Switching the substrate specificity of lysoplasmalogen‐specific phospholipase D

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 A(2) (Lp‐PLA(2)) 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‐PLA(2) 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‐PLA(2) 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 3.1.4.2 (currently assigned).