A site-directed mutagenesis study at Lys-113 of NAD(P)H:quinone-acceptor oxidoreductase: an involvement of Lys-113 in the binding of the flavin adenine dinucleotide prosthetic group.
NAD(P)H: quinone-acceptor oxidoreductase (EC 1.6.99.2), also referred to as DT-diaphorase, is a flavoprotein that catalyzes the two-electron reduction of quinones and quinonoid compounds to hydroquinones, using either NADH or NADPH as the electron donor. Using an Escherichia coli expression system developed previously, we prepared three mutants of the rat liver quinone reductase. These mutants are Lys-113-His (K113H), Lys-113-Asp (K113D), and Lys-113-Ala (K113A). While the mutant K113H was readily purified using the same procedure as for the purification of the wild-type quinone reductase and found to have an activity similar to that of the wild-type enzyme, K113D and K113A were purified only in very small quantities, mainly in the form of apoprotein, and had very low activities. The results suggest that a positively charged amino acid at this position is important for the binding of the flavin adenine dinucleotide (FAD) prosthetic group. Flavin spectral studies of 6-mercapto-FAD-reconstituted mutants revealed that mutation at Lys-113 affects the protein environment around position-6 of the isoalloxazine ring.
Tedeschi
G
G
Division of Immunology, Beckman Research Institute of the City of Hope, Duarte, California 91010, USA.
Deng
P S
PS
Chen
H H
HH
Forrest
G L
GL
Massey
V
V
Chen
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D007700:Kinetics; D008239:Lysine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014812:Vitamin K / Q000378:metabolism
D017931:DNA Primers; D011994:Recombinant Proteins; D009243:NAD; D014812:Vitamin K; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D016660:NAD(P)H Dehydrogenase (Quinone); D008239:Lysine