Phospholipid N-methyltransferases (PLMTs) synthesize phosphatidylcholine by methylating phosphatidylethanolamine using S-adenosylmethionine as a methyl donor. Eukaryotic PLMTs are integral membrane enzymes located in the endoplasmic reticulum (ER). Recently Opi3, a PLMT of the yeast Saccharomyces cerevisiae was proposed to perform in trans catalysis, i.e. while localized in the ER, Opi3 would methylate lipid substrates located in the plasma membrane at membrane contact sites. Here, we tested whether the Opi3 active site is located at the cytosolic side of the ER membrane, which is a prerequisite for in trans catalysis. The membrane topology of Opi3 (and its human counterpart, phosphatidylethanolamine N-methyltransferase, expressed in yeast) was addressed by topology prediction algorithms and by the substituted cysteine accessibility method. The results of these analyses indicated that Opi3 (as well as phosphatidylethanolamine N-methyltransferase) has an N-out C-in topology and contains four transmembrane domains, with the fourth forming a re-entrant loop. On the basis of the sequence conservation between the C-terminal half of Opi3 and isoprenyl cysteine carboxyl methyltransferases with a solved crystal structure, we identified amino acids critical for Opi3 activity by site-directed mutagenesis. Modeling of the structure of the C-terminal part of Opi3 was consistent with the topology obtained by the substituted cysteine accessibility method and revealed that the active site faces the cytosol. In conclusion, the location of the Opi3 active site identified here is consistent with the proposed mechanism of in trans catalysis, as well as with conventional catalysis in cis.

中文翻译：

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酿酒酵母内质网驻留磷脂甲基转移酶 Opi3 的拓扑结构与反式催化一致。

磷脂 N-甲基转移酶 (PLMT) 使用 S-腺苷甲硫氨酸作为甲基供体，通过甲基化磷脂酰乙醇胺来合成磷脂酰胆碱。真核 PLMT 是位于内质网 (ER) 中的整合膜酶。最近，酿酒酵母的 PLMT Opi3 被提议进行反式催化，即当定位于内质网时，Opi3 会甲基化位于膜接触位点的质膜中的脂质底物。在这里，我们测试了 Opi3 活性位点是否位于 ER 膜的胞质侧，这是反式催化的先决条件。Opi3（及其在酵母中表达的人类对应物磷脂酰乙醇胺 N-甲基转移酶）的膜拓扑结构通过拓扑预测算法和取代的半胱氨酸可及性方法来解决。这些分析结果表明，Opi3（以及磷脂酰乙醇胺 N-甲基转移酶）具有 N-out C-in 拓扑结构，并包含四个跨膜结构域，其中第四个跨膜结构域形成重入环。基于 Opi3 的 C 端半部和异戊二烯基半胱氨酸羧基甲基转移酶之间的序列保守性以及已解决的晶体结构，我们通过定点诱变鉴定了对 Opi3 活性至关重要的氨基酸。Opi3 C 端部分结构的建模与取代半胱氨酸可达性方法获得的拓扑结构一致，表明活性位点面向细胞质。总之，这里确定的 Opi3 活性位点的位置与所提出的反式催化机制以及顺式催化的传统机制一致。