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.