Discovery and in vitro biosynthesis of haloduracin, a two-component lantibiotic
Discovery and in vitro biosynthesis of haloduracin, a two-component lantibiotic
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DOI：
10.1073/pnas.0606088103
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发表时间：
2006-11-14
影响因子：
11.1
通讯作者：
van der Donk, Wilfred A.
中科院分区：
文献类型：
--
作者：
McClerren, Amanda L.;Cooper, Lisa E.;van der Donk, Wilfred A.
羊毛硫抗生素是核糖体合成的肽，经过翻译后修饰成为其成熟的抗菌形式。它们的特征在于独特的氨基酸羊毛硫氨酸和甲基羊毛硫氨酸，其通过Ser/Thr残基的脱水，随后所得的半胱氨酸与半胱氨酸反应形成硫醚键而引入。双组分羊毛硫抗生素使用两种肽，其各自经后修饰以产生两种功能不同的产物，其协同作用以提供杀菌活性。利用遗传数据代替分离，在革兰氏阳性嗜碱菌嗜盐芽孢杆菌C-125的基因组中鉴定出一种双组分羊毛硫醚抗生素--卤度拉辛。我们表明，异源表达和纯化的前体肽HalA 1和HalA 2的纯化的修饰酶HalM 1和HalM 2在体外重建的双组分lantibiotic的生物合成处理。每种HalM酶的活性具有底物特异性，并且测定产物在工程化因子Xa切割位点处去除其前导序列后表现出抗微生物活性，表明已发生正确的硫醚形成。卤度拉新的生物活性取决于两种修饰肽的存在。研究了两种成熟的卤度拉辛肽Hal alpha和Hal beta的结构，表明它们与其他双组分羊毛硫抗生素相比既有相似之处，又有一些明显的差异。
Lantibiotics are ribosomally synthesized peptides that undergo posttranslational modifications to their mature, antimicrobial form. They are characterized by the unique amino acids lanthionine and methyllanthionine, introduced by means of dehydration of Ser/Thr residues followed by reaction of the resulting dehydro amino acids with cysteines to form thioether linkages. Two-component lantibiotics use two pepticles that are each posttranslationally modified to yield two functionally distinct products that act in synergy to provide bactericidal activity. By Using genetic data instead of isolation, a two-component lantibiotic, haloduracin, was identified in the genome of the Gram-positive alkaliphilic bacterium Bacillus halodurans C-125. We show that heterologously expressed and purified precursor peptides HalA1 and HalA2 are processed by the purified modification enzymes HalM1 and HalM2 in an in vitro reconstitution of the biosynthesis of a two-component lantibiotic. The activity of each HalM enzyme is substratespecific, and the assay products exhibit antimicrobial activity after removal of their leader sequences at an engineered Factor Xa cleavage site, indicating that correct thioether formation has occurred. Haloduracin's biological activity depends on the presence of both modified pepticles. The structures of the two mature haloduracin pepticles Hal alpha and Hal beta were investigated, indicating that they have similarities as well as some distinct differences compared with other two-component lantibiotics.