Direct Enzymatic Synthesis of 1-Phosphatidyl-β-D-glucose by Engineered Phospholipase D

A simple, direct method for the synthesis of 1-phosphatidyl-β-D-glucose (1-PGlc) was established, being the first example of direct enzymatic synthesis of this recently discovered, highly important bioactive phospholipid. The method employs phospholipase D (PLD, E.C. 3.1.4.4)-catalyzed transphosphatidylation, in which the polar head group of phosphatidylcholine is exchanged to glucose. Although wild-type PLD can catalyze the transfer reaction, it provides only 6-phosphatidyl-glucose, a positional isomer of 1-PGlc, due to its strong preference towards the primary hydroxyl group. To synthesize 1-PGlc, engineered PLD variants, previously isolated as the ones active on secondary hydroxyls of inositol, were screened for the ability to catalyze the reaction. One of the variants, 187K/191W/385Y (KWY), was identified as the best-performing in 1-PGlc synthesis, however, in addition to 1-PGlc the reaction with KWY also afforded undesired positional isomers as byproducts. To facilitate the isolation of 1-PGlc, the isomers were converted into the corresponding amines by reductive amination. Following the column chromatography, the desired product was isolated with the overall yield of 12.5 %. The structure of the product was confirmed by ¹H-NMR analysis.