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 dehydrogenases fold into two domains in each subunit, a substrate-binding domain and an 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 elucidate the function and folding process of these two domains, we have genetically constructed a fragmentary form of thermostable leucine dehydrogenase of Bacillus stearothermophilus consisting of an N-terminal polypeptide fragment corresponding to the substrate-binding domain including an N-terminus, and a C-terminal fragment corresponding to the NAD(+)-binding domain. The two peptide fragments were expressed in separate host cells and purified. When both fragments were mixed, the leucine dehydrogenase activity with a specific activity of 1.4% of that of the wild-type enzyme appeared. This suggests that both peptide fragments mutually recognize each other, associate and fold correctly to be catalytically active, although the activity is low. However, the fragmentary form of enzyme produced catalyzed the oxidative deamination of l-leucine, l-isoleucine, and l-valine with broad substrate specificity compared to that of the wild-type enzyme. The fragmentary enzyme retained more than 75% of the initial activity after heating at 50 degrees C for 60 min. The fragmentary enzyme was more stable on heating than separate peptide fragments. These results suggest that the two domains of leucine dehydrogenase probably fold independently, and the two peptide fragments interact and associate with each other to form a functional active site.
Oikawa
T
T
Department of Biotechnology, Kansai University, Suita, Osaka-fu, 564-8680, Japan.
Kataoka
K
K
Jin
Y
Y
Suzuki
S
S
Soda
K
K
D000594:Amino Acid Oxidoreductases / Q000737:chemistry*; D000595:Amino Acid Sequence; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007532:Isoleucine / Q000737:chemistry; D007930:Leucine / Q000737:chemistry; D050803:Leucine Dehydrogenase; D008969:Molecular Sequence Data; D010100:Oxygen / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000737:chemistry; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D013696:Temperature; D014633:Valine / Q000737:chemistry
D010455:Peptides; D007532:Isoleucine; D000594:Amino Acid Oxidoreductases; D050803:Leucine Dehydrogenase; D007930:Leucine; D014633:Valine; D010100:Oxygen