{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,14]],"date-time":"2026-07-14T19:09:09Z","timestamp":1784056149290,"version":"3.55.0"},"reference-count":32,"publisher":"Wiley","license":[{"start":{"date-parts":[[2026,7,14]],"date-time":"2026-07-14T00:00:00Z","timestamp":1783987200000},"content-version":"am","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,7,14]],"date-time":"2026-07-14T00:00:00Z","timestamp":1783987200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,7,14]],"date-time":"2026-07-14T00:00:00Z","timestamp":1783987200000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"funder":[{"DOI":"10.13039\/501100002241","name":"Japan Science and Technology Agency","doi-asserted-by":"publisher","award":["JPMJCR20E1"],"award-info":[{"award-number":["JPMJCR20E1"]}],"id":[{"id":"10.13039\/501100002241","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100002241","name":"Japan Science and Technology Agency","doi-asserted-by":"publisher","award":["JPMJSP2138"],"award-info":[{"award-number":["JPMJSP2138"]}],"id":[{"id":"10.13039\/501100002241","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["febs.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["The FEBS Journal"],"abstract":"<jats:p>\n                    Carbonic anhydrase (CA) catalyzes the reversible hydration of carbon dioxide (CO\n                    <jats:sub>2<\/jats:sub>\n                    ) to bicarbonate (HCO\n                    <jats:sub>3<\/jats:sub>\n                    <jats:sup>\u2212<\/jats:sup>\n                    ) and plays an essential role in carbon fixation in marine diatoms. Here we report the structural and functional characterization of a novel CA, \u03b8\u2010CA3, from the diatom\n                    <jats:italic>Phaeodactylum tricornutum<\/jats:italic>\n                    , elucidating its physiological role and catalytic mechanism. AlphaFold prediction, sequence alignment, and metal analysis showed that \u03b8\u2010CA3 is a dimeric enzyme, with each monomer composed of two zinc\u2010binding catalytic domains. High\u2010resolution X\u2010ray crystallographic structures of domain 2 of \u03b8\u2010CA3 in the CO\n                    <jats:sub>2<\/jats:sub>\n                    \u2010bound form revealed the detailed substrate binding pattern in the active site. Site\u2010directed mutagenesis showed that Asp49 and Arg117 in the active site are essential for catalysis. Notably, introducing a negative charge near the active\u2010site entrance resulted in a mutant enzyme with markedly increased activity under acidic pH, suggesting that electrostatic modulation of the active\u2010site environment regulates proton transfer and catalysis. Furthermore, we identified an HCO\n                    <jats:sub>3<\/jats:sub>\n                    <jats:sup>\u2212<\/jats:sup>\n                    ion at the dimer interface that contributes to enzyme activation. Collectively, our findings provide fundamental structural insight into how the active\u2010site electrostatic charges and metal environment govern the catalytic efficiency of \u03b8\u2010CA3, offering a new perspective on the molecular basis of carbon fixation in diatoms.\n                  <\/jats:p>","DOI":"10.1111\/febs.70654","type":"journal-article","created":{"date-parts":[[2026,7,14]],"date-time":"2026-07-14T18:48:35Z","timestamp":1784054915000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Structural insights into \u03b8\u2010type carbonic anhydrases 3 and 4: Tuning the directionality of\n                    <scp>\n                      CO\n                      <sub>2<\/sub>\n                    <\/scp>\n                    hydration in a diatom"],"prefix":"10.1111","author":[{"ORCID":"https:\/\/orcid.org\/0009-0000-8868-7086","authenticated-orcid":false,"given":"Hiroto","family":"Negoro","sequence":"first","affiliation":[{"name":"Department of Biotechnology, Graduate School of Engineering The University of Osaka  Japan"},{"name":"Institute for Protein Research The University of Osaka  Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Atsuki","family":"Ohsawa","sequence":"additional","affiliation":[{"name":"Department of Bioscience, School of Biological and Environmental Sciences Kwansei Gakuin University  Sanda Hyogo Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ginga","family":"Shimakawa","sequence":"additional","affiliation":[{"name":"Department of Bioscience, School of Biological and Environmental Sciences Kwansei Gakuin University  Sanda Hyogo Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hideaki","family":"Tanaka","sequence":"additional","affiliation":[{"name":"Institute for Protein Research The University of Osaka  Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yusuke","family":"Matsuda","sequence":"additional","affiliation":[{"name":"Department of Bioscience, School of Biological and Environmental Sciences Kwansei Gakuin University  Sanda Hyogo Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5354-0807","authenticated-orcid":false,"given":"Genji","family":"Kurisu","sequence":"additional","affiliation":[{"name":"Department of Biotechnology, Graduate School of Engineering The University of Osaka  Japan"},{"name":"Institute for Protein Research The University of Osaka  Japan"},{"name":"Institute for Open and Transdisciplinary Research Initiatives The University of Osaka  Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,7,14]]},"reference":[{"key":"e_1_2_10_2_1","doi-asserted-by":"crossref","first-page":"291","DOI":"10.1126\/science.290.5490.291","article-title":"The global carbon cycle: a test of our knowledge of earth as a system","volume":"290","author":"Falkowski P","year":"2000","journal-title":"Science"},{"key":"e_1_2_10_3_1","doi-asserted-by":"crossref","first-page":"237","DOI":"10.1126\/science.281.5374.237","article-title":"Primary production of the biosphere: integrating terrestrial and oceanic components","volume":"281","author":"Field CB","year":"1998","journal-title":"Science"},{"key":"e_1_2_10_4_1","doi-asserted-by":"crossref","first-page":"99","DOI":"10.1146\/annurev.arplant.56.032604.144052","article-title":"CO2 concentrating mechanisms in algae: mechanisms, environmental modulation, and evolution","volume":"56","author":"Giordano M","year":"2005","journal-title":"Annu Rev Plant Biol"},{"key":"e_1_2_10_5_1","doi-asserted-by":"crossref","first-page":"1767","DOI":"10.1073\/pnas.1216234110","article-title":"SLC4 family transporters in a marine diatom directly pump bicarbonate from seawater","volume":"110","author":"Nakajima K","year":"2013","journal-title":"Proc Natl Acad Sci USA"},{"key":"e_1_2_10_6_1","doi-asserted-by":"crossref","first-page":"1432","DOI":"10.1093\/plphys\/kiae137","article-title":"Low\u2010CO2\u2010inducible bestrophins outside the pyrenoid sustain high photosynthetic efficacy in diatoms","volume":"195","author":"Nigishi M","year":"2024","journal-title":"Plant Physiol"},{"key":"e_1_2_10_7_1","doi-asserted-by":"crossref","first-page":"5919","DOI":"10.1016\/j.cell.2024.09.013","article-title":"Diatom pyrenoids are encased in a protein shell that enables efficient CO2 fixation","volume":"187","author":"Shimakawa G","year":"2024","journal-title":"Cell"},{"key":"e_1_2_10_8_1","doi-asserted-by":"crossref","first-page":"105","DOI":"10.1186\/s12915-021-01039-8","article-title":"Characterization of a novel type of carbonic anhydrase that acts without metal cofactors","volume":"19","author":"Hirakawa Y","year":"2021","journal-title":"BMC Biol"},{"key":"e_1_2_10_9_1","doi-asserted-by":"crossref","first-page":"48615","DOI":"10.1074\/jbc.R100045200","article-title":"Carbonic anhydrase: new insights for an ancient enzyme","volume":"276","author":"Tripp BC","year":"2001","journal-title":"J Biol Chem"},{"key":"e_1_2_10_10_1","doi-asserted-by":"crossref","first-page":"817","DOI":"10.1021\/bi802246s","article-title":"The archetype \u03b3\u2010class carbonic anhydrase (cam) contains iron when synthesized in vivo","volume":"48","author":"MacAuley SR","year":"2009","journal-title":"Biochemistry"},{"key":"e_1_2_10_11_1","doi-asserted-by":"crossref","first-page":"1041","DOI":"10.1104\/pp.105.065185","article-title":"Regulation of the expression of intracellular \u03b2\u2010carbonic anhydrase in response to CO2 and light in the marine diatom Phaeodactylum tricornutum","volume":"139","author":"Harada H","year":"2005","journal-title":"Plant Physiol"},{"key":"e_1_2_10_12_1","doi-asserted-by":"crossref","first-page":"207","DOI":"10.1104\/pp.104.058982","article-title":"Localization of soluble \u03b2\u2010carbonic anhydrase in the marine diatom Phaeodactylum tricornutum. Sorting to the chloroplast and cluster formation on the girdle lamellae","volume":"138","author":"Tanaka Y","year":"2005","journal-title":"Plant Physiol"},{"key":"e_1_2_10_13_1","doi-asserted-by":"crossref","first-page":"205","DOI":"10.1007\/s11120-011-9634-4","article-title":"Localization of putative carbonic anhydrases in two marine diatoms, Phaeodactylum tricornutum and Thalassiosira pseudonana","volume":"109","author":"Tachibana M","year":"2011","journal-title":"Photosynth Res"},{"key":"e_1_2_10_14_1","doi-asserted-by":"crossref","first-page":"9828","DOI":"10.1073\/pnas.1603112113","article-title":"Thylakoid luminal \u03b8\u2010carbonic anhydrase critical for growth and photosynthesis in the marine diatom Phaeodactylum\u00a0tricornutum","volume":"113","author":"Kikutani S","year":"2016","journal-title":"Proc Natl Acad Sci USA"},{"key":"e_1_2_10_15_1","doi-asserted-by":"crossref","first-page":"1453","DOI":"10.1093\/pcp\/pcq105","article-title":"Light and low\u2010CO2\u2010dependent LCIBLCIC complex localization in the chloroplast supports the carbon\u2010concentrating mechanism in Chlamydomonas reinhardtii","volume":"51","author":"Yamano T","year":"2010","journal-title":"Plant Cell Physiol"},{"key":"e_1_2_10_16_1","doi-asserted-by":"crossref","first-page":"676","DOI":"10.1107\/S2059798320007202","article-title":"Structural and biochemical characterization of novel carbonic anhydrases from Phaeodactylum tricornutum","volume":"76","author":"Jin S","year":"2020","journal-title":"Acta Crystallogr D Struct Biol"},{"key":"e_1_2_10_17_1","doi-asserted-by":"crossref","first-page":"14716","DOI":"10.1073\/pnas.1616294113","article-title":"Structural insights into the LCIB protein family reveals a new group of \u03b2\u2010carbonic anhydrases","volume":"113","author":"Jin S","year":"2016","journal-title":"Proc Natl Acad Sci USA"},{"key":"e_1_2_10_18_1","doi-asserted-by":"crossref","first-page":"2505","DOI":"10.1073\/pnas.70.9.2505","article-title":"The catalytic mechanism of carbonic anhydrase","volume":"70","author":"Lindskog S","year":"1973","journal-title":"Proc Natl Acad Sci USA"},{"key":"e_1_2_10_19_1","doi-asserted-by":"crossref","first-page":"362","DOI":"10.1016\/j.bbapap.2009.08.002","article-title":"Structure and catalytic mechanism of the \u03b2\u2010carbonic anhydrases","volume":"1804","author":"Rowlett RS","year":"2010","journal-title":"Biochim Biophys Acta Proteins Proteom"},{"key":"e_1_2_10_20_1","doi-asserted-by":"crossref","first-page":"611","DOI":"10.1046\/j.1365-3040.2001.00702.x","article-title":"Regulation of the induction of bicarbonate uptake by dissolved CO2 in the marine diatom, Phaeodactylum tricornutum","volume":"24","author":"Matsuda Y","year":"2001","journal-title":"Plant Cell Environ"},{"key":"e_1_2_10_21_1","doi-asserted-by":"crossref","first-page":"67","DOI":"10.1007\/s10126-025-10446-9","article-title":"Overexpression of plastid acetyl\u2010CoA carboxylase confers stress tolerances with increased levels of unsaturated fatty acids in the marine diatom Phaeodactylum tricornutum","volume":"27","author":"Toyoshima M","year":"2025","journal-title":"Mar Biotechnol"},{"key":"e_1_2_10_22_1","doi-asserted-by":"crossref","first-page":"W462","DOI":"10.1093\/nar\/gkq319","article-title":"ZiFiT (zinc finger Targeter): an updated zinc finger engineering tool","volume":"38","author":"Sander JD","year":"2010","journal-title":"Nucleic Acids Res"},{"key":"e_1_2_10_23_1","doi-asserted-by":"crossref","first-page":"4014","DOI":"10.1093\/bioinformatics\/btv537","article-title":"Cas\u2010designer: a web\u2010based tool for choice of CRISPR\u2010Cas9 target sites","volume":"31","author":"Park J","year":"2015","journal-title":"Bioinformatics"},{"key":"e_1_2_10_24_1","doi-asserted-by":"crossref","first-page":"347","DOI":"10.1038\/nbt.2842","article-title":"CRISPR\u2010Cas systems for editing, regulating and targeting genomes","volume":"32","author":"Sander JD","year":"2014","journal-title":"Nat 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anhydrase","volume":"176","author":"Wilbur KM","year":"1948","journal-title":"J Biol Chem"},{"key":"e_1_2_10_29_1","doi-asserted-by":"crossref","first-page":"125","DOI":"10.1107\/S0907444909047337","article-title":"XDS","volume":"66","author":"Kabsch W","year":"2010","journal-title":"Acta Crystallogr D"},{"key":"e_1_2_10_30_1","doi-asserted-by":"crossref","first-page":"458","DOI":"10.1107\/S0907444905001617","article-title":"Likelihood\u2010enhanced fast translation functions","volume":"61","author":"McCoy AJ","year":"2005","journal-title":"Acta Crystallogr D Biol Crystallogr"},{"key":"e_1_2_10_31_1","doi-asserted-by":"crossref","first-page":"61","DOI":"10.1107\/S090744490705024X","article-title":"Iterative model building, structure refinement and density modification with the PHENIX AutoBuild wizard","volume":"64","author":"Terwilliger TC","year":"2008","journal-title":"Acta Crystallogr 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Journal"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/febs.onlinelibrary.wiley.com\/doi\/pdf\/10.1111\/febs.70654","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/febs.onlinelibrary.wiley.com\/doi\/full-xml\/10.1111\/febs.70654","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/febs.onlinelibrary.wiley.com\/doi\/pdf\/10.1111\/febs.70654","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/febs.onlinelibrary.wiley.com\/doi\/pdf\/10.1111\/febs.70654","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,7,14]],"date-time":"2026-07-14T18:48:40Z","timestamp":1784054920000},"score":1,"resource":{"primary":{"URL":"https:\/\/febs.onlinelibrary.wiley.com\/doi\/10.1111\/febs.70654"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,7,14]]},"references-count":32,"alternative-id":["10.1111\/febs.70654"],"URL":"https:\/\/doi.org\/10.1111\/febs.70654","archive":["Portico"],"relation":{},"ISSN":["1742-464X","1742-4658"],"issn-type":[{"value":"1742-464X","type":"print"},{"value":"1742-4658","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,7,14]]},"assertion":[{"value":"2026-01-27","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-07-01","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-07-14","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"febs.70654"}}