Functional and Structural Analysis of the Siderophore Synthetase AsbB through Reconstitution of the Petrobactin Biosynthetic Pathway from Bacillus anthracis
Functional and Structural Analysis of the Siderophore Synthetase AsbB through Reconstitution of the Petrobactin Biosynthetic Pathway from Bacillus anthracis
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DOI：
10.1074/jbc.m112.359349
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发表时间：
2012-05-04
影响因子：
4.8
通讯作者：
Sherman, David H.
中科院分区：
文献类型：
--
作者：
Nusca, Tyler D.;Kim, Youngchang;Sherman, David H.
岩杆菌素是一种儿茶酚-羧酸铁载体的混合物，它是炭疽杆菌（炭疽的病原体）完全毒力所必需的。asbABCDEF操纵子编码这种次生代谢物的生物合成机制。在这里，我们证明了asb操纵子编码的五个基因产物的功能对于在体外系统中将内源性前体转化为石油藻蛋白是必要和充分的。在这一途径中，铁载体合成酶AsbB通过使用生物合成中间体（而不是初级代谢物）作为羧酸供体，催化酰胺键的形成，这对石油胶蛋白的组装至关重要。为了解决炭疽杆菌铁载体生物合成蛋白B （AsbB）的晶体结构，我们揭示了非核糖体肽合成酶非依赖性铁载体（NIS）合成酶的三维模型。结构特征为这种双功能凝聚酶如何结合和腺苷化多种含柠檬酸盐的底物以及天然和非天然多胺亲核试剂的结合提供了新的见解。该活动能够形成多个最终阶段产品，从而最终组装成油凝蛋白。随后对非核糖体肽合成酶样AsbC、AsbD和AsbE多肽进行的酶促分析表明，AsbB的替代产物进一步转化为油杆菌蛋白，验证了之前提出的形成这种铁载体的聚合途径。这些研究确定了潜在的治疗靶点，以阻止由炭疽杆菌和其他致病菌引起的致命感染，并为化学酶合成新化合物提供了新的途径。
Petrobactin, a mixed catechol-carboxylate siderophore, is required for full virulence of Bacillus anthracis, the causative agent of anthrax. The asbABCDEF operon encodes the biosynthetic machinery for this secondary metabolite. Here, we show that the function of five gene products encoded by the asb operon is necessary and sufficient for conversion of endogenous precursors to petrobactin using an in vitro system. In this pathway, the siderophore synthetase AsbB catalyzes formation of amide bonds crucial for petrobactin assembly through use of biosynthetic intermediates, as opposed to primary metabolites, as carboxylate donors. In solving the crystal structure of the B. anthracis siderophore biosynthesis protein B (AsbB), we disclose a three-dimensional model of a nonribosomal peptide synthetase-independent siderophore (NIS) synthetase. Structural characteristics provide new insight into how this bifunctional condensing enzyme can bind and adenylate multiple citrate-containing substrates followed by incorporation of both natural and unnatural polyamine nucleophiles. This activity enables formation of multiple end-stage products leading to final assembly of petrobactin. Subsequent enzymatic assays with the nonribosomal peptide synthetase-like AsbC, AsbD, and AsbE polypeptides show that the alternative products of AsbB are further converted to petrobactin, verifying previously proposed convergent routes to formation of this siderophore. These studies identify potential therapeutic targets to halt deadly infections caused by B. anthracis and other pathogenic bacteria and suggest new avenues for the chemoenzymatic synthesis of novel compounds.