Overexpression and mutagenesis of the catalytic domain of dihydrolipoamide acetyltransferase from Saccharomyces cerevisiae.
Overexpression and mutagenesis of the catalytic domain of dihydrolipoamide acetyltransferase from Saccharomyces cerevisiae.
复制标题
酿酒酵母二氢硫辛酰胺乙酰转移酶催化结构域的过度表达和诱变。
DOI：
10.1021/bi00489a017
复制
发表时间：
1990
期刊：
影响因子：
2.9
通讯作者：
Reed,LJ
中科院分区：
文献类型：
--
作者：
Niu,XD;Stoops,JK;Reed,LJ
摘要：通过表达载体pKK 233 -2在大肠杆菌菌株JM 105中过表达了来自酿酒酵母的丙酮酸脱氢酶复合物的二氢硫辛酰胺乙酰转移酶组分（E2 p）的内核结构域（残基-221-454）。将截短的E2 p纯化至表观均一性。它表现出与野生型E2 p非常相似的催化活性（从[1 - 14 C]乙酰基-CoA到二氢硫辛酰胺的乙酰基转移）。截短型和野生型E2 p的外观也非常相似，通过负染电子显微镜观察到，即五角十二面体。这些结果表明，S.酿酒酵母存在于内核结构域，即催化结构域，并且该结构域单独可以进行自组装。纯化的截短的E2 p显示出聚集的趋势。通过基因工程将结构域间接头区段（残基-181-220）连接至催化结构域来防止聚集。所有二氢硫辛酰胺酰基转移酶在其羧基末端附近含有序列His-Xaa-Xaa-Xaa-Asp-Gly。通过与氯霉素乙酰转移酶类比，假定高度保守的His和Asp残基参与催化机制[Guest，JR（1987）FEMS Microbiol. Lett. 44，417-422]，S.通过定点诱变，用Asn或Ala对酿酒酵母截短的E2 p（His-427）进行诱变，对截短的E2 p的fccat或Km值没有显著影响。然而，Asp-431-Asn、Ala或Glu取代分别导致kM降低16倍、24倍和3.7倍，而Km值几乎没有变化。这些结果表明，His残基不参与S.但Asp-431起着重要作用。这种作用是结构性的还是催化性的，还有待确定。11种二氢硫辛酰胺酰基转移酶具有独特的多结构域结构（Reed & Hackert，1990; Guest等人，
Revised Manuscript Received June 26, 1990 abstract: The inner core domain (residues—221—454) of the dihydrolipoamide acetyltransferase component (E2p) of the pyruvate dehydrogenase complex from Saccharomyces cerevisiae has been overexpressed in Escherichia coli strain JM105 via theexpression vector pKK233-2. The truncated E2p was purified to apparent homogeneity. It exhibited catalytic activity (acetyl transfer from [l-14C] acetyl-CoA to dihydrolipoamide) very similar to that of wild-type E2p. The appearance of the truncated and wild-type E2p was also very similar, as observed by negative-stain electron microscopy, namely, a pentagonal dodecahedron. These findings demonstrate that the activesite of E2p from S. cerevisiae resides in the inner core domain, ie, catalytic domain, and that this domain alone can undergo self-assembly. The purified truncated E2p showed a tendency to aggregate. Aggregation was prevented by genetically engineered attachment of the interdomain linker segment (residues—181—220) to the catalytic domain. All dihydrolipoamide acyl-transferases contain the sequence His-Xaa-Xaa-Xaa-Asp-Gly near their carboxyl termini. By analogy with chloramphenicol acetyltransferase, the highly conserved His and Asp residues were postulated to be involved in the catalytic mechanism [Guest, JR (1987) FEMS Microbiol. Lett. 44, 417-422], Substitution of the sole His residue in the S. cerevisiae truncated E2p, His-427, by Asn or Ala by site-directed mutagenesis did not have a significant effect on the fccat or Km values of the truncated E2p. However, the Asp-431-Asn, Ala, or Glu substitutionsresulted in a 16-, 24-, and 3.7-fold reduction, respectively, in kM, with little change in Km values. These findings indicate that a His residue is not involved in the catalytic mechanism of E2p from S. cerevisiae but that Asp-431 plays an important role. Whether this role is structural or catalytic remains to be established.^^. 11 dihydrolipoamide acyltransferases possess a unique multidomain structure (Reed & Hackert, 1990; Guest et al.,
登录
查看更多内容
DOI：
10.1016/s0021-9258(18)61070-1
发表时间：
1987-07
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
P. Matsudaira
通讯作者：
P. Matsudaira
2-含氧酸脱氢酶多酶复合物：结构域、动力学和设计
影响因子：
5.2
作者：
R. Perham;L. Packman
通讯作者：
L. Packman
制备噬菌体和质粒 DNA 的实际问题：细菌和噬菌体的生长、维持和储存。
影响因子：
--
作者：
H. Miller
通讯作者：
H. Miller
大肠杆菌 2-含氧酸脱氢酶复合物的二氢硫辛酰酰基转移酶成分中的链折叠
影响因子：
3.5
作者：
L. Packman;R. Perham
通讯作者：
R. Perham
影响因子：
2.9
作者：
LEWENDON, A;MURRAY, IA;SHAW, WV
通讯作者：
SHAW, WV