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This study investigates whether this species has evolved mechanisms to store Cd complexed with metallothioneins (MTs), proteins that bind heavy metal ions via cysteinyl (Cys)\u2013thiolate bonds, how these MTs originated, and how similar mechanisms are present in other fungal species. Size exclusion chromatography revealed that a substantial fraction of Cd in <jats:italic>A. crocodilinus<\/jats:italic> sporocarps was sequestered in a 3.4\u00a0kDa complex containing Cys-rich peptides. Screening a sporocarp cDNA expression library in a Cd-sensitive <jats:italic>Saccharomyces cerevisiae<\/jats:italic> strain identified two MT transcripts, Ac<jats:italic>MT1<\/jats:italic> and Ac<jats:italic>MT2<\/jats:italic>, encoding 49-amino acid (AA) AcMT1 with 10 Cys and 32-AA AcMT2 with 7 Cys. The presence of AcMT2 in the 3.4\u00a0kDa Cd\u2013peptide complex isolated from sporocarp was confirmed by mass spectrometry. In mycelial isolates exposed to heavy metals, Ac<jats:italic>MT1<\/jats:italic> was more strongly upregulated, while Ac<jats:italic>MT2<\/jats:italic> was more expressed under normal conditions. Sequence comparisons revealed that Ac<jats:italic>MT2<\/jats:italic> is closer to the ancestral gene, whereas Ac<jats:italic>MT1<\/jats:italic> is a more recent duplicate. Combined bioinformatic and functional evidence supports Ac<jats:italic>MT2<\/jats:italic> as a constitutively expressed MT involved in Cd binding in the sporocarp, while Ac<jats:italic>MT1<\/jats:italic>, though more inducible in mycelia and more protective in yeast, appears to serve a transient detoxification role. Moreover, the gene duplication and domain rearrangement mechanism underlying this MT diversification was also identified in other <jats:italic>Agaricales<\/jats:italic> and <jats:italic>Boletales<\/jats:italic> species.<\/jats:p>","DOI":"10.1007\/s10534-025-00721-6","type":"journal-article","created":{"date-parts":[[2025,7,18]],"date-time":"2025-07-18T09:24:24Z","timestamp":1752830664000},"page":"1569-1585","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Identification of two metallothioneins in Agaricus crocodilinus reveals gene duplication and domain expansion, a pattern conserved across fungal species"],"prefix":"10.1007","volume":"38","author":[{"given":"Jan","family":"S\u00e1ck\u00fd","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anna","family":"Chaloupeck\u00e1","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ji\u0159\u00ed","family":"\u0160antr\u016f\u010dek","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anton\u00edn","family":"Ka\u0148a","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tereza","family":"Leonhardt","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jan","family":"Borovi\u010dka","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pavel","family":"Kotrba","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,7,18]]},"reference":[{"issue":"22","key":"721_CR1","doi-asserted-by":"publisher","first-page":"7499","DOI":"10.1128\/jb.186.22.7499-7507.2004","volume":"186","author":"A Anton","year":"2004","unstructured":"Anton A, Weltrowski A, Haney CJ, Franke S, Grass G, Rensing C, Nies DH (2004) Characteristics of zinc transport by two bacterial cation diffusion facilitators from Ralstonia metallidurans CH34 and Escherichia coli. 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