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Here, we report the discovery and characterization of a new FAD-dependent D-lactate dehydrogenase (PdG-D-LDH) from the gut microbiome of the isopod\n                      <jats:italic toggle=\"yes\">Porcellio dilatatus<\/jats:italic>\n                      . The enzyme was identified through\n                      <jats:italic toggle=\"yes\">in silico<\/jats:italic>\n                      screening using BLAST and AlphaFold3 and functionally characterized as a homodimeric, thermoactive, and thermostable protein, demonstrating the robustness required for biotechnological applications. PdG-D-LDH exhibits a strong catalytic preference toward D-lactate and preferentially reduces quinones over cytochrome\n                      <jats:italic toggle=\"yes\">c<\/jats:italic>\n                      or molecular oxygen. X-ray crystallography revealed a VAO\/PCMH-like fold with a solvent-accessible active site that harbors both a FAD cofactor and an Fe(II) ion. Molecular docking studies provided insights into the structural determinants of its stereoselective substrate recognition. Under mild conditions, the enzyme catalyzed the oxidation of D-lactate to pyruvate with a 90% yield after 24 h of reaction, using molecular oxygen as the electron acceptor.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>This study illustrates how metagenomics, structural biology, and computational tools can jointly drive the discovery of new enzymes with valuable biotechnological applications aligned with circular economic principles. The newly identified D-lactate dehydrogenase, PdG-D-LDH, exhibits thermostability, stereoselectivity, and high catalytic efficiency, providing new insights into the structure-function relationships of lactate-metabolizing enzymes.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/aem.01480-25","type":"journal-article","created":{"date-parts":[[2025,11,13]],"date-time":"2025-11-13T19:02:39Z","timestamp":1763060559000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Shotgun metagenomic mining reveals a new FAD-dependent D-lactate dehydrogenase in an isopod gut microbiome"],"prefix":"10.1128","volume":"91","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6760-367X","authenticated-orcid":false,"given":"Catarina","family":"Coelho","sequence":"first","affiliation":[{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier, Universidade Nova de Lisboa","place":["Oeiras, 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