Fragmentary form of thermostable leucine dehydrogenase of Bacillus stearothermophilus: Its construction and reconstitution of active fragmentary enzyme

X-ray crystallographic studies revealed that various amino acid dehydrogena ses fold into two domains in each subunit, a substrate-binding domain and a n NAD(P)(+)-binding domain (Baker, P. J., Turnbull, A. P., Sedelnikova, S. E., Stillman, T. J., and Rice, D. W. (1995) Structure 3, 693-705). To eluci date the function and folding process of these two domains, we have genetic ally constructed a fragmentary form of thermostable leucine dehydrogenase o f Bacillus stearothermophilus consisting of an N-terminal polypeptide fragm ent corresponding to the substrate-binding domain including an N-terminus, and a C-terminal fragment corresponding to the NAD(+)-binding domain. The t wo peptide fragments were expressed in separate host cells and purified. Wh en both fragments were mixed, the leucine dehydrogenase activity with a spe cific activity of 1.4% of that of the wild-type enzyme appeared. This sugge sts that both peptide fragments mutually recognize each other, associate an d fold correctly to be catalytically active, although the activity is low. However, the fragmentary form of enzyme produced catalyzed the oxidative de amination of L-leucine, L-isoleucine, and L-valine with broad substrate spe cificity compared to that of the wild-type enzyme. The fragmentary enzyme r etained more than 75% of the initial activity after heating at 50 degreesC for 60 min. The fragmentary enzyme was more stable on heating than separate peptide fragments. These results suggest that the two domains of leucine d ehydrogenase probably fold independently, and the two peptide fragments int eract and associate with each other to form a functional active site, (C) 2 001 Academic Press.