{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,2]],"date-time":"2026-10-02T13:40:45Z","timestamp":1790948445737,"version":"4.1.0"},"reference-count":45,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2025,6,29]],"date-time":"2025-06-29T00:00:00Z","timestamp":1751155200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Science Centre (NCN, Poland)","award":["2020\/37\/B\/NZ1\/03250"],"award-info":[{"award-number":["2020\/37\/B\/NZ1\/03250"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomolecules"],"abstract":"<jats:p>The ReAV enzyme from Rhizobium etli, a representative of Class 3 L-asparaginases, is sequentially and structurally different from other known L-asparaginases. This distinctiveness makes ReAV a candidate for novel antileukemic therapies. ReAV is a homodimeric protein, with each subunit containing a highly specific zinc-binding site created by two cysteines, a lysine, and a water molecule. Two Ser-Lys tandems (Ser48-Lys51, Ser80-Lys263) are located in the close proximity of the metal binding site, with Ser48 hypothesized to be the catalytic nucleophile. To further investigate the catalytic process of ReAV, site-directed mutagenesis was employed to introduce alanine substitutions at residues from the Ser-Lys tandems and at Arg47, located near the Ser48-Lys51 tandem. These mutational studies, along with enzymatic assays and X-ray structure determinations, demonstrated that substitution of each of these highly conserved residues abolished the catalytic activity, confirming their essential role in enzyme mechanism.<\/jats:p>","DOI":"10.3390\/biom15070944","type":"journal-article","created":{"date-parts":[[2025,7,2]],"date-time":"2025-07-02T07:55:43Z","timestamp":1751442943000},"page":"944","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Probing the Active Site of Class 3 L-Asparaginase by Mutagenesis: Mutations of the Ser-Lys Tandems of ReAV"],"prefix":"10.3390","volume":"15","author":[{"given":"Kinga","family":"Pokrywka","sequence":"first","affiliation":[{"name":"Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12\/14, 61-704 Poznan, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7605-1563","authenticated-orcid":false,"given":"Marta","family":"Grzechowiak","sequence":"additional","affiliation":[{"name":"Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12\/14, 61-704 Poznan, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1153-6380","authenticated-orcid":false,"given":"Joanna","family":"Sliwiak","sequence":"additional","affiliation":[{"name":"Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12\/14, 61-704 Poznan, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Paulina","family":"Worsztynowicz","sequence":"additional","affiliation":[{"name":"Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12\/14, 61-704 Poznan, Poland"},{"name":"Department of Biotechnology and Food Microbiology, Poznan University of Life Science, Wojska Polskiego 48, 60-627 Poznan, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7345-4527","authenticated-orcid":false,"given":"Joanna I.","family":"Loch","sequence":"additional","affiliation":[{"name":"Department of Crystal Chemistry and Crystal Physics, Faculty of Chemistry, Jagiellonian University, Gronostajowa 2, 30-387 Krakow, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4415-3100","authenticated-orcid":false,"given":"Milosz","family":"Ruszkowski","sequence":"additional","affiliation":[{"name":"Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12\/14, 61-704 Poznan, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3941-9947","authenticated-orcid":false,"given":"Miroslaw","family":"Gilski","sequence":"additional","affiliation":[{"name":"Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12\/14, 61-704 Poznan, Poland"},{"name":"Department of Crystallography, Faculty of Chemistry, Adam Mickiewicz University, Uniwersytetu Poznanskiego 8, 61-614 Poznan, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1587-6489","authenticated-orcid":false,"given":"Mariusz","family":"Jaskolski","sequence":"additional","affiliation":[{"name":"Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12\/14, 61-704 Poznan, Poland"},{"name":"Department of Crystallography, Faculty of Chemistry, Adam Mickiewicz University, Uniwersytetu Poznanskiego 8, 61-614 Poznan, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,6,29]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"514","DOI":"10.1107\/S2052252521006011","article-title":"Structural and biophysical aspects of L-asparaginases: A growing family with amazing diversity","volume":"8","author":"Loch","year":"2021","journal-title":"IUCrJ"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"503","DOI":"10.1002\/bab.2127","article-title":"Structural and functional diversity of asparaginases: Overview and recommendations for a revised nomenclature","volume":"69","author":"Doonan","year":"2022","journal-title":"Biotechnol. 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