title: Threonine dehydratases of Corynebacterium glutamicum with altered allosteric control: their generation and biochemical and structural analysis.
abstract: Threonine dehydratase is the key enzyme in L-isoleucine synthesis, since it is allosterically feedback-inhibited by L-isoleucine. With the aim of obtaining regulatorily altered mutants of the threonine dehydratase of Corynebacterium glutamicum, amino acids were specifically exchanged and a new biological system of mutant selection was developed, based on the intoxication of Escherichia coli by ketobutyrate, which is the dehydratase reaction product. A collection of 19 mutant enzymes was generated and genetically and biochemically characterized comprising a whole range of regulatorily and catalytically altered enzymes. Of particular interest is the mutant Val-323-Ala, which is characterized by the fact that the L-isoleucine inhibition is entirely abolished so that the enzyme is always present in a relaxed, high-activity state. Correspondingly, the Hill coefficient is 1.4, in contrast to the value of 3.4 characteristic of the wild-type enzyme. Another peculiar mutant generated is the double mutant His-278-Arg-Leu-351-Ser. Here, again, L-isoleucine no longer inhibits catalytic activity, but the effector still promotes major structural changes of the protein, as ascertained from the L-isoleucine-dependent loss of pyridoxal-5'-phosphate from this mutant enzyme. Further enzymes obtained are reduced in L-isoleucine inhibition to a varying degree. Detailed studies on the structure of the enzyme revealed a partially very high similarity of the secondary structure to the mechanistically identical beta-subunit of the tryptophan synthase. This provides further indications concerning the localization of the regulatory and catalytic domain of the threonine dehydratase.
authors: Institut für Biotechnologie 1, Forschungszentrum Jülich, Germany.
authors: B
authors: B
authors: Möckel
authors: L
authors: L
authors: Eggeling
authors: H
authors: H
authors: Sahm
mesh_terms: D000494:Allosteric Regulation; D000495:Allosteric Site; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002087:Butyrates / Q000494:pharmacology; D003352:Corynebacterium / Q000201:enzymology*; D004259:DNA-Directed DNA Polymerase; D007532:Isoleucine / Q000031:analogs & derivatives; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011732:Pyridoxal Phosphate / Q000502:physiology*; D015203:Reproducibility of Results; D012641:Selection, Genetic; D016415:Sequence Alignment; D019914:Taq Polymerase; D013913:Threonine Dehydratase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D002087:Butyrates; D007532:Isoleucine; D011732:Pyridoxal Phosphate; C005087:alpha-ketobutyric acid; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase; D013913:Threonine Dehydratase