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 phosp hohydroxypyruvate to phosphoserine in the phosphorylated pathway of se rine biosynthesis. A cDNA clone encoding PSA was isolated from the cDN A library of spinach (Spinacia oleracea L.) green leaves. Determinatio n of the nucleotide sequence revealed the presence of an open reading frame encoding 430 amino acids, exhibiting 38-50% homology with the am ino acid sequences of bacterial, yeast and animal PSA. It contains an N-terminal extension of ca. 60 amino acids in addition to the sequence s from other organisms. The general features of plastidic transit pept ide are observed in this N-terminal sequence, suggesting the plastid l ocalization of the PSA protein encoded by this cDNA. The bacterial exp ression 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-expr essing the cDNA. Southern blot analysis indicated the presence of a co uple of related RNA blot hybridization suggested the preferential expr ession of the Psa gene in the roots of green seedlings and in the susp ension cells cultured under a dark condition.