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Secreted proteins of the pathogen govern important steps in the intracellular life cycle including bacterial egress. Among these is the type II secreted PlaA which, together with PlaC and PlaD, belongs to the GDSL phospholipase family found in <jats:italic>L. pneumophila<\/jats:italic>. PlaA shows lysophospholipase A (LPLA) activity which increases after secretion and subsequent processing by the zinc metalloproteinase ProA within a disulfide loop. Activity of PlaA contributes to the destabilization of the LCV in the absence of the type IVB\u2010secreted effector SdhA. We here present the 3D structure of PlaA which shows a typical \u03b1\/\u03b2\u2010hydrolase fold and reveals that the uncleaved disulfide loop forms a lid structure covering the catalytic triad S30\/D278\/H282. This leads to reduction of substrate access before activation; however, the catalytic site gets more accessible when the disulfide loop is processed. After structural modeling, a similar activation process is suggested for the GDSL hydrolase PlaC, but not for PlaD. Furthermore, the size of the PlaA substrate\u2010binding site indicated preference toward phospholipids comprising ~16 carbon fatty acid residues which was verified by lipid hydrolysis, suggesting a molecular ruler mechanism. Indeed, mutational analysis changed the substrate profile with respect to fatty acid chain length. In conclusion, our analysis revealed the structural basis for the regulated activation and substrate preference of PlaA.<\/jats:p>","DOI":"10.1111\/mmi.15201","type":"journal-article","created":{"date-parts":[[2023,12,22]],"date-time":"2023-12-22T08:27:11Z","timestamp":1703233631000},"page":"497-512","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Structure\u2013function relationships underpin disulfide loop cleavage\u2010dependent activation of <i>Legionella pneumophila<\/i> lysophospholipase A <scp>PlaA<\/scp>"],"prefix":"10.1111","volume":"121","author":[{"given":"Miriam","family":"Hiller","sequence":"first","affiliation":[{"name":"Division of Enteropathogenic Bacteria and Legionella (FG11) Robert Koch Institute  Wernigerode Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maurice","family":"Diwo","sequence":"additional","affiliation":[{"name":"Structure and Function of Proteins Helmholtz 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