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Hpo, a non-heme Fe(II)-dependent dioxygenase from\n                      <jats:italic toggle=\"yes\">Pseudomonas putida<\/jats:italic>\n                      S16, catalyzes ring cleavage of 2,5-DHP, yet its structure has remained uncharacterized. We determined the crystal structure of Hpo at 2.56 \u00c5 resolution, revealing a catalytic Fe center coordinated by His257, His310, and Asp312 in a conserved 2-His-1-carboxylate motif, with Ser294 positioned directly below the iron and essential for iron binding. The substrate-binding pocket, formed primarily by Phe94, Phe97, Phe186, Phe188, and Arg285, incorporates potential hydrogen-bonding interactions from Glu169 and His181. Hpo employs a distinctive Phe94-Leu95-Leu96-Phe97 motif for substrate recognition, contrasting with the Met102-Leu103-Leu104-His105 motif in the isoenzyme NicX. Molecular dynamics simulations spanning 200 ns demonstrated that Phe94 and Phe97 mutations destabilized substrate binding, confirming their role in substrate recognition, and explaining the corresponding loss of catalytic activity. These findings reveal the active-site architecture and substrate engagement mechanism of Hpo, advancing our understanding of pyridine ring-cleaving non-heme Fe(II)-dependent dioxygenases.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>Pyridine compounds are common environmental contaminants that pose risks to human health. The microbial degradation of pyridine compounds generates toxic intermediates, including 2,5-dihydroxypyridine (2,5-DHP), which require enzymatic detoxification. Prior to this study, only one 2,5-DHP dioxygenase had been structurally characterized, leaving the structural basis of this rare enzyme subgroup unresolved. By determining the Hpo crystal structure and identifying residues critical for iron coordination and catalysis, this work addresses a significant knowledge gap and establishes a structural framework for understanding pyridine ring-cleaving non-heme Fe(II)-dependent dioxygenases and may also provide an enzymatic perspective on the removal of pyridine contaminants from the environment.<\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/aem.01467-26","type":"journal-article","created":{"date-parts":[[2026,10,14]],"date-time":"2026-10-14T15:10:31Z","timestamp":1791990631000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Structural and catalytic characterization of the 2,5-dihydroxypyridine ring-cleaving dioxygenase Hpo from\n                    <i>Pseudomonas putida<\/i>\n                    S16"],"prefix":"10.1128","author":[{"given":"Zhenyu","family":"Cheng","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/04t3q5q03","id-type":"ROR","asserted-by":"publisher"}],"name":"State Key Laboratory of Microbial Metabolism and School of Life Sciences & Biotechnology, Shanghai Jiao Tong University","place":["Shanghai, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xu","family":"Han","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/042pyga86","id-type":"ROR","asserted-by":"publisher"}],"name":"National Engineering Research Center of Industrial Enzyme, National Technology Innovation Center for Synthetic Biology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences","place":["Tianjin, 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