The biochemistry of hemolysin toxin activation: characterization of HlyC, an internal protein acyltransferase.
The biochemistry of hemolysin toxin activation: characterization of HlyC, an internal protein acyltransferase.
复制标题
溶血素毒素激活的生物化学：HlyC（一种内部蛋白质酰基转移酶）的表征。
DOI：
10.1021/bi971588y
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Ernst-Fonberg,ML
中科院分区：
文献类型：
--
作者：
Trent,MS;Worsham,LM;Ernst-Fonberg,ML
溶血素毒素是由致病性大肠杆菌产生和分泌的一种溶细胞的、结构同源的蛋白质毒素，称为RTX（毒素中的重复序列）毒素。RTX毒素是CABD基因簇的产物。A基因产物无毒溶血素（proHlyA）通过两个内部赖氨酸残基的翻译后脂肪酰化而具有毒性。C基因产物HlyC是酰化所必需的，酰基-酰基载体蛋白（acyl-acyl carrier protein，ACP）是酰基供体。HlyB和HlyD参与毒素的分泌。分别亚克隆、表达和纯化ProHlyA和HlyC，并合成具有不同放射性酰基的酰基-ACP。用这些蛋白质，测定通过酰基转移将proHlyA转化为HlyA。酰基-ACP是专性酰基供体。HlyC单体催化酰基转移，并显示酰基酶中间体。HlyA的Km和Vmax分别为0.94 μM和7.5pmol酰基转移/min，肉豆蔻酰-ACP的Km和Vmax分别为0.48 μM和6.9pmol酰基转移/min。具有不同酰基的ACP在酰基转移酶反应中的不同动力学效力与它们产生的相应酰基毒素的裂解能力形成显著对比。
Hemolysin toxin produced and secreted by pathogenicEscherichia coliis one of a family of cytolytic, structurally homologous protein toxins known as RTX (repeats in toxin) toxins. RTX toxins are products of a gene cluster,CABD. TheAgene product, nontoxic hemolysin (proHlyA), is made toxic by posttranslational fatty acylation of two internal lysine residues. HlyC, theCgene product, is essential for acylation, and acyl-acyl carrier protein (ACP) is the acyl donor. HlyB and HlyD are involved in secretion of the toxin. ProHlyA and HlyC were separately subcloned, expressed, and purified, and acyl-ACPs with diverse radioactive acyl groups were synthesized. With these proteins, the conversion of proHlyA to HlyA by acyl transfer was assayed. Acyl-ACP was the obligate acyl donor. Acyl transfer was catalyzed by HlyC monomer, and an acyl-enzyme intermediate was shown. Reaction was inhibited by ACPSH but not by fatty acid or fatty-acyl CoA.KmandVmaxfor HlyA were 0.94 μM and 7.5 pmol of acyl group transferred/min, respectively;KmandVmaxfor myristoyl-ACP were 0.48 μM and 6.9 pmol/min. The kinetic parameters of different acyl-ACPs resembled a competitive inhibition as acyl group carbon chain length increased;Km's increased whileVmax's remained unchanged. The different kinetic efficacies in the acyltransferase reaction of the ACPs with different acyl groups contrasted notably with the lytic powers of the corresponding acyl-toxins that they generated.