Fatty acid synthases (FASs) and polyketide synthases (PKSs) iteratively elongate and often reduce two-carbon ketide units in de novo fatty acid and polyketide biosynthesis. Cycles of chain extensions in FAS and PKS are initiated by an acyltransferase (AT), which loads monomer units onto acyl carrier proteins (ACPs), small, flexible proteins that shuttle covalently linked intermediates between catalytic partners. Formation of productive ACP–AT interactions is required for catalysis and specificity within primary and secondary FAS and PKS pathways. Here, we use the Escherichia coli FAS AT, FabD, and its cognate ACP, AcpP, to interrogate type II FAS ACP–AT interactions. We utilize a covalent crosslinking probe to trap transient interactions between AcpP and FabD to elucidate the X-ray crystal structure of a type II ACP–AT complex. Our structural data are supported using a combination of mutational, crosslinking, and kinetic analyses, and long-timescale molecular dynamics (MD) simulations. Together, these complementary approaches reveal key catalytic features of FAS ACP–AT interactions. These mechanistic inferences suggest that AcpP adopts multiple, productive conformations at the AT binding interface, allowing the complex to sustain high transacylation rates. Furthermore, MD simulations support rigid body subdomain motions within the FabD structure that may play a key role in AT activity and substrate selectivity.

脂肪酸合酶（FASs）和聚酮化合物合酶（PKSs）反复拉长，并经常减少从头脂肪酸和聚酮化合物生物合成中的二碳酮化合物单元。FAS和PKS中扩链的周期由酰基转移酶（AT）启动，该酶将单体单元加载到酰基载体蛋白（ACP）上，这些小而柔软的蛋白在催化伙伴之间共价连接中间体。在初级和次级FAS和PKS途径内的催化作用和特异性要求形成有效的ACP-AT相互作用。在这里，我们使用大肠杆菌FAS AT，FabD及其同类ACP AcpP询问II型FAS ACP-AT相互作用。我们利用共价交联探针捕获AcpP和FabD之间的瞬时相互作用，以阐明II型ACP-AT复合物的X射线晶体结构。我们的结构数据结合了突变，交联和动力学分析以及长时间尺度的分子动力学（MD）模拟得到了支持。这些互补的方法一起揭示了FAS ACP-AT相互作用的关键催化特征。这些机理推断表明，AcpP在AT结合界面处采用了多种有效的构象，从而使复合物能够维持较高的酰化率。此外，MD模拟支持FabD结构内的刚体子域运动，这可能在AT活性和底物选择性方面发挥关键作用。