TY - JOUR
T1 - Switching the substrate specificity of lysoplasmalogen-specific phospholipase D
AU - Oyama, Takayuki
AU - Murayama, Kazutaka
AU - Sugimori, Daisuke
N1 - Funding Information:
We would like to thank Editage for English language editing. This work was supported by grants from JSPS KAKENHI (grant numbers JP15K05557 and JP20K05822), Takahashi Industrial and Economic Research Foundation, and Koyanagi Foundation to DS. The funding source(s) played no role in our study design, in the collection, analysis, and interpretation of data, or in the decision to submit the article for publication.
Publisher Copyright:
© 2021 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.
PY - 2021/4
Y1 - 2021/4
N2 - 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 3.1.4.2 (currently assigned).
AB - 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 3.1.4.2 (currently assigned).
KW - lipoprotein-associated phospholipase A
KW - LysoPAF-specific PLD
KW - lysoplasmalogen-specific PLD
KW - phospholipase D
KW - substrate-specificity switch
KW - Thermocrispum sp.
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U2 - 10.1002/2211-5463.13123
DO - 10.1002/2211-5463.13123
M3 - Article
C2 - 33599379
AN - SCOPUS:85102646198
SN - 2211-5463
VL - 11
SP - 1132
EP - 1143
JO - FEBS Open Bio
JF - FEBS Open Bio
IS - 4
ER -
