Purification and characterization of serine acetyltransferase from Escherichia coli partially truncated at the C-terminal region
Purification and characterization of serine acetyltransferase from Escherichia coli partially truncated at the C-terminal region
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DOI：
10.1271/bbb.63.168
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发表时间：
1999-01-01
影响因子：
1.6
通讯作者：
Nakanishi, K
中科院分区：
文献类型：
--
作者：
Mino, K;Yamanoue, T;Nakanishi, K
在不存在蛋白酶抑制剂的情况下，将来自大肠杆菌的丝氨酸乙酰转移酶（SAT）在25 ° C下孵育产生截短的SAT。截短的SAT比野生型SAT对反馈抑制的敏感性低得多。对SAT的N-和C-末端氨基酸序列分析发现，被命名为SAT Δ C20的截短SAT是野生型SAT在Ser 253和Met 254之间切割的产物形式，从C-末端缺失20个氨基酸残基。基于这些发现，我们构建了含有编码截短SAT的改变的cysE基因的质粒。SAT Delta C20是用大肠杆菌细胞生产的。转化大肠杆菌JM 70，经SDS-聚丙烯酰胺凝胶电泳纯化。与野生型SAT和Met-256-Ile突变体SAT（其由Denk和Bock分离但未纯化（J. Gen，Microbiol.，133，515-525（1987）），SAT Delta C20由四个相同的亚基组成，如野生型SAT和Met-256-Ile突变体SAT。SAT Delta C20的比活性、反应的最适pH、热稳定性和对试剂的稳定性与野生型SAT和Met-256-Ile突变体SAT相似。然而，SAT Delta C20没有与O-乙酰丝氨酸巯基化酶-A（OASS-A）（半胱氨酸合成酶的对应物）形成复合物，并且与野生型SAT和Met-256-Ile突变体SAT相比没有降低OASS活性。
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 Bock 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 to 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.