Enzymatic Synthesis of 1‐Alkyl‐2‐hydroxy‐sn‐glycero‐2,3‐cyclic‐phosphate Using a Novel Lysoplasmalogen‐Specific Phopholipase D

Abstract

Many phospholipase Ds (PLDs) are known to catalyze transphosphatidylation as well as hydrolysis of phospholipids. Transphosphatidylation of lysoplasmalogen (LyPls)‐specific phospholipase D (LyPls‐PLD), which catalyzes hydrolysis of ether lysophospholipids such as LyPls and 1‐hexadecyl‐2‐hydroxy‐sn‐glycero‐3‐phosphocholine (Lyso‐PAF), still remains unclear. This study aims to reveal the transphosphatidylation activity of LyPls‐PLD, that is, the production of cyclic ether lysophospholipid. The enzymatic reaction is conducted in a buffer system, and the reaction products of a novel LyPls‐PLD from Thermocrispum sp. are investigated using mass spectrometry (MS). MS analyses demonstrate the reaction products to consist of 100% 1‐hexadecyl‐2‐hydroxy‐sn‐glycero‐2,3‐cyclic‐phosphate (cLyPA) and choline from Lyso‐PAF; however, 1‐alkenyl‐2‐hydroxy‐sn‐glycero‐2,3‐cyclic‐phosphate from 1‐O‐1′‐(Z)‐octadecenyl‐2‐hydroxy‐sn‐glycero‐3‐phosphocholine and 1‐O‐1′‐(Z)‐octadecenyl‐2‐hydroxy‐sn‐glycero‐3‐phosphoethanolamine is not produced. These results are expected to help in elucidating the catalytic mechanism of LyPls‐PLD, that is, the rate‐limiting step, and indicate LyPls‐PLD to be useful for the one‐pot synthesis of cLyPA. Practical Applications: A novel phospholipase D, LyPls‐PLD, can exclusively synthesize cLyPA from Lyso‐PAF using a one‐step enzymatic reaction without an organic solvent. cLyPA could be expected to show bioactivities similar to those of cyclic phosphatidic acid, which promotes normal cell differentiation, hyaluronic acid synthesis, antiproliferative activity in fibroblasts, and inhibitory activity toward cancer cell invasion and metastasis.

Switching the substrate specificity of lysoplasmalogen‐specific phospholipase D

“…However, such flexibility may also make it more difficult for the intermediate or product to dissociate from the catalytic pocket. This conclusion is supported by the fact that hydrolytic reaction with WT allowed the 100% cyclic product, 1‐hexadecyl‐2‐hydroxy‐ sn ‐glycero‐2,3‐cyclic‐phosphate, to be generated from LysoPAF, but not from LyPlsCho [7]. We also reported that the conformation of the sn ‐1 chain of the substrate was likely involved in substrate binding and product release, which represented the rate‐limiting steps of the reaction, thereby suggesting that substrate recognition may be strongly influenced by the conformation of sn ‐1.…”

“…A previous study of ours indicated that LyPls‐PLD shows 53% relative activity with 1‐hexadecyl‐2‐hydroxy‐ sn ‐glycero‐3‐phosphocholine (LysoPAF) than with LyPlsCho [7]. This increased activity level may be useful for conducting a lipoprotein‐associated phospholipase A 2 (Lp‐PLA 2 ) activity assay, as described below [8].…”