{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,5,13]],"date-time":"2025-05-13T13:58:09Z","timestamp":1747144689798,"version":"3.40.5"},"reference-count":33,"publisher":"Wiley","issue":"4","license":[{"start":{"date-parts":[[2021,3,19]],"date-time":"2021-03-19T00:00:00Z","timestamp":1616112000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100008965","name":"Takahashi Industrial and Economic Research Foundation","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100008965","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["JP15K05557","JP20K05822"],"award-info":[{"award-number":["JP15K05557","JP20K05822"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["febs.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["FEBS Open Bio"],"published-print":{"date-parts":[[2021,4]]},"abstract":"<jats:sec><jats:label\/><jats:p>Lysoplasmalogen\u2010specific phospholipase D (LyPls\u2010PLD) catalyzes reactions in a manner similar to those catalyzed by glycerophosphodiester phosphodiesterase (GDPD) and other well\u2010known PLDs. Although these enzymes hydrolyze the glycerophosphodiester bond, their substrate specificities are completely different. Previously, we reported that LyPls\u2010PLD from <jats:italic>Thermocrispum<\/jats:italic> sp. RD004668 shows only 53% activity with 1\u2010hexadecyl\u20102\u2010hydroxy\u2010<jats:italic>sn<\/jats:italic>\u2010glycero\u20103\u2010phosphocholine (LysoPAF) relative to the 100% activity it shows with choline lysoplasmalogen (LyPlsCho). Lipoprotein\u2010associated phospholipase A<jats:sub>2<\/jats:sub> (Lp\u2010PLA<jats:sub>2<\/jats:sub>) activity can be used to evaluate for cardiovascular disease. Hence, development of a point\u2010of\u2010care testing kit requires a LysoPAF\u2010specific PLD (LysoPAF\u2010PLD) to measure Lp\u2010PLA<jats:sub>2<\/jats:sub> activity. Rational site\u2010directed mutagenesis and kinetic analysis were applied to generate LysoPAF\u2010PLD from LyPls\u2010PLD and to clarify the mechanisms underlying the substrate\u2010recognition ability of LyPls\u2010PLD. Our results suggest that LyPls\u2010PLD 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\u2010fold higher than that corresponding to the wild\u2010type enzyme. Thus, we succeeded in switching from LyPlsCho\u2010 to LysoPAF\u2010PLD. These results suggest that the F211L variant may be utilized to measure Lp\u2010PLA<jats:sub>2<\/jats:sub> activity. Kinetic analyses demonstrated that product release was the rate\u2010limiting step of the reaction, with flexibility of the <jats:italic>sn<\/jats:italic>\u20101 ether\u2010linked 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\u2010phosphodiesterase superfamily) in relation to substrate recognition.<\/jats:p><\/jats:sec><jats:sec><jats:title>Enzyme<\/jats:title><jats:p>\n\nEC\u00a0<jats:ext-link xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"https:\/\/www.brenda-enzymes.org\/literature.php?e=3.1.4.2%26r=750552\">3.1.4.2<\/jats:ext-link> (currently assigned).<\/jats:p><\/jats:sec>","DOI":"10.1002\/2211-5463.13123","type":"journal-article","created":{"date-parts":[[2021,2,18]],"date-time":"2021-02-18T13:16:13Z","timestamp":1613654173000},"page":"1132-1143","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Switching the substrate specificity of lysoplasmalogen\u2010specific phospholipase 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