HlyC, the internal protein acyltransferase that activates hemolysin toxin: role of conserved histidine, serine, and cysteine residues in enzymatic activity as probed by chemical modification and site-directed mutagenesis.
HlyC, the internal protein acyltransferase that activates hemolysin toxin: role of conserved histidine, serine, and cysteine residues in enzymatic activity as probed by chemical modification and site-directed mutagenesis.
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HlyC，激活溶血素毒素的内部蛋白酰基转移酶：通过化学修饰和定点诱变探测保守的组氨酸、丝氨酸和半胱氨酸残基在酶活性中的作用。
DOI：
10.1021/bi982491u
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发表时间：
1999
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
HlyC是一种内部蛋白质酰基转移酶，可以激活溶血素，溶血素是一种由致病性大肠杆菌产生的有毒蛋白质。酰基−酰基载体蛋白(ACP)是必需的酰基供体。分别亚克隆、表达和纯化了原溶血素A(ProHlyA)、HlyC和[1-14C]肉豆蔻酰-ACP，研究了proHlyA向HlyA的转化[Trent，M.S.，Worsham，L.M.和Ernst-Fonberg，M.L.(1998)生化37,4644−4655]。HlyC和溶血素属于由革兰氏阴性细菌产生的至少13种毒素家族。该家族的同源酰基转移酶显示了许多可能参与酰基转移的保守残基。特定的化学试剂和定点诱变表明，酶活性不需要单一保守的半胱氨酸或三个保守的丝氨酸残基。可逆组氨酸修饰的焦碳酸二乙酯(DEPC)处理抑制了酰基转移酶的活性，羟胺处理后酰基转移酶的活性恢复。底物肉豆蔻酰-ACP保护HlyC免受DEPC的抑制。这些发现和光谱吸光度的变化表明，组氨酸，特别是底物结合部位附近的组氨酸，是酶活性所必需的。单一保守的组氨酸残基His23对丙氨酸、半胱氨酸或丝氨酸的定点突变导致酶完全失活。
HlyC is an internal protein acyltransferase that activates hemolysin, a toxic protein produced by pathogenicEscherichia coli. Acyl−acyl carrier protein (ACP) is the essential acyl donor. Separately subcloned, expressed, and purified prohemolysin A (proHlyA), HlyC, and [1-14C]myristoyl-ACP have been used to study the conversion of proHlyA to HlyA [Trent, M. S., Worsham, L. M., and Ernst-Fonberg, M. L. (1998)Biochemistry 37, 4644−4655]. HlyC and hemolysin belong to a family of at least 13 toxins produced by Gram-negative bacteria. The homologous acyltransferases of the family show a number of conserved residues that are possible candidates for participation in acyl transfer. Specific chemical reagents and site-directed mutagenesis showed that neither the single conserved cysteine nor the three conserved serine residues were required for enzyme activity. Treatment with the reversible histidine-modifying diethyl pyrocarbonate (DEPC) inhibited acyltransferase activity, and acyltransferase activity was restored following hydroxylamine treatment. The substrate myristoyl-ACP protected HlyC from DEPC inhibition. These findings and spectral absorbance changes suggested that histidine, particularly a histidine proximal to the substrate binding site, was essential for enzyme activity. Site-directed mutageneses of the single conserved histidine residue, His23, to alanine, cysteine, or serine resulted in each instance in complete inactivation of the enzyme.