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In this work, directed evolution was used to expand the substrate specificity of <jats:italic>P. aerophilum<\/jats:italic> McoP for organic substrates. Six rounds of error-prone PCR and DNA shuffling followed by high-throughput screening lead to the identification of a hit variant with a 220-fold increased efficiency (<jats:italic>k<\/jats:italic><jats:sub>cat<\/jats:sub>\/K<jats:sub>m<\/jats:sub>) than the wild-type for 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) without compromising its intrinsic activity for metal ions. The analysis of the X-ray crystal structure reveals four proximal mutations close to the T1Cu active site. One of these mutations is within the 23-residues loop that occludes this site, a distinctive feature of prokaryotic MCOs. The increased flexibility of this loop results in an enlarged tunnel and one additional pocket that facilitates bulky substrate-enzyme interactions. These findings underscore the synergy between mutations that modulate the dynamics of the active-site loop enabling enhanced catalytic function. This study highlights the potential of targeting loops close to the T1Cu for engineering improvements suitable for biotechnological applications.<\/jats:p>\n                <jats:p><jats:bold>Graphical Abstract<\/jats:bold><\/jats:p>","DOI":"10.1007\/s00775-023-02040-y","type":"journal-article","created":{"date-parts":[[2024,1,16]],"date-time":"2024-01-16T09:02:18Z","timestamp":1705395738000},"page":"339-351","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":16,"title":["Flexible active-site loops fine-tune substrate specificity of hyperthermophilic metallo-oxidases"],"prefix":"10.1007","volume":"29","author":[{"given":"V\u00e2nia","family":"Brissos","sequence":"first","affiliation":[]},{"given":"Patr\u00edcia T.","family":"Borges","sequence":"additional","affiliation":[]},{"given":"Ferran","family":"Sancho","sequence":"additional","affiliation":[]},{"given":"Maria F\u00e1tima","family":"Lucas","sequence":"additional","affiliation":[]},{"given":"Carlos","family":"Fraz\u00e3o","sequence":"additional","affiliation":[]},{"given":"Felipe","family":"Conzuelo","sequence":"additional","affiliation":[]},{"given":"L\u00edgia O.","family":"Martins","sequence":"additional","affiliation":[]}],"member":"297","published-online":{"date-parts":[[2024,1,16]]},"reference":[{"key":"2040_CR1","doi-asserted-by":"publisher","first-page":"2563","DOI":"10.1021\/cr950046o","volume":"96","author":"EI Solomon","year":"1996","unstructured":"Solomon EI, Sundaram UM, Machonkin TE (1996) Multicopper oxidases and oxygenases. 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