{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,21]],"date-time":"2026-02-21T05:24:56Z","timestamp":1771651496241,"version":"3.50.1"},"reference-count":37,"publisher":"MDPI AG","issue":"13","license":[{"start":{"date-parts":[[2019,6,28]],"date-time":"2019-06-28T00:00:00Z","timestamp":1561680000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100002261","name":"Russian Foundation for Basic Research","doi-asserted-by":"publisher","award":["18-04-00270\u0430"],"award-info":[{"award-number":["18-04-00270\u0430"]}],"id":[{"id":"10.13039\/501100002261","id-type":"DOI","asserted-by":"publisher"}]},{"name":"RAS Program for Molecular and Cell Biology and Post-genomic Technologies","award":["\u0410\u0410\u0410\u0410-\u041019-119012890117-0"],"award-info":[{"award-number":["\u0410\u0410\u0410\u0410-\u041019-119012890117-0"]}]},{"DOI":"10.13039\/501100006769","name":"Russian Science Foundation","doi-asserted-by":"publisher","award":["17-14-01207"],"award-info":[{"award-number":["17-14-01207"]}],"id":[{"id":"10.13039\/501100006769","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Laccases (EC 1.10.3.2) are multicopper oxidoreductases acting on diphenols and related substances. Laccases are highly important for biotechnology and environmental remediation. These enzymes contain mononuclear one T2 copper ion and two T3 copper ions (Cu3\u03b1 and Cu3\u03b2), which form the so-called trinuclear center (TNC). Along with the typical three-domain laccases Bacteria produce two-domain (2D) enzymes, which are active at neutral and basic pH, thermostable, and resistant to inhibitors. In this work we present the comparative analysis of crystal structures and catalytic properties of recombinant 2D laccase from Streptomyces griseoflavus Ac-993 (SgfSL) and its four mutant forms with replacements of two amino acid residues, located at the narrowing of the presumable T3-solvent tunnels. We obtained inactive enzymes with substitutions of His165, with Phe, and Ile170 with Ala or Phe. His165Ala variant was more active than the wild type. We suggest that His165 is a \u201cgateway\u201d at the O2-tunnel leading from solvent to the Cu3\u03b2 of the enzyme. The side chain of Ile170 could be indirectly involved in the coordination of copper ions at the T3 center by maintaining the position of the imidazole ring of His157 that belongs to the first coordination sphere of Cu3\u03b1.<\/jats:p>","DOI":"10.3390\/ijms20133184","type":"journal-article","created":{"date-parts":[[2019,6,28]],"date-time":"2019-06-28T11:20:26Z","timestamp":1561720826000},"page":"3184","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":27,"title":["Investigations of Accessibility of T2\/T3 Copper Center of Two-Domain Laccase from Streptomyces griseoflavus Ac-993"],"prefix":"10.3390","volume":"20","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-1016-5936","authenticated-orcid":false,"given":"Azat","family":"Gabdulkhakov","sequence":"first","affiliation":[{"name":"Institute of Protein Research RAS, Institutskaya 4, Pushchino, Moscow 142290, Russia"}]},{"given":"Ilya","family":"Kolyadenko","sequence":"additional","affiliation":[{"name":"Institute of Protein Research RAS, Institutskaya 4, Pushchino, Moscow 142290, Russia"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9937-8841","authenticated-orcid":false,"given":"Olga","family":"Kostareva","sequence":"additional","affiliation":[{"name":"Institute of Protein Research RAS, Institutskaya 4, Pushchino, Moscow 142290, Russia"}]},{"given":"Alisa","family":"Mikhaylina","sequence":"additional","affiliation":[{"name":"Institute of Protein Research RAS, Institutskaya 4, Pushchino, Moscow 142290, Russia"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0938-152X","authenticated-orcid":false,"given":"Paulo","family":"Oliveira","sequence":"additional","affiliation":[{"name":"i3S\u2014Instituto de Investiga\u00e7\u00e3o e Inova\u00e7\u00e3o em Sa\u00fade, Universidade do Porto, 4200-135 Porto, Portugal"},{"name":"IBMC\u2014Instituto de Biologia Molecular e Celular, Universidade do Porto, 4200-135 Porto, Portugal"}]},{"given":"Paula","family":"Tamagnini","sequence":"additional","affiliation":[{"name":"i3S\u2014Instituto de Investiga\u00e7\u00e3o e Inova\u00e7\u00e3o em Sa\u00fade, Universidade do Porto, 4200-135 Porto, Portugal"},{"name":"IBMC\u2014Instituto de Biologia Molecular e Celular, Universidade do Porto, 4200-135 Porto, Portugal"},{"name":"Departamento de Biologia, Faculdade de Ci\u00eancias da Universidade do Porto, 4169-007 Porto, Portugal"}]},{"given":"Alexander","family":"Lisov","sequence":"additional","affiliation":[{"name":"G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino 142292, Moscow Region, Russia"}]},{"given":"Svetlana","family":"Tishchenko","sequence":"additional","affiliation":[{"name":"Institute of Protein Research RAS, Institutskaya 4, Pushchino, Moscow 142290, Russia"}]}],"member":"1968","published-online":{"date-parts":[[2019,6,28]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"10327","DOI":"10.1007\/s00253-018-9404-8","article-title":"Bifunctional in vivo role of laccase exploited in multiple biotechnological applications","volume":"102","author":"Sharma","year":"2018","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"2563","DOI":"10.1021\/cr950046o","article-title":"Multicopper Oxidases and Oxygenases","volume":"96","author":"Solomon","year":"1996","journal-title":"Chem. 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