Molecular characterization of lysine 6-dehydrogenase from Achromobacter denitrificans.
An inducible lysine 6-dehydrogenase (Lys 6-DH), which catalyzes the oxidative deamination of the 6-amino group of L-lysine in the presence of NAD(+), was purified to homogeneity from Achromobacter denitrificans, yielding a homodimeric protein of 80 kDa. The enzyme was specific for the substrate L-lysine and NAD(+) served as a cofactor. The dimeric enzyme associated into a hexamer in the presence of 10 mM L-lysine. The K(m) values for L-lysine and NAD(+) were 5.0 and 0.09 mM, respectively. The lys 6-dh gene was cloned and overexpressed in E. coli. The open reading frame was 1,107 nucleotides long and encoded a peptide containing 368 amino acids with 39,355 Da. The recombinant enzyme was purified to homogeneity and characterized. Enzyme activities and kinetic properties of the recombinant enzyme were almost the same as those of the endogenous enzyme obtained from A. denitrificans. Crystals of the enzyme were obtained using the hanging drop method.
Ruldeekulthamrong
Prakarn
P
Biological Sciences Program, Faculty of Science, Chulalongkorn University, Bangkok, Thailand.
Maeda
Sayaka
S
Kato
Shin-ichiro
S
Shinji
Nagata
N
Sittipraneed
Siriporn
S
Packdibamrung
Kanoktip
K
Misono
Haruo
H
D042441:Achromobacter denitrificans / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D000594:Amino Acid Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D021122:Protein Subunits / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
D021122:Protein Subunits; D011994:Recombinant Proteins; D000594:Amino Acid Oxidoreductases; C034168:lysine epsilon-dehydrogenase