Switching the substrate specificity of lysoplasmalogen-specific phospholipase D

Takayuki Oyama, Kazutaka Murayama, Daisuke Sugimori

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1 Citation (Scopus)


Lysoplasmalogen-specific phospholipase D (LyPls-PLD) catalyzes reactions in a manner similar to those catalyzed by glycerophosphodiester phosphodiesterase (GDPD) and other well-known PLDs. Although these enzymes hydrolyze the glycerophosphodiester bond, their substrate specificities are completely different. Previously, we reported that LyPls-PLD from Thermocrispum sp. RD004668 shows only 53% activity with 1-hexadecyl-2-hydroxy-sn-glycero-3-phosphocholine (LysoPAF) relative to the 100% activity it shows with choline lysoplasmalogen (LyPlsCho). Lipoprotein-associated phospholipase A2 (Lp-PLA2) activity can be used to evaluate for cardiovascular disease. Hence, development of a point-of-care testing kit requires a LysoPAF-specific PLD (LysoPAF-PLD) to measure Lp-PLA2 activity. Rational site-directed mutagenesis and kinetic analysis were applied to generate LysoPAF-PLD from LyPls-PLD and to clarify the mechanisms underlying the substrate-recognition ability of LyPls-PLD. Our results suggest that LyPls-PLD variants A47, M71, N173, F211, and W282 are possibly involved in substrate recognition and that F211L may substantially alter substrate preference. Moreover, the specific activity ratio LysoPAF/LyPlsCho corresponding to F211L was up to 25-fold higher than that corresponding to the wild-type enzyme. Thus, we succeeded in switching from LyPlsCho- to LysoPAF-PLD. These results suggest that the F211L variant may be utilized to measure Lp-PLA2 activity. Kinetic analyses demonstrated that product release was the rate-limiting step of the reaction, with flexibility of the sn-1 ether-linked vinyl/alkyl chain of the substrate being essential for substrate binding and product release. Our findings may lead to a better understanding of the differences between homologous enzymes (such as PLD, sphingomyelinase D, and GDPD of the phosphatidylinositol-phosphodiesterase superfamily) in relation to substrate recognition. Enzyme: EC (currently assigned).

Original languageEnglish
Pages (from-to)1132-1143
Number of pages12
JournalFEBS Open Bio
Issue number4
Publication statusPublished - 2021 Apr


  • lipoprotein-associated phospholipase A
  • LysoPAF-specific PLD
  • lysoplasmalogen-specific PLD
  • phospholipase D
  • substrate-specificity switch
  • Thermocrispum sp.


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