The first archaeal L-aspartate dehydrogenase from the hyperthermophile Archaeoglobus fulgidus: gene cloning and enzymological characterization.
A gene encoding an L-aspartate dehydrogenase (EC 1.4.1.21) homologue was identified in the anaerobic hyperthermophilic archaeon Archaeoglobus fulgidus. After expression in Escherichia coli, the gene product was purified to homogeneity, yielding a homodimeric protein with a molecular mass of about 48 kDa. Characterization revealed the enzyme to be a highly thermostable L-aspartate dehydrogenase, showing little loss of activity following incubation for 1 h at up to 80 degrees C. The optimum temperature for L-aspartate dehydrogenation was about 80 degrees C. The enzyme specifically utilized L-aspartate as the electron donor, while either NAD or NADP could serve as the electron acceptor. The Km values for L-aspartate were 0.19 and 4.3 mM when NAD or NADP, respectively, served as the electron acceptor. The Km values for NAD and NADP were 0.11 and 0.32 mM, respectively. For reductive amination, the Km values for oxaloacetate, NADH and ammonia were 1.2, 0.014 and 167 mM, respectively. The enzyme showed pro-R (A-type) stereospecificity for hydrogen transfer from the C4 position of the nicotinamide moiety of NADH. This is the first report of an archaeal L-aspartate dehydrogenase. Within the archaeal domain, homologues of this enzyme occurred in many Methanogenic species, but not in Thermococcales or Sulfolobales species.
Yoneda
Kazunari
K
Department of Biological Science and Technology, Faculty of Engineering, The University of Tokushima, 2-1 Tokushima 770-8506, Japan.
Kawakami
Ryushi
R
Tagashira
Yuya
Y
Sakuraba
Haruhiko
H
Goda
Shuichiro
S
Ohshima
Toshihisa
T
D000594:Amino Acid Oxidoreductases / Q000737:chemistry*; D000595:Amino Acid Sequence; D019611:Archaeoglobus fulgidus / Q000201:enzymology*; D003001:Cloning, Molecular; D019281:Dimerization; D004795:Enzyme Stability; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D062907:Oxaloacetic Acid / Q000737:chemistry; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
D062907:Oxaloacetic Acid; D000594:Amino Acid Oxidoreductases; C025745:aspartate dehydrogenase