Catalytic properties of NAD(P)H:quinone acceptor oxidoreductase: study involving mouse, rat, human, and mouse-rat chimeric enzymes.
NAD(P):quinone acceptor oxidoreductase (quinone reductase) (DT-diaphorase, EC 1.6.99.2) is involved in the process of reductive activation of cytotoxic antitumor quinones and nitrobenzenes. In this study, we initially examined the relative abilities of mouse, rat, and human quinone reductases to reduce two prodrugs, CB 1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide] and EO9 [5-(1-aziridinyl)-3-(hydroxymethyl)-2-(3-hydroxy-1-propenyl)-1- methyl-1H-indole-4,7-dione]. By using Escherichia coli-expressed quinone reductases and evaluating them under identical conditions, we confirmed previous finding showing that the human enzyme is not as effective as the rat enzyme in reducing CB 1954 and EO9, although the two enzymes have similar NAD(P)H-menadione reductase activities. Interestingly, although the amino acid sequence of mouse quinone reductase is more homologous to that of the rat enzyme, we found that the mouse enzyme behaves similarly to the human enzyme in its ability to reduce these compounds and to generate drug-induced DNA damage. To determine the region of quinone reductase that is responsible for the catalytic differences, two mouse-rat chimeric enzymes were generated. MR-P, a chimeric enzyme that has mouse amino-terminal and rat carboxy-terminal segments of quinone reductase, was shown to have catalytic properties resembling those of rat quinone reductase, and RM-P, a chimeric enzyme that has rat amino-terminal and mouse carboxyl-terminal segments of quinone reductase, was shown to have catalytic properties resembling those of mouse quinone reductase. In addition, MR-P and RM-P were found to be inhibited by flavones with Ki values similar to those for rat and mouse quinone reductases, respectively. Based on these results, we propose that the carboxyl-terminal portion of the enzyme plays an important role in the reduction of cytotoxic drugs and the binding of flavones.
Chen
S
S
Division of Immunology, Beckman Research Institute of the City of Hope, Duarte, California 91010, USA.
Knox
R
R
Lewis
A D
AD
Friedlos
F
F
Workman
P
P
Deng
P S
PS
Fung
M
M
Ebenstein
D
D
Wu
K
K
Tsai
T M
TM
D000595:Amino Acid Sequence; D000818:Animals; D001388:Aziridines / Q000378:metabolism / Q000633:toxicity; D001483:Base Sequence; D004249:DNA Damage; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D045563:Indolequinones; D007211:Indoles / Q000378:metabolism / Q000633:toxicity; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000235:genetics / Q000378:metabolism*; D011355:Prodrugs / Q000378:metabolism / Q000633:toxicity; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D001388:Aziridines; D017931:DNA Primers; D045563:Indolequinones; D007211:Indoles; D011355:Prodrugs; D011993:Recombinant Fusion Proteins; C100099:tretazicar; D016660:NAD(P)H Dehydrogenase (Quinone); C060817:apaziquone