Department of Biochemistry and Biomedical Sciences and Michael G. DeGroote Institute for Infectious Disease Research, McMaster University, Hamilton, Ontario, Canada L8N 3Z5.
Biochemistry. 2010 Mar 23;49(11):2368-79. doi: 10.1021/bi901669q.
The Escherichia coli outer membrane phospholipid:lipid A palmitoyltransferase PagP exhibits remarkable selectivity because its binding pocket for lipid acyl chains excludes those differing in length from palmitate by a solitary methylene unit. This narrow detergent-binding hydrophobic pocket buried within the eight-strand antiparallel beta-barrel is known as the hydrocarbon ruler. Gly88 lines the acyl chain binding pocket floor, and its substitution can raise the floor to correspondingly shorten the selected acyl chain. An aromatic exciton interaction between Tyr26 and Trp66 provides an intrinsic spectroscopic probe located immediately adjacent to Gly88. The Gly88Cys PagP enzyme was engineered to function as a dedicated myristoyltransferase, but the mutant enzyme instead selected both myristoyl and pentadecanoyl groups, was devoid of the exciton, and displayed a 21 degrees C reduction in thermal stability. We now demonstrate that the structural perturbation results from a buried thiolate anion attributed to suppression of the Cys sulfhydryl group pK(a) from 9.4 in aqueous solvent to 7.5 in the hydrocarbon ruler microenvironment. The Cys thiol is sandwiched at the interface between a nonpolar and a polar beta-barrel interior milieu, suggesting that local electrostatics near the otherwise hydrophobic hydrocarbon ruler pocket serve to perturb the thiol pK(a). Neutralization of the Cys thiolate anion by protonation restores wild-type exciton and thermal stability signatures to Gly88Cys PagP, which then functions as a dedicated myristoyltransferase at pH 7. Gly88Cys PagP assembled in bacterial membranes recapitulates lipid A myristoylation in vivo. Hydrocarbon ruler-exciton coupling in PagP thus reveals a thiol-thiolate ionization mechanism for modulating lipid acyl chain selection.
脂 A 棕榈酰转移酶 PagP 表现出显著的选择性，因为其脂质酰基链结合口袋排除了与棕榈酸相差一个亚甲基单元的那些。这个位于八链反平行β-桶内的狭窄去污剂结合疏水性口袋被称为烃标尺。Gly88 排列在酰基链结合口袋的底部，其取代可以相应地升高底部，从而缩短所选酰基链。Tyr26 和 Trp66 之间的芳香族激子相互作用提供了一个位于 Gly88 附近的固有光谱探针。Gly88Cys PagP 酶被设计为专门的肉豆蔻酰转移酶，但突变酶反而选择了肉豆蔻酰基和十五烷酰基，没有激子，并且热稳定性降低了 21°C。我们现在证明，结构扰动是由于硫醇阴离子的埋藏引起的，这归因于抑制 Cys 巯基的 pK(a)从水溶剂中的 9.4 降低到烃标尺微环境中的 7.5。Cys 硫醇夹在非极性和极性β-桶内部环境之间的界面处，表明烃标尺口袋的局部静电作用会干扰硫醇的 pK(a)。Cys 硫醇阴离子的中和通过质子化恢复了 Gly88Cys PagP 的野生型激子和热稳定性特征，使其在 pH 7 下作为专门的肉豆蔻酰转移酶发挥作用。在细菌膜中组装的 Gly88Cys PagP 再现了体内脂 A 的豆蔻酰化。因此，PagP 中的烃标尺-激子偶联揭示了一种用于调节脂质酰基链选择的硫醇-硫醇盐离子化机制。
