Molecular cloning and functional characterization of cDNAs encoding cysteine synthase and serine acetyltransferase that may be responsible for high cellular cysteine content in Allium tuberosum.
The plants belonging to the genus Allium are known to accumulate sulfur-containing secondary compounds that are derived from cysteine. Here, we report on molecular cloning and functional characterization of two cDNAs that encode serine acetyltransferase and cysteine synthase from A. tuberosum (Chinese chive). The cDNA for serine acetyltransferase encodes an open reading frame of 289 amino acids, of which expression could complement the lacking of cysE gene for endogenous serine acetyltransferase in Escherichia coli. The cDNA for cysteine synthase encodes an open reading frame of 325 amino acids, of which expression in the E. coli lacking endogenous cysteine synthase genes could functionally rescue the growth without addition of cysteine. Both deduced proteins seem to be localized in cytosol, judging from their primary structures. Northern blot analysis indicated that both transcripts accumulated in almost equal levels in leaves and root of green and etiolated seedlings of A. tuberosum. The activity of recombinant serine acetyltransferase produced from the cDNA was inhibited by L-cysteine, which is the end-product of the pathway; however, the sensitivity to cysteine (48.7 microM of the concentration for 50% inhibition, IC(50)) was fairly low compared with that of previously reported serine acetyltransferases ( approximately 5 microM IC(50)) from various plants. In A. tuberosum, the cellular content of cysteine was several-fold higher than those in Arabidopsis thaliana and tobacco. This higher concentration of cysteine in A. tuberosum is likely due to the lower sensitivity of feedback inhibition of serine acetyltransferase to cysteine.
Urano
Y
Y
Laboratory of Molecular Biology and Biotechnology, Research Center of Medicinal Resources, Faculty of Pharmaceutical Sciences, Chiba University, Inage-ku, 263-8522, Chiba, Japan.
Manabe
T
T
Noji
M
M
Saito
K
K
D000123:Acetyltransferases / Q000235:genetics*; D000490:Allium / Q000201:enzymology / Q000235:genetics*; D000595:Amino Acid Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D003545:Cysteine / Q000378:metabolism*; D003547:Cysteine Synthase / Q000235:genetics*; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D005978:Glutathione / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D051050:Serine O-Acetyltransferase; D014018:Tissue Distribution
D018076:DNA, Complementary; D012333:RNA, Messenger; D000123:Acetyltransferases; D051050:Serine O-Acetyltransferase; D003547:Cysteine Synthase; D005978:Glutathione; D003545:Cysteine