Purification and characterization of serine acetyltransferase from Escherichia coli partially truncated at the C-terminal region.
Incubation of serine acetyltransferase (SAT) from Escherichia coli at 25 degrees C in the absence of protease inhibitors yielded a truncated SAT. The truncated SAT was much less sensitive to feedback inhibition than the wild-type SAT. Analyses of the N- and C-terminal amino acid sequences found that the truncated SAT designated as SAT delta C20 was a resultant form of the wild-type SAT cleaved between Ser 253 and Met 254, deleting 20 amino acid residues from the C-terminus. Based on these findings, we constructed a plasmid containing an altered cysE gene encoding the truncated SAT. SAT delta C20 was produced using the cells of E. coli JM70 transformed with the plasmid and purified to be homogeneous on an SDS-polyacrylamide gel. Properties of the purified SAT delta C20 were investigated in comparison with those of the wild-type SAT and Met-256-Ile mutant SAT, which was isolated by Denk and Böck but not purified (J. Gen. Microbiol., 133, 515-525 (1987)). SAT delta C20 was composed of four identical subunits like the wild-type SAT and Met-256-Ile mutant SAT. Specific activity, optimum pH for reaction, thermal stability, and stability to reagents for SAT delta C20 were similar those for the wild-type SAT and Met-256-Ile mutant SAT. However, SAT delta C20 did not form a complex with O-acetylserine sulfhydrylase-A (OASS-A), a counterpart of the cysteine synthetase and did not reduce OASS activity in contrast to the wild-type SAT and Met-256-Ile mutant SAT.
Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, Japan.
K
K
Mino
T
T
Yamanoue
T
T
Sakiyama
N
N
Eisaki
A
A
Matsuyama
K
K
Nakanishi
D000123:Acetyltransferases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D003547:Cysteine Synthase / Q000302:isolation & purification; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000302:isolation & purification; D010957:Plasmids / Q000235:genetics; D017354:Point Mutation; D017384:Sequence Deletion; D051050:Serine O-Acetyltransferase
D017931:DNA Primers; D009097:Multienzyme Complexes; D000123:Acetyltransferases; D051050:Serine O-Acetyltransferase; D003547:Cysteine Synthase