PDF(Pubmed)
Abstract：
The acyl carrier protein of Escherichia coli, termed AcpP, is a prototypical example of type II fatty acid synthase systems found in many bacteria. It serves as a central hub by accepting diverse acyl moieties (4-18 carbons) and shuttling them between its multiple enzymatic partners to generate fatty acids. Prior structures of acyl-AcpPs established that thioester-linked acyl cargos are sequestered within AcpP\'s hydrophobic lumen. In contrast, structures of enzyme-bound acyl-AcpPs showed translocation of AcpP-tethered acyl chains into the active sites of enzymes. The mechanistic underpinnings of this conformational interplay, termed chain-flipping, are unclear. Here, using heteronuclear NMR spectroscopy, we reveal that AcpP-tethered acyl chains (6-10 carbons) spontaneously adopt lowly populated solvent-exposed conformations. To this end, we devised a new strategy to replace AcpP\'s thioester linkages with 15N-labeled amide bonds, which facilitated direct \"visualization\" of these excited states using NMR chemical exchange saturation transfer and relaxation dispersion measurements. Global fitting of the corresponding data yielded kinetic rate constants of the underlying equilibrium and populations and lifetimes of solvent-exposed states. The latter were influenced by acyl chain composition and ranged from milliseconds to submilliseconds for chains containing six, eight, and ten carbons, owing to their variable interactions with AcpP\'s hydrophobic core. Although transient, the exposure of AcpP-tethered acyl chains to the solvent may allow relevant enzymes to gain access to its active thioester, and the enzyme-induced selection of this conformation will culminate in the production of fatty acids.
摘要：
大肠杆菌的酰基载体蛋白，被称为AcpP，是在许多细菌中发现的II型脂肪酸合酶系统的典型实例。它通过接受不同的酰基部分（4-18个碳）并在其多个酶伴侣之间穿梭以产生脂肪酸而充当中心中心。酰基-AcpP的先前结构确定硫酯连接的酰基货物被隔离在AcpP的疏水内腔中。相比之下,酶结合的酰基-AcpP的结构显示AcpP连接的酰基链易位到酶的活性位点。这种构象相互作用的机制基础，称为链条翻转，不清楚。这里,使用异核核磁共振光谱，我们发现AcpP连接的酰基链（6-10个碳）自发采用低填充的溶剂暴露构象。为此,我们设计了一种新的策略，用15N标记的酰胺键代替AcpP的硫酯键，这有助于使用NMR化学交换饱和转移和弛豫色散测量对这些激发态进行直接“可视化”。相应数据的全局拟合产生了基本平衡的动力学速率常数以及溶剂暴露状态的种群和寿命。后者受酰基链组成的影响，对于包含六个，八,和十个碳，由于它们与AcpP疏水核的可变相互作用。虽然是短暂的，AcpP连接的酰基链暴露于溶剂可能会使相关酶获得其活性硫酯，酶诱导的这种构象的选择将最终导致脂肪酸的产生。
