Involvement of Lys-308 in the FAD-dependent oxidase activity of NADH dehydrogenase from an alkaliphilic Bacillus.
It has been suggested that the oxidase activity of NADH dehydrogenase of an alkaliphilic Bacillus YN-1 is markedly increased by the addition of free FAD. Site-directed mutagenesis of Lys-306, Lys-308, Arg-317, Arg-319 and Lys-332 of the enzyme was attempted to determine whether the basic amino acid residues are involved in FAD-dependent oxidase activity. Replacement of Arg-317, Arg-319 and Lys-332 by Ala had almost no effect on activity. Substitution of Lys-306 by Ala caused complete loss of the activity. When Lys-308 was replaced by Ala, the extent of FAD stimulation of the oxidase activity of the mutant (K308A) was only one-third that of the wild-type enzyme. FAD stimulation of oxidase activity of the wild-type enzyme was competitively inhibited by NAD. Although the K308A enzyme was also inhibited by NAD, this inhibition was significantly lower than that of the wild-type enzyme. It is likely that Lys-308 plays an important role in regulation of oxidase activity.
Kitazume
Yayoi
Y
Department of Chemistry, Faculty of Science, Chiba University, Yayoi, Chiba, Japan.
Mutoh
Mamiko
M
Shiraki
Masato
M
Koyama
Noriyuki
N
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001407:Bacillus / Q000201:enzymology*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009245:NADH Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment
D005182:Flavin-Adenine Dinucleotide; D009245:NADH Dehydrogenase; D008239:Lysine