Abstract
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 30 mol%. 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.
Original language | English |
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Article number | 1800089 |
Journal | European Journal of Lipid Science and Technology |
Volume | 120 |
Issue number | 6 |
DOIs | |
Publication status | Published - 2018 Jun |
Externally published | Yes |
Keywords
- phosphatidylthreonine
- phospholipase D
- protein engineering
- transphosphatidylation
ASJC Scopus subject areas
- Biotechnology
- Food Science
- Chemistry(all)
- Industrial and Manufacturing Engineering