{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T07:16:04Z","timestamp":1788419764535,"version":"build-2803163510"},"reference-count":58,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T00:00:00Z","timestamp":1788393600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Microbiol."],"abstract":"<jats:p>[NiFe] hydrogenases reversibly catalyze hydrogen oxidation and proton reduction at a Ni\u2013Fe active site coordinated by four cysteine (Cys) residues in two CXXC motifs within the catalytic large subunit. A subset of these enzymes contains selenocysteine (Sec) in place of Cys in the C-terminal CXXC motif, forming [NiFeSe] hydrogenases with enhanced oxygen tolerance and hydrogen production activity. However, such Sec-containing variants have been reported only in Groups 1 and 3 [NiFe] hydrogenases. Here, we present a comprehensive dataset of Group 4 [NiFeSe] hydrogenases based on a genomic survey combined with UGA stop-codon read-through analysis. The dataset comprises 47 non-redundant protein sequences from nine bacterial phyla. Sec substitutions were exclusively identified in the N-terminal CXXC motif, including 27 CXXU-, 4 UXXC-, and 16 UXXU-type sequences, indicating the emergence of non-canonical Sec-containing motifs in Group 4. Phylogenetic analysis revealed a sporadic distribution across three distinct lineages, indicating at least three independent origins of Sec-containing variants from Cys-type ancestors. Such substitutions were found across diverse ecosystems. Genomic context analysis further suggests that these Sec-containing enzymes form energy-converting complexes, similar to those formed by other Group 4 enzymes. The Sec-containing variants co-occur with Sec biosynthesis genes and a conserved guanine residue in the apical loop of Sec insertion sequence elements. These findings provide new insights into the evolution of Sec utilization in [NiFe] hydrogenases.<\/jats:p>","DOI":"10.3389\/fmicb.2026.1925269","type":"journal-article","created":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T06:41:18Z","timestamp":1788417678000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["In silico discovery of selenocysteine-containing variants of Group 4 [NiFe] hydrogenases"],"prefix":"10.3389","volume":"17","author":[{"given":"Masamitsu","family":"Takano","sequence":"first","affiliation":[{"name":"College of Life Sciences, Ritsumeikan University","place":["Kusatsu, Shiga, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Masao","family":"Inoue","sequence":"additional","affiliation":[{"name":"College of Life Sciences, Ritsumeikan University","place":["Kusatsu, Shiga, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Riku","family":"Aono","sequence":"additional","affiliation":[{"name":"College of Life Sciences, Ritsumeikan University","place":["Kusatsu, Shiga, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anna","family":"Ochi","sequence":"additional","affiliation":[{"name":"College of Life Sciences, Ritsumeikan University","place":["Kusatsu, Shiga, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hisaaki","family":"Mihara","sequence":"additional","affiliation":[{"name":"College of Life Sciences, Ritsumeikan University","place":["Kusatsu, Shiga, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,9,3]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"493","DOI":"10.1038\/s41586-024-07487-w","article-title":"Accurate structure prediction of biomolecular interactions with AlphaFold 3","volume":"630","author":"Abramson","year":"2024","journal-title":"Nature"},{"key":"B2","doi-asserted-by":"publisher","first-page":"948","DOI":"10.1002\/ejic.201001127","article-title":"Nickel\u2013iron\u2013selenium hydrogenases \u2013 an overview","volume":"2011","author":"Baltazar","year":"2011","journal-title":"Eur. 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