Switching the substrate specificity of lysoplasmalogen-specific phospholipase D.

摘要: 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 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 100% it choline lysoplasmalogen (LyPlsCho). Lipoprotein-associated A2 (Lp-PLA2 ) can be used evaluate for cardiovascular disease. Hence, development of point-of-care testing kit requires LysoPAF-specific PLD (LysoPAF-PLD) measure Lp-PLA2 activity. Rational site-directed mutagenesis kinetic analysis were applied generate LysoPAF-PLD clarify mechanisms underlying substrate-recognition ability LyPls-PLD. Our results suggest variants A47, M71, N173, F211, W282 possibly involved recognition F211L may substantially alter preference. Moreover, specific ratio LysoPAF/LyPlsCho corresponding was up 25-fold higher than wild-type enzyme. Thus, succeeded switching LyPlsCho- LysoPAF-PLD. These variant utilized Kinetic analyses demonstrated product release rate-limiting step reaction, flexibility sn-1 ether-linked vinyl/alkyl chain being essential binding release. findings lead better understanding differences between homologous (such as PLD, sphingomyelinase D, GDPD phosphatidylinositol-phosphodiesterase superfamily) relation recognition. ENZYME: EC 3.1.4.2 (currently assigned).