Chloroplastic monodehydroascorbate radical reductase from spinach,Chloroplastic monodehydroascorbate radical reductase from spinach

Abstract

Active oxygens are inevitably produced in chloroplats with photosynthesis. In the water-water cycle, ascorbate (AsA) is univalently oxidized to monodehydroascorbate (MDA) radical with scavenging hydrogen peroxide by AsA peroxidase and regenerated via three pathways. MDA radical reductase (MDAR), which contributes to one of these pathways, catalyzes the reduction of MDA radical to AsA using NAD(P)H as the electron donor. Cytosolic MDARs have been purified and characterized, though chloroplastic isoform has never been. We purified MDAR from stromal fraction of intact chloroplasts isolated from spinach. And then we also succeeded to purify chloroplastic isoform from crude extract of spinach leaves, which contained two major MDARs. Its molecular mass was 51 kDa as determined by SDS-PAGE, being 4 kDa larger than that of cytosolic MDAR. Its Km values for NADH and NADPH were 5 µM and 12 µM, respectively. The chloroplastic isoform showed the similar inhibition by SH-modifier and dicumarol to those of cytosolic isoform. The amino acid sequence of the chloroplastic MDAR was determined from the amino termini to 25th residues. The cDNA cloned with oligonucleotide corresponded to the amino-terminal region contained an open reading frame of 1,494 bp length. The open reading frame encoded the region expected to transit peptide consisting of 56 residues. The predicted amino acid sequence of the region expected to mature chloroplastic MDAR contains the FAD- and NAD(P)H-binding domains of flavoproteins and shows partial homology with cytosolic isozymes.