Henrich Erik, Löhr Frank, Pawlik Grzegorz, Peetz Oliver, Dötsch Volker, Morgner Nina, de Kroon Anton I, Bernhard Frank
Institute of Biophysical Chemistry, Centre for Biomolecular Magnetic Resonance , J.W.-Goethe-University , 60438 Frankfurt am Main , Germany.
Membrane Biochemistry & Biophysics, Bijvoet Center for Biomolecular Research and Institute of Biomembranes , Utrecht University , 3584 CH Utrecht , The Netherlands.
Biochemistry. 2018 Oct 9;57(40):5780-5784. doi: 10.1021/acs.biochem.8b00807. Epub 2018 Sep 24.
Biomembranes composed of lipids and proteins play central roles in physiological processes, and the precise balance between different lipid species is crucial for maintaining membrane function. One pathway for the biosynthesis of the abundant lipid phosphatidylcholine in eukaryotes involves a membrane-integrated phospholipid methyltransferase named Opi3 in yeast. A still unanswered question is whether Opi3 can catalyze phosphatidylcholine synthesis in trans, at membrane contact sites. While evidence for this activity was obtained from studies with complex in vitro-reconstituted systems based on endoplasmic reticulum membranes, isolated and purified Opi3 could not be analyzed. We present new insights into Opi3 activity by characterizing the in vitro-synthesized enzyme in defined hydrophobic environments. Saccharomyces cerevisiae Opi3 was cell-free synthesized and either solubilized in detergent micelles or co-translationally inserted into preformed nanodisc membranes of different lipid compositions. While detergent-solubilized Opi3 was inactive, the enzyme inserted into nanodisc membranes showed activity and stayed monomeric as revealed by native mass spectrometry. The methylation of its lipid substrate dioleoylphosphatidylmonomethylethanolamine to phosphatidylcholine was monitored by one-dimensional P nuclear magnetic resonance. Phosphatidylcholine formation was observed not only in nanodiscs containing inserted Opi3 but also in nanodiscs devoid of the enzyme containing the lipid substrate. This result gives a clear indication for in trans catalysis by Opi3; i.e., it acts on the substrate in juxtaposed membranes, while in cis lipid conversion may also contribute. Our established system for the characterization of pure Opi3 in defined lipid environments may be applicable to other lipid biosynthetic enzymes and help in understanding the subcellular organization of lipid synthesis.
由脂质和蛋白质组成的生物膜在生理过程中起着核心作用，不同脂质种类之间的精确平衡对于维持膜功能至关重要。真核生物中丰富的脂质磷脂酰胆碱生物合成的一条途径涉及酵母中一种名为Opi3的膜整合磷脂甲基转移酶。一个尚未得到解答的问题是，Opi3是否能在膜接触位点进行反式催化磷脂酰胆碱的合成。虽然基于内质网膜的复杂体外重组系统的研究获得了这种活性的证据，但无法对分离和纯化的Opi3进行分析。我们通过在确定的疏水环境中表征体外合成的酶，对Opi3的活性有了新的认识。酿酒酵母Opi3通过无细胞合成，要么溶解在去污剂胶束中，要么共翻译插入到不同脂质组成的预制纳米盘膜中。虽然去污剂溶解的Opi3没有活性，但插入纳米盘膜中的酶显示出活性，并且如通过原生质谱所揭示的那样保持单体状态。通过一维磷核磁共振监测其脂质底物二油酰磷脂单甲基乙醇胺甲基化为磷脂酰胆碱的过程。不仅在含有插入Opi3的纳米盘中观察到了磷脂酰胆碱的形成，而且在不含该酶但含有脂质底物的纳米盘中也观察到了。这一结果清楚地表明Opi3进行了反式催化；即，它作用于并列膜中的底物，而顺式脂质转化也可能起作用。我们建立的在确定脂质环境中表征纯Opi3的系统可能适用于其他脂质生物合成酶，并有助于理解脂质合成的亚细胞组织。
