A novel NADPH : diamide oxidoreductase activity in Arabidopsis thaliana P1zeta-crystallin

The zeta-crystallin (ZCr) gene P1 of Arabidopsis thaliana, known to confer tolerance toward the oxidizing drug 1,1'-azobis(N,N-dimethylformamide) (dia mide) to yeast [Babiychuk, E., Kushnir, S., Belles-Boix, E., Van Montagu, M . & Inze, D. (1995) J. Biol. Chem. 270, 26224], was expressed in Escherichi a coli to characterize biochemical properties of the P1-zeta-crystallin (P1 -ZCr). Recombinant P1-ZCr, a noncovalent dimer, showed NADPH:quinone oxidor eductase activity with specificity to quinones similar to that of guinea-pi g ZCr. P1-ZCr also catalyzed the divalent reduction of diamide to 1,2-bis(N ,N-dimethylcarbamoyl)hydrazine, with a k(cat) comparable with that for quin ones. Two other azodicarbonyl compounds also served as substrates of P1-ZCr . Guinea-pig ZCr, however, did not catalyze the azodicarbonyl reduction. He nce, plant ZCr is distinct from mammalian ZCr, and can be referred to as NA DPH:azodicarbonyl/quinone reductase. The quinone-reducing reaction was acco mpanied by radical chain reactions to produce superoxide radicals, while th e azodicarbonyl-reducing reaction was not. Specificity to NADPH, as judged by k(cat)/K-m, was >1000-fold higher than that to NADH both for quinones an d diamide. N-Ethylmaleimide and p-chloromercuribenzoic acid inhibited both quinone-reducing and diamide-reducing activities. Both NADPH and NADP(+) su ppressed the inhibition, but NADH did not, suggesting that sulfhydryl group s reside in the binding site for the phosphate group on the adenosine moiet y of NADPH. The diamide-reducing activity of P1-ZCr accounts for the tolera nce of P1-overexpressing yeast to diamide. Other possible physiological fun ctions of P1-ZCr in plants are discussed.