Highly stable L-lysine 6-dehydrogenase from the thermophile Geobacillus stearothermophilus isolated from a Japanese hot spring: characterization, gene cloning and sequencing, and expression.
L-Lysine dehydrogenase, which catalyzes the oxidative deamination of L-lysine in the presence of NAD, was found in the thermophilic bacterium Geobacillus stearothermophilus UTB 1103 and then purified about 3,040-fold from a crude extract of the organism by using four successive column chromatography steps. This is the first report showing the presence of a thermophilic NAD-dependent lysine dehydrogenase. The product of the enzyme catalytic activity was determined to be Delta1-piperideine-6-carboxylate, indicating that the enzyme is L-lysine 6-dehydrogenase (LysDH) (EC 1.4.1.18). The molecular mass of the purified protein was about 260 kDa, and the molecule was determined to be a homohexamer with subunit molecular mass of about 43 kDa. The optimum pH and temperature for the catalytic activity of the enzyme were about 10.1 and 70 degrees C, respectively. No activity was lost at temperatures up to 65 degrees C in the presence of 5 mM L-lysine. The enzyme was relatively selective for L-lysine as the electron donor, and either NAD or NADP could serve as the electron acceptor (NADP exhibited about 22% of the activity of NAD). The Km values for L-lysine, NAD, and NADP at 50 degrees C and pH 10.0 were 0.73, 0.088, and 0.48 mM, respectively. When the gene encoding this LysDH was cloned and overexpressed in Escherichia coli, a crude extract of the recombinant cells had about 800-fold-higher enzyme activity than the extract of G. stearothermophilus. The nucleotide sequence of the LysDH gene encoded a peptide containing 385 amino acids with a calculated molecular mass of 42,239 Da.
Heydari
Mojgan
M
Department of Biological Science and Technology, Faculty of Engineering, The University of Tokushima, Tokushima 770-8506, Japan.
Ohshima
Toshihisa
T
Nunoura-Kominato
Naoki
N
Sakuraba
Haruhiko
H
D000594:Amino Acid Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D005618:Fresh Water / Q000382:microbiology*; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D006358:Hot Temperature; D007564:Japan; D008239:Lysine / Q000378:metabolism*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA
D011994:Recombinant Proteins; D000594:Amino Acid Oxidoreductases; D008239:Lysine