Stable ammonia-specific NAD synthetase from Bacillus stearothermophilus: purification, characterization, gene cloning, and applications.
Bacillus stearothermophilus H-804 isolated from a hot spring in Beppu, Japan, produced an ammonia-specific NAD synthetase (EC 6.3.1.5). The enzyme specifically used NH3 as an amide donor for the synthesis of NAD as it formed AMP and pyrophosphate from deamide-NAD and ATP. None of the l-amino acids tested, such as l-asparagine or l-glutamine, or other amino compounds such as urea, uric acid, or creatinine was used instead of NH3. Mg2+ was needed for the activity, and the maximum enzyme activity was obtained with 3 mM MgCl2. The molecular mass of the native enzyme was 50 kDa by gel filtration, and SDS-PAGE showed a single protein band at the molecular mass of 25 kDa. The optimum pH and temperature for the activity were from 9.0 to 10.0 and 60 degrees C, respectively. The enzyme was stable at a pH range of 7.5 to 9.0 and up to 60 degrees C. The Km for NH3, ATP, and deamide-NAD were 0.91, 0.052, and 0.028 mM, respectively. The gene encoding the enzyme consisted of an open reading frame of 738 bp and encoded a protein of 246 amino acid residues. The deduced amino acid sequence of the gene had about 32% homology to those of Escherichia coli and Bacillus subtilis NAD synthetases. We caused the NAD synthetase gene to be expressed in E. coli at a high level; the enzyme activity (per liter of medium) produced by the recombinant E. coli was 180-fold that of B. stearothermophilus H-804. The specific assay of ammonia and ATP (up to 25 microM) with this stable NAD synthetase was possible.
Yamaguchi
Fumihiko
F
Planova Technology Development, Asahi-Kasei Corporation, 6-4158 Asahimachi, Nobeoka, Miyazaki-ken 882-0847, Japan. yamaguchi.fb@om.asahi-kasei.co.jp
Koga
Shinji
S
Yoshioka
Issei
I
Takahashi
Mamoru
M
Sakuraba
Haruhiko
H
Ohshima
Toshihisa
T
D000255:Adenosine Triphosphate / Q000378:metabolism; D000465:Algorithms; D019732:Amide Synthases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000641:Ammonia / Q000378:metabolism*; D003001:Cloning, Molecular; D015342:DNA Probes; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005798:Genes, Bacterial / Q000235:genetics; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics / Q000648:ultrastructure; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013696:Temperature
D015342:DNA Probes; D004791:Enzyme Inhibitors; D008670:Metals; D000641:Ammonia; D000255:Adenosine Triphosphate; D019732:Amide Synthases; C038479:NAD+ synthase