Serine acetyltransferase involved in cysteine biosynthesis from spinach: molecular cloning, characterization and expression analysis of cDNA encoding a plastidic isoform.
A cDNA clone that encodes a chloroplast-localizing isoform of serine acetyltransferase (SATase) (EC 2.3.1.30) was isolated from spinach (Spinacia oleracea L.). The cDNA encodes a polypeptide of 347 amino acids containing a putative transit peptide of ca. 60-70 amino acids at the N-terminal. Deduced amino acid sequence of SATase from spinach exhibited homology with other SATases from plants. DNA blot hybridization analysis showed the presence of 2-3 copies of Sat gene in the genome of spinach. RNA blot hybridization analysis indicated the constitutive expression of Sat gene in green and etiolated seedlings of spinach. Bacterial expression of the cDNA could directly rescue the cysteine auxotrophy of Escherchia coli caused by a lack of SATase locus (cysE). Catalytically active SATase protein was produced in E. coli cells. L-Cysteine, an end product of the cysteine biosynthetic pathway, inhibited the activity of recombinant spinach SATase, indicating the regulatory function of SATase in this metabolic pathway. A chloroplastic localization of this spinach SATase was revealed by the analyses of transgenic plant expressing transit peptide of SATase-beta-glucuronidase (GUS) fusion protein, and transient expression using the transit peptide-green fluorescent protein (GFP) fusion protein. The result from in vitro translation analysis suggests that this cDNA may encode both plastidic and cytosolic SATases.
Noji
M
M
Department of Molecular Biology and Biotechnology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, 263-8522 Japan.
Takagi
Y
Y
Kimura
N
N
Inoue
K
K
Saito
M
M
Horikoshi
M
M
Saito
F
F
Takahashi
H
H
Saito
K
K
D000123:Acetyltransferases / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002384:Catalysis; D003001:Cloning, Molecular; D018387:Codon, Initiator; D003545:Cysteine / Q000096:biosynthesis*; D018076:DNA, Complementary; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins; D015870:Gene Expression; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D018087:Plastids / Q000201:enzymology; D014176:Protein Biosynthesis; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D051050:Serine O-Acetyltransferase; D018724:Spinacia oleracea / Q000201:enzymology* / Q000235:genetics; D013347:Subcellular Fractions; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D018387:Codon, Initiator; D018076:DNA, Complementary; D029968:Escherichia coli Proteins; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D000123:Acetyltransferases; C070785:CysE protein, E coli; D051050:Serine O-Acetyltransferase; D003545:Cysteine