Touvrey Cédric, Courageux Charlotte, Guillon Virginia, Terreux Raphael, Nachon Florian, Brazzolotto Xavier
Institut de Biologie et Chimie des Protéines, 7 Passage du Vercors, 69367 Lyon Cedex 07, France.
Institut de Recherche Biomédicale des Armées, Département de Toxicologie et Risques Chimiques, Brétigny sur Orge, 91220, France.
Toxicology. 2019 Jan 1;411:15-23. doi: 10.1016/j.tox.2018.10.015. Epub 2018 Oct 22.
The efficiency of human butyrylcholinesterase (BChE) as a stoichiometric bioscavenger of nerve agents is well established. However, wide use is currently limited by production and purification costs. Aiming at identifying an alternative human protein bioscavenger, we looked for an original scaffold candidate by virtual screening of the Protein Data Bank for functional similarity using the "Surfing the Molecules" software (sumo-pbil.ibcp.fr) and a search model based on the BChE active site topology. Besides the expected acetylcholinesterase and butyrylcholinesterase, we identified a set of bile salt activated lipases structures, among which the human pancreatic lipase (hBAL) that shares 34% identity with BChE. We produced the recombinant enzyme in mammalian cells, purified it, and measured the inhibition constants for paraoxon and surrogates of VX, sarin and tabun. We solved the X-ray structure of apo hBAL and conjugates with paraoxon and the surrogates at resolutions in the 2-Å range. These structures allow the assessment of hBAL for scavenging nerve agents. They revealed that hBAL has inverted stereoselectivity for the surrogates of nerve agent compared to human cholinesterases. We observed a remarkable flip of the catalytic histidine driven by the chelation of Zn. Dealkylation of the conjugate, aka aging, was solely observed for paraoxon.
人类丁酰胆碱酯酶（BChE）作为神经毒剂的化学计量生物清除剂的效率已得到充分证实。然而，目前其广泛应用受到生产和纯化成本的限制。为了寻找一种替代的人类蛋白质生物清除剂，我们使用“Surfing the Molecules”软件（sumo-pbil.ibcp.fr）通过对蛋白质数据库进行功能相似性虚拟筛选，并基于BChE活性位点拓扑结构建立搜索模型，寻找一种原始的支架候选物。除了预期的乙酰胆碱酯酶和丁酰胆碱酯酶外，我们还鉴定出一组胆汁盐激活脂肪酶结构，其中人类胰腺脂肪酶（hBAL）与BChE有34%的序列同一性。我们在哺乳动物细胞中生产了重组酶，进行了纯化，并测量了对氧磷以及VX、沙林和塔崩替代物的抑制常数。我们解析了脱辅基hBAL以及与对氧磷和替代物结合物在2埃分辨率范围内的X射线结构。这些结构有助于评估hBAL对神经毒剂的清除能力。结果显示，与人类胆碱酯酶相比，hBAL对神经毒剂替代物具有相反的立体选择性。我们观察到由锌螯合驱动的催化组氨酸发生了显著翻转。仅在对氧磷的结合物中观察到了共轭物的脱烷基化，即老化现象。
