ISSN:
1365-2958
Source:
Blackwell Publishing Journal Backfiles 1879-2005
Topics:
Biology
,
Medicine
Notes:
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.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1111/j.1365-2958.1994.tb00475.x
Permalink
