a high frequency, as shown by transduction experiments; and (ii) the isoleucine requirement is suppressed by lysogenization with a phage carrying the amber suppressor su-3. Cell extracts of the mutant strain contain a low activity of threonine deaminase. The possibility that this activity is biodegradative was ruled out by kinetic experiments. The mutant threonine deaminase was purified to homogeneity by conventional procedures. The enzyme is a dimer of identical subunits of an approximate molecular weight of 43,000 (Grimminger and Feldner, 1974), whereas the wild-type enzyme is a tetramer of 50,000-dalton subunits (Calhoun et al., 1973; Grimminger et al., 1973). The mutant enzyme is not inhibited by isoleucine and does not bind isoleucine, as shown by equilibrium dialysis experiments. Pyridoxal phosphate enhances the maximum catalytic activity of the mutant enzyme by a factor of five, whereas the wild-type enzyme is not affected. In wild-type and mutant threonine deaminase the ratio of protein subunits and bound pyridoxal phosphate is 2:1. The activation of threonine deaminase from strain IP7 is due to a second coenzyme binding site, as shown by (i) spectrophotometric titration of the enzyme with pyridoxal phosphate and by (ii) measurement the pyridoxal phosphate content of the enzyme after sodium borohydride reduction of the protein. The observation of one pyridoxal phosphate binding site per peptide dimer in the wild-type enzyme and of two binding sites per dimer in the mutant strongly suggests that one of the potential sites in the wild-type enzyme is masked by allosteric effects. The factors responsible for the half-of-the-sites reactivity of the coenzyme sites appear to be nonoperative in the mutant protein.

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“…TD generally forms a homotetramer with four PLP molecules bound. 10,18 Only the three-dimensional structure of the E. coli ilvA-encoded biosynthetic TD in complex with PLP has been determined to date. 19,20 The structure displays a type II fold of PLP-dependent enzymes.…”

mentioning

confidence: 99%

“…TD generally forms a homotetramer with four PLP molecules bound. , Only the three-dimensional structure of the E. coli ilvA -encoded biosynthetic TD in complex with PLP has been determined to date. , The structure displays a type II fold of PLP-dependent enzymes . The 2.8 Å structure includes two domains connected by a thin necklike region: a catalytic PLP-bound N-terminal domain and a C-terminal regulatory domain.…”

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confidence: 99%

Biochemical Characterization of the Mycobacterium smegmatis Threonine Deaminase

2018

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The biosynthesis of branched-chain amino acids or BCAAs (l-isoleucine, l-leucine, and l-valine) is essential in eubacteria, but mammals are branched-chain amino acid auxotrophs, making the enzymes in the pathway excellent targets for antibacterial drug development. The biosynthesis of l-isoleucine, l-leucine, and l-valine is very efficient, requiring only eight enzymes. Threonine dehydratase (TD), a pyridoxal 5'-phosphate (PLP)-dependent enzyme encoded by the ilvA gene, is the enzyme responsible for the conversion of l-threonine (l-Thr) to α-ketobutyrate, ammonia, and water, which is the first step in the biosynthesis of l-isoleucine. We have cloned, expressed, and biochemically characterized the reaction catalyzed by Mycobacterium smegmatis TD (abbreviated as MsIlvA) using steady-state kinetics and kinetic isotope effects. We show here that in addition to l-threonine, l-allo-threonine and l-serine are also used as substrates by TD, and all exhibit sigmoidal, non-Michaelis-Menten kinetics. Curiously, β-chloro-l-alanine was also a substrate rather than an inhibitor as expected. The enzymatic activity of TD is sensitive to the presence of allosteric regulators, including the activator l-valine or the end product feedback inhibitor of the BCAA pathway in which TD is involved, l-isoleucine. Primary deuterium kinetic isotopes are small, suggesting Cα proton abstraction is only partially rate-limiting. Solvent kinetic isotopes were significantly larger, indicating that a proton transfer occurring during the reaction is also partially rate-limiting. Finally, we demonstrate that l-cycloserine, a general inhibitor of PLP-dependent enzymes, is an excellent inhibitor of threonine deaminase.

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Purification of biosynthetic threonine deaminase from Escherichia coli

Cited by 20 publications

References 25 publications

An Expanded Two-State Model Accounts for Homotropic Cooperativity in Biosynthetic Threonine Deaminase from Escherichia coli

An Expanded Two-State Model Accounts for Homotropic Cooperativity in Biosynthetic Threonine Deaminase from Escherichia coli

Threonine deaminase from a nonsense mutant of Escherichia coli requiring isoleucine or pyridoxine: evidence for half-of-the-sites reactivity

Biochemical Characterization of the Mycobacterium smegmatis Threonine Deaminase

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