Threonine dehydratases of Corynebacterium glutamicum with altered allosteric control: their generation and biochemical and structural analysis

Summary 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 β‐subunit of the tryptophan synthase. This provides further indications concerning the localization of the regulatory and catalytic domain of threonine dehydratase.