Molecular and biochemical characterisation of a serine acetyltransferase of onion, Allium cepa (L.).
We have previously cloned a cDNA, designated SAT1, corresponding to a gene coding for a serine acetyltransferase (SAT) from onion (Allium cepa L.). The SAT1 locus was mapped to chromosome 7 of onion using a single-stranded conformation polymorphism (SSCP) in the 3' UTR of the gene. Northern analysis has demonstrated that expression of the SAT1 gene is induced in leaf tissue in response to low S-supply. Phylogenetic analysis has placed SAT1 in a strongly supported group (100% bootstrap) that comprises sequences that have been characterised biochemically, including Allium tuberosum, Spinacea oleracea, Glycine max, Citrullus vulgaris, and SAT5 (AT5g56760) of Arabidopsis thaliana. This group can be divided further with the SAT1 of A. cepa sequence grouping strongly with the A. tuberosum sequence. Translation of SAT1 from onion generates a protein of 289 amino acids with a calculated molecular mass of 30,573 Da and pI of 6.52. The conserved G277 and H282 residues that have been identified as critical for L-cysteine inhibition are observed at G272 and H277. SAT1 has been cloned into the pGEX plasmid, expressed in E. coli and SAT activity of the recombinant enzyme has been measured as acetyl-CoA hydrolysis detected at 232 nm. A Km of 0.72 mM was determined for l-serine as substrate, a Km of 92 microM was calculated with acetyl-CoA as substrate, and an inhibition curve for L-cysteine generated an IC50 value of 3.1 microM. Antibodies raised against the recombinant SAT1 protein recognised a protein of ca. 33 kDa in whole leaf onion extracts. These properties of the SAT1 enzyme from onion are compared with other SAT enzymes characterised from closely related species.
McManus
Michael T
MT
Institute of Molecular BioSciences, Massey University, Private Bag 11222, Palmerston North, New Zealand. M.T.McManus@massey.ac.nz
Leung
Susanna
S
Lambert
Anya
A
Scott
Richard W
RW
Pither-Joyce
Meeghan
M
Chen
Balance
B
McCallum
John
J
D000123:Acetyltransferases / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D002736:Chloroplasts / Q000378:metabolism; D002874:Chromosome Mapping; D032461:Chromosomes, Plant / Q000235:genetics; D003001:Cloning, Molecular; D003545:Cysteine / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D008969:Molecular Sequence Data; D019697:Onions / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D010802:Phylogeny; D018515:Plant Leaves / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D051050:Serine O-Acetyltransferase
D011993:Recombinant Fusion Proteins; D000123:Acetyltransferases; D051050:Serine O-Acetyltransferase; D003545:Cysteine