Khan M Adil, Neale Chris, Michaux Catherine, Pomès Régis, Privé Gilbert G, Woody Robert W, Bishop Russell E
Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada M5S 1A8.
Biochemistry. 2007 Apr 17;46(15):4565-79. doi: 10.1021/bi602526k. Epub 2007 Mar 22.
The structural basis of lipid acyl-chain selection by membrane-intrinsic enzymes is poorly understood because most integral membrane enzymes of lipid metabolism have proven refractory to structure determination; however, robust enzymes from the outer membranes of gram-negative bacteria are now providing a first glimpse at the underlying mechanisms. The methylene unit resolution of the phospholipid:lipid A palmitoyltransferase PagP is determined by the hydrocarbon ruler, a 16-carbon saturated acyl-chain-binding pocket buried within the transmembrane beta-barrel structure. Substitution of Gly88 lining the floor of the hydrocarbon ruler with Ala or Met makes the enzyme select specifically 15- or 12-carbon saturated acyl chains, respectively, indicating that hydrocarbon ruler depth determines acyl-chain selection. However, the Gly88Cys PagP resolution does not diminish linearly because it selects both 14- and 15-carbon saturated acyl chains. We discovered that an exciton, emanating from a buried Tyr26-Trp66 phenol-indole interaction, is extinguished by a local structural perturbation arising from the proximal Gly88Cys PagP sulfhydryl group. Site-specific S-methylation of the single Cys afforded Gly88Cys-S-methyl PagP, which reasserted both the exciton and methylene unit resolution by specifically selecting 13-carbon saturated acyl chains for transfer to lipid A. Unlike the other Gly88 substitutions, the Cys sulfhydryl group recedes from the hydrocarbon ruler floor and locally perturbs the subjacent Tyr26 and Trp66 aromatic rings. The resulting hydrocarbon ruler expansion thus occurs at the exciton's expense and accommodates an extra methylene unit in the selected acyl chain. The hydrocarbon ruler-exciton juxtaposition endows PagP with a molecular gauge for probing the structural basis of lipid acyl-chain selection in a membrane-intrinsic environment.
膜内在酶对脂质酰基链的选择结构基础仍知之甚少，因为大多数脂质代谢的整合膜酶已被证明难以进行结构测定；然而，革兰氏阴性菌外膜中的强大酶类现在让我们首次得以窥见其潜在机制。磷脂：脂多糖棕榈酰转移酶PagP的亚甲基单元分辨率由烃尺决定，烃尺是一个位于跨膜β桶结构内的16碳饱和酰基链结合口袋。用丙氨酸或甲硫氨酸取代烃尺底部的Gly88，会使该酶分别特异性选择15碳或12碳饱和酰基链，这表明烃尺深度决定酰基链选择。然而，Gly88Cys PagP的分辨率并非呈线性降低，因为它同时选择14碳和15碳饱和酰基链。我们发现，由埋藏的Tyr26-Trp66酚-吲哚相互作用产生的激子，会因近端Gly88Cys PagP巯基引起的局部结构扰动而熄灭。对单个半胱氨酸进行位点特异性S-甲基化得到Gly88Cys-S-甲基PagP，它通过特异性选择13碳饱和酰基链转移到脂多糖上，恢复了激子和亚甲基单元分辨率。与其他Gly88取代不同，半胱氨酸巯基从烃尺底部后退，并局部扰动相邻的Tyr26和Trp66芳香环。由此产生的烃尺扩展是以激子为代价的，并在所选酰基链中容纳一个额外的亚甲基单元。烃尺与激子的并置赋予PagP一种分子尺度，用于探究膜内在环境中脂质酰基链选择的结构基础。
