Gene cloning and sequence determination of leucine dehydrogenase from Bacillus stearothermophilus and structural comparison with other NAD(P)+-dependent dehydrogenases.
The gene for leucine dehydrogenase (EC 1.4.1.9) from Bacillus stearothermophilus was cloned and expressed in Escherichia coli. The selection for the cloned gene was based upon activity staining of the replica printed E. coli cells. A transformant showing high leucine dehydrogenase activity was found to carry an about 9 kilobase pair plasmid, which contained 4.6 kilobase pairs of B. stearothermophilus DNA. The nucleotide sequence including the 1287 base pair coding region of the leucine dehydrogenase gene was determined by the dideoxy chain termination method. The translated amino acid sequence was confirmed by automated Edman degradation of several peptide fragments produced from the purified enzyme by trypsin digestion. The polypeptide contained 429 amino acid residues corresponding to the subunit (Mr 49,000) of the hexameric enzyme. Comparison of the amino acid sequence of leucine dehydrogenase with those of other pyridine nucleotide dependent oxidoreductases registered in a protein data bank revealed significant sequence similarity, particularly between leucine and glutamate dehydrogenases, in the regions containing the coenzyme binding domain and certain specific residues with catalytic importance.
Nagata
S
S
Institute for Chemical Research, Kyoto University, Japan.
Tanizawa
K
K
Esaki
N
N
Sakamoto
Y
Y
Ohshima
T
T
Tanaka
H
H
Soda
K
K
D000594:Amino Acid Oxidoreductases / Q000235:genetics* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D005969:Glutamate Dehydrogenase / Q000235:genetics; D050803:Leucine Dehydrogenase; D008969:Molecular Sequence Data; D009249:NADP / Q000494:pharmacology*; D010088:Oxidoreductases / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D014170:Transformation, Genetic
D000596:Amino Acids; D009249:NADP; D010088:Oxidoreductases; D000594:Amino Acid Oxidoreductases; D005969:Glutamate Dehydrogenase; D050803:Leucine Dehydrogenase