Liu Ye, Zheng Tengfei, Bruner Steven D
Department of Chemistry, Boston College, Chestnut Hill, MA 02167, USA.
Chem Biol. 2011 Nov 23;18(11):1482-8. doi: 10.1016/j.chembiol.2011.09.018.
Phosphopantetheine-modified carrier domains play a central role in the template-directed, biosynthesis of several classes of primary and secondary metabolites. Fatty acids, polyketides, and nonribosomal peptides are constructed on multidomain enzyme assemblies using phosphopantetheinyl thioester-linked carrier domains to traffic and activate building blocks. The carrier domain is a dynamic component of the process, shuttling pathway intermediates to sequential enzyme active sites. Here, we report an approach to structurally fix carrier domain/enzyme constructs suitable for X-ray crystallographic analysis. The structure of a two-domain construct of Escherichia coli EntF was determined with a conjugated phosphopantetheinyl-based inhibitor. The didomain structure is locked in an active orientation relevant to the chemistry of nonribosomal peptide biosynthesis. This structure provides details into the interaction of phosphopantetheine arm with the carrier domain and the active site of the thioesterase domain.
磷酸泛酰巯基乙胺修饰的载体结构域在几类初级和次级代谢产物的模板导向生物合成中起着核心作用。脂肪酸、聚酮化合物和非核糖体肽是在多结构域酶组装体上构建的，利用磷酸泛酰巯基乙胺硫酯连接的载体结构域来运输和激活构建模块。载体结构域是该过程的动态组成部分，将途径中间体穿梭到连续的酶活性位点。在这里，我们报告了一种在结构上固定适合X射线晶体学分析的载体结构域/酶构建体的方法。利用一种基于共轭磷酸泛酰巯基乙胺的抑制剂确定了大肠杆菌EntF的双结构域构建体的结构。双结构域结构被锁定在与非核糖体肽生物合成化学相关的活性取向。该结构提供了磷酸泛酰巯基乙胺臂与载体结构域和硫酯酶结构域活性位点相互作用的详细信息。
