{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,1]],"date-time":"2026-06-01T18:46:13Z","timestamp":1780339573519,"version":"3.54.1"},"reference-count":47,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2025,2,27]],"date-time":"2025-02-27T00:00:00Z","timestamp":1740614400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Sao Paulo Research Foundation (FAPESP)","award":["2020\/08615-8"],"award-info":[{"award-number":["2020\/08615-8"]}]},{"name":"Sao Paulo Research Foundation (FAPESP)","award":["2024\/00876-8"],"award-info":[{"award-number":["2024\/00876-8"]}]},{"name":"Sao Paulo Research Foundation (FAPESP)","award":["2024\/13327-2"],"award-info":[{"award-number":["2024\/13327-2"]}]},{"name":"Sao Paulo Research Foundation (FAPESP)","award":["2024\/16184-8"],"award-info":[{"award-number":["2024\/16184-8"]}]},{"name":"Sao Paulo Research Foundation (FAPESP)","award":["2024\/01956-5"],"award-info":[{"award-number":["2024\/01956-5"]}]},{"name":"Sao Paulo Research Foundation (FAPESP)","award":["309940\/2019-2"],"award-info":[{"award-number":["309940\/2019-2"]}]},{"name":"Sao Paulo Research Foundation (FAPESP)","award":["401415\/2023-6"],"award-info":[{"award-number":["401415\/2023-6"]}]},{"name":"Brazilian Council for Scientific and Technological Development (CNPq)","award":["2020\/08615-8"],"award-info":[{"award-number":["2020\/08615-8"]}]},{"name":"Brazilian Council for Scientific and Technological Development (CNPq)","award":["2024\/00876-8"],"award-info":[{"award-number":["2024\/00876-8"]}]},{"name":"Brazilian Council for Scientific and Technological Development (CNPq)","award":["2024\/13327-2"],"award-info":[{"award-number":["2024\/13327-2"]}]},{"name":"Brazilian Council for Scientific and Technological Development (CNPq)","award":["2024\/16184-8"],"award-info":[{"award-number":["2024\/16184-8"]}]},{"name":"Brazilian Council for Scientific and Technological Development (CNPq)","award":["2024\/01956-5"],"award-info":[{"award-number":["2024\/01956-5"]}]},{"name":"Brazilian Council for Scientific and Technological Development (CNPq)","award":["309940\/2019-2"],"award-info":[{"award-number":["309940\/2019-2"]}]},{"name":"Brazilian Council for Scientific and Technological Development (CNPq)","award":["401415\/2023-6"],"award-info":[{"award-number":["401415\/2023-6"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Toxins"],"abstract":"<jats:p>Phospholipase D (PLD) enzymes from Loxosceles spider venom mediate envenomation pathology by cleaving phospholipid headgroups. We revisited the crystal structure of Loxosceles intermedia PLD (PDB: 3RLH) to evaluate two alternative mechanisms\u2014covalent and non-covalent\u2014for headgroup cleavage. The covalent mechanism involves a nucleophilic attack on the substrate\u2019s P atom by catalytic histidine, forming a phosphohistidine intermediate. It was originally suggested that this intermediate hydrolyzes, leading to linear phosphates. The non-covalent mechanism relies on the substrate\u2019s hydroxyl group performing an intramolecular attack on the P atom, thereby generating a cyclic phosphate. Structural refinement of the crystal structure revealed a cyclic phosphate bound at the active site, replacing previously assigned PEG molecules. This cyclic product, stabilized by His12, His47, and Mg2+, provides structural evidence that supports phosphate cyclization. The results of computational analyses, including molecular dynamics and quantum mechanics\/molecular mechanics simulations, further support the non-covalent mechanism as the energetically preferred pathway, with a significantly lower activation barrier. Our findings highlight the role of substrate orientation and of the catalytic His residues in transphosphatidylation, advancing our understanding of PLD enzymology and providing insights for the design of inhibitors against Loxosceles envenomation.<\/jats:p>","DOI":"10.3390\/toxins17030111","type":"journal-article","created":{"date-parts":[[2025,2,27]],"date-time":"2025-02-27T03:37:57Z","timestamp":1740627477000},"page":"111","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Structural and Energetic Evidence Supports the Non-Covalent Phosphate Cyclization by the Class II Phospholipase D from Loxosceles intermedia"],"prefix":"10.3390","volume":"17","author":[{"given":"Carolina","family":"Gismene","sequence":"first","affiliation":[{"name":"Biological Structures Group, Multiuser Center for Biomolecular Innovation (CMIB), S\u00e3o Paulo State University\u2014UNESP, S\u00e3o Jos\u00e9 do Rio Preto CEP 15054-000, SP, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jos\u00e9 Fernando","family":"Ruggiero Bachega","sequence":"additional","affiliation":[{"name":"Graduate Program in Molecular and Cellular Biology, Federal University of Rio Grande do Sul (UFRGS), Porto Alegre CEP 90050-170, RS, Brazil"},{"name":"Departament of Farmacosciences, Federal University of Health Sciences of Porto Alegre (UFCSPA), Porto Ale-gre, CEP 90050-170, RS, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daniel Z.","family":"Doherty","sequence":"additional","affiliation":[{"name":"Biological Structures Group, Multiuser Center for Biomolecular Innovation (CMIB), S\u00e3o Paulo State University\u2014UNESP, S\u00e3o Jos\u00e9 do Rio Preto CEP 15054-000, SP, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Silvio Sanches","family":"Veiga","sequence":"additional","affiliation":[{"name":"Department of Cell Biology, Federal University of Paran\u00e1 (UFPR), Curitiba CEP 81531-980, PR, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2460-1145","authenticated-orcid":false,"given":"Raghuvir K.","family":"Arni","sequence":"additional","affiliation":[{"name":"Biological Structures Group, Multiuser Center for Biomolecular Innovation (CMIB), S\u00e3o Paulo State University\u2014UNESP, S\u00e3o Jos\u00e9 do Rio Preto CEP 15054-000, SP, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4770-8677","authenticated-orcid":false,"given":"Jorge Enrique","family":"Hern\u00e1ndez Gonz\u00e1lez","sequence":"additional","affiliation":[{"name":"Biological Structures Group, Multiuser Center for Biomolecular Innovation (CMIB), S\u00e3o Paulo State University\u2014UNESP, S\u00e3o Jos\u00e9 do Rio Preto CEP 15054-000, SP, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,2,27]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"6064","DOI":"10.1021\/cr200296t","article-title":"Phospholipase D: Enzymology, Functionality, and Chemical Modulation","volume":"111","author":"Selvy","year":"2011","journal-title":"Chem. 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