cDNA cloning of monodehydroascorbate radical reductase from cucumber: a high degree of homology in terms of amino acid sequence between this enzyme and bacterial flavoenzymes.
The FAD-enzyme monodehydroascorbate (MDA) reductase catalyzes the regeneration of ascorbate from the MDA radical using NAD(P)H as the electron donor [Hossain and Asada (1985) J. Biol. Chem. 260: 12920]. We cloned a cDNA of MDA reductase from cucumber seedlings and deduced its entire sequence of amino acid residues. The cDNA library from cucumber seedlings in the expression vector was screened with an antiserum against cucumber MDA reductase. Inserts from three immunoscreened clones hybridized with two oligonucleotide probes designed on the basis of the sequences of two peptide fragments from the cucumber enzyme. The nucleotide sequences of these three clones were determined and the longest one contained an open reading frame of 1,302 bp in length. The molecular mass of the translation product predicted from the open reading frame was 47 kDa, the same as that determined for the purified enzyme. The amino acid sequences determined from fragments of lysyl endopeptidase-digested MDA reductase could be aligned with that deduced from the open reading frame, although substitution of several residues was apparent. Thus, the open reading frame encoded an isozyme of MDA reductase of cucumber different from the purified enzyme. MDA reductase has the FAD- and NAD(P)H-binding domains of flavoproteins but shares only limited homology in terms of amino acid sequence with flavoenzymes from eukaryotes.
Sano
S
S
Research Institute for Food Science, Kyoto University, Japan.
Asada
K
K
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D004247:DNA; D005420:Flavoproteins / Q000235:genetics*; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D014675:Vegetables / Q000201:enzymology*
D001426:Bacterial Proteins; D005420:Flavoproteins; D010940:Plant Proteins; D004247:DNA; D009247:NADH, NADPH Oxidoreductases; C023927:monodehydroascorbate reductase (NADH)