Facile Enzymatic Synthesis of Phosphatidylthreonine Using an Engineered Phospholipase D
Facile Enzymatic Synthesis of Phosphatidylthreonine Using an Engineered Phospholipase D
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DOI：
10.1002/ejlt.201800089
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发表时间：
2018-06-01
影响因子：
2.7
通讯作者：
Iwasaki, Yugo
中科院分区：
文献类型：
--
作者：
Damnjanovic, Jasmina;Matsunaga, Nozomi;Iwasaki, Yugo
本文介绍了一种新建立的直接酶法合成磷脂酰苏氨酸（PtdThr）的方法。这是首次报道酶法合成这种稀有磷脂。它利用磷脂酶D（PLD）催化的转磷脂酰化，其中磷脂酰胆碱（PtdCho）的头部基团交换为L-苏氨酸（L-Thr）。使用野生型PLD催化PtdCho和L-Thr之间的反应的尝试没有成功，可能是因为L-Thr的仲羟基基团对于酶是不可接近的。为了合成天然PtdThr异构体Ptd-L-Thr，筛选对肌醇的仲羟基有活性的工程化PLD变体接受L-Thr的能力。六个变体被鉴定为阳性，其中187 F/191 Y/385 L（FYL）显示出最高活性。通过优化反应参数，Ptd-L-Thr含量达到30mol%左右。产物经柱层析分离，总收率为5.2%，其结构经NMR确证。此外，FYL变体还可以与苏氨酸、L-别-Thr和D-别-Thr以及L-Thr的一些立体异构体反应，但不与D-Thr反应。配体对接模拟解释了酶对这些立体异构体的偏好; L-、L-allo-和D-allo-Thr可以以生产方向结合到酶的活性位点，而D-Thr结合的位置使反应不可能进行。实际应用：酶促方法能够从PtdCho和L-苏氨酸一步合成PtdThr，而无需任何保护/脱保护步骤，从而比目前使用的化学方法简单得多并且危险性更小。合成的PtdThr可以以纯的形式分离，并用作阐明其生物学功能的试剂。描述了一步酶法合成磷脂酰苏氨酸（PtdThr）的方法。它使用由工程化磷脂酶D（PLD）催化的磷脂酰胆碱（PtdCho）与苏氨酸（Thr）的头基交换。该酶能接受Thr的某些立体异构体，即L-Thr、L-allo-Thr和D-allo-Thras作为底物，生成相应的PtdThr，但不能生成D-Thr。对接模拟解释了酶对这些酯对映异构体的偏好; L-、L-allo-和D-allo-Thr可以以生产方向结合到酶的活性位点，而D-Thr以非生产方向结合，这使得反应不可能进行。
Here is described the newly established method for direct enzymatic synthesis of phosphatidylthreonine (PtdThr). It is the first report on enzymatic synthesis of this rare phospholipid. It utilizes phospholipase D (PLD)-catalyzed transphosphatidylation, in which the head group of phosphatidylcholine (PtdCho) is exchanged to L-Threonine (L-Thr). An attempt to catalyze the reaction between PtdCho and L-Thr using wild-type PLD is not successful, possibly because the secondary hydroxyl group of L-Thr is not accessible to the enzyme. To synthesize Ptd-L-Thr, the natural PtdThr isomer, engineered PLD variants active toward secondary hydroxyls of inositol are screened for their ability to accept L-Thr. Six variants are identified as positive, among which 187F/191Y/385L (FYL) shows highest activity. After optimizing the reaction parameters, Ptd-L-Thr content reaches approximately 30mol%. The product is isolated by column chromatography with the overall yield of 5.2%, and its structure is confirmed by NMR. In addition, the FYL variant can also react on some stereoisomers of threonine, L-allo-Thr, and D-allo-Thr as well as L-Thr, but not D-Thr. Ligand docking simulation explains the enzyme's preference toward these stereoisomers; L-, L-allo-, and D-allo-Thr can bind to the enzyme's active site in a productive orientation, whereas D-Thr binds in a position which makes the reaction impossible to proceed.Practical Applications: The enzymatic method enables one-step synthesis of PtdThr from PtdCho and L-Threonine without any protection/deprotection steps, thereby being much more simple and less hazardous than the currently used chemical methods. The synthesized PtdThr can be isolated in pure form and used as a reagent for elucidation of its biological functions. A method for one-step enzymatic synthesis of phosphatidylthreonine (PtdThr) is described. It uses the head group exchange of phosphatidylcholine (PtdCho) with Threonine(Thr) catalyzed by an engineered phospholipase D (PLD). The enzyme can accept some stereoisomers of Thr, that is, L-Thr, L-allo-Thr, and D-allo-Thras the substrates to give the corresponding PtdThr, but notD-Thr. Docking simulation explainsthe enzyme's preference toward thesestereoisomers; L-, L-allo-, and D-allo-Thr can bind to the enzyme's active site in a productive orientation, whereas D-Thrbinds in a non-productive orientation which makes the reaction impossible to proceed.