A novel NADPH:diamide oxidoreductase activity in arabidopsis thaliana P1 zeta-crystallin.
The zeta-crystallin (ZCr) gene P1 of Arabidopsis thaliana, known to confer tolerance toward the oxidizing drug 1,1'-azobis(N, N-dimethylformamide) (diamide) to yeast [Babiychuk, E., Kushnir, S., Belles-Boix, E., Van Montagu, M. & Inzé, D. (1995) J. Biol. Chem. 270, 26224], was expressed in Escherichia coli to characterize biochemical properties of the P1-zeta-crystallin (P1-ZCr). Recombinant P1-ZCr, a noncovalent dimer, showed NADPH:quinone oxidoreductase activity with specificity to quinones similar to that of guinea-pig ZCr. P1-ZCr also catalyzed the divalent reduction of diamide to 1,2-bis(N,N-dimethylcarbamoyl)hydrazine, with a kcat comparable with that for quinones. Two other azodicarbonyl compounds also served as substrates of P1-ZCr. Guinea-pig ZCr, however, did not catalyze the azodicarbonyl reduction. Hence, plant ZCr is distinct from mammalian ZCr, and can be referred to as NADPH:azodicarbonyl/quinone reductase. The quinone-reducing reaction was accompanied by radical chain reactions to produce superoxide radicals, while the azodicarbonyl-reducing reaction was not. Specificity to NADPH, as judged by kcat/Km, was > 1000-fold higher than that to NADH both for quinones and diamide. N-Ethylmaleimide and p-chloromercuribenzoic acid inhibited both quinone-reducing and diamide-reducing activities. Both NADPH and NADP+ suppressed the inhibition, but NADH did not, suggesting that sulfhydryl groups reside in the binding site for the phosphate group on the adenosine moiety of NADPH. The diamide-reducing activity of P1-ZCr accounts for the tolerance of P1-overexpressing yeast to diamide. Other possible physiological functions of P1-ZCr in plants are discussed.
Mano
J
J
Research Institute for Food Science, Kyoto University, Uji, Japan. mano@soya.food.kyoto-u.ac.jp
Babiychuk
E
E
Belles-Boix
E
E
Hiratake
J
J
Kimura
A
A
Inzé
D
D
Kushnir
S
S
Asada
K
K
D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000378:metabolism*; D003459:Crystallins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D003958:Diamide / Q000378:metabolism*; D001728:Dicumarol / Q000494:pharmacology; D019281:Dimerization; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D006168:Guinea Pigs; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D009582:Nitrofurantoin / Q000494:pharmacology; D009711:Nucleotides / Q000378:metabolism; D018384:Oxidative Stress; D010088:Oxidoreductases / Q000378:metabolism*; D010940:Plant Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011808:Quinone Reductases / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry
D003459:Crystallins; D004791:Enzyme Inhibitors; D009711:Nucleotides; D010940:Plant Proteins; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D003958:Diamide; D009249:NADP; D001728:Dicumarol; D009582:Nitrofurantoin; D010088:Oxidoreductases; D011808:Quinone Reductases