Molecular cloning, characterization and expression of cDNA encoding phosphoserine aminotransferase involved in phosphorylated pathway of serine biosynthesis from spinach.
Phosphoserine aminotransferase (PSA) catalyzes the conversion of phosphohydroxypyruvate to phosphoserine in the phosphorylated pathway of serine biosynthesis. A cDNA clone encoding PSA was isolated from the cDNA library of spinach (Spinacia oleracea L.) green leaves. Determination of the nucleotide sequence revealed the presence of an open reading frame encoding 430 amino acids, exhibiting 38-50% homology with the amino acid sequences of bacterial, yeast and animal PSA. It contains an N-terminal extension of ca. 60 amino acids in addition to the sequences from other organisms. The general features of plastidic transit peptide are observed in this N-terminal sequence, suggesting the plastid localization of the PSA protein encoded by this cDNA. The bacterial expression of the cDNA could functionally rescue the auxotrophy of serine in the serC- mutant, Escherichia coli KL282. The enzymatic activity of PSA was demonstrated in vitro in the extracts of E. coli over-expressing the cDNA. Southern blot analysis indicated the presence of a couple of related genes (Psa) in the spinach genome. RNA blot hybridization suggested the preferential expression of the Psa gene in the roots of green seedlings and in the suspension cells cultured under a dark condition.
Saito
K
K
Faculty of Pharmaceutical Sciences, Chiba University, Yayoi-cho, Japan.
Takagi
Y
Y
Ling
H C
HC
Takahashi
H
H
Noji
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001419:Bacteria / Q000201:enzymology; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D018744:DNA, Plant / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D015723:Gene Library; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010766:Phosphorylation; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000096:biosynthesis; D018724:Spinacia oleracea / Q000201:enzymology*; D000637:Transaminases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics
D018076:DNA, Complementary; D018744:DNA, Plant; D011994:Recombinant Proteins; D012694:Serine; D000637:Transaminases; C022393:phosphoserine aminotransferase