{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,27]],"date-time":"2026-04-27T05:08:31Z","timestamp":1777266511287,"version":"3.51.4"},"reference-count":18,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2022,5,25]],"date-time":"2022-05-25T00:00:00Z","timestamp":1653436800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Associazione Gian Franco Lupo\u2014Un sorriso alla vita\u2014ONLUS"},{"name":"Italian Ministry of Education, University and Research (MIUR)"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Bacterial L-asparaginases are amidohydrolases (EC 3.5.1.1) capable of deaminating L-asparagine and, with reduced efficiency, L-glutamine. Interest in the study of L-asparaginases is driven by their use as biodrugs for the treatment of acute lymphoblastic leukemia. Here, we report for the first time the description of the molecular structure of type II asparaginase from Escherichia coli in complex with its secondary product, L-glutamate. To obtain high-quality crystals, we took advantage of the N24S variant, which has structural and functional features similar to the wild-type enzyme, but improved stability, and which yields more ordered crystals. Analysis of the structure of the N24S-L\u2013glutamate complex (N24S\u2013GLU) and comparison with its apo and L-aspartate-bound form confirmed that the enzyme-reduced catalytic efficiency in the presence of L-glutamine is due to L-glutamine misfitting into the enzyme-binding pocket, which causes a local change in the catalytic center geometry. Moreover, a tight interaction between the two protomers that form the enzyme active site limits the capability of L-glutamine to fit into (and to exit from) the binding pocket of E. coli L-asparaginase, explaining why the enzyme has lower glutaminolytic activity compared to other enzymes of the same family, in particular the Erwinia chrysanthemi one.<\/jats:p>","DOI":"10.3390\/ijms23115942","type":"journal-article","created":{"date-parts":[[2022,5,26]],"date-time":"2022-05-26T08:50:22Z","timestamp":1653555022000},"page":"5942","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":8,"title":["Structural Aspects of E. coli Type II Asparaginase in Complex with Its Secondary Product L-Glutamate"],"prefix":"10.3390","volume":"23","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9075-8143","authenticated-orcid":false,"given":"Maristella","family":"Maggi","sequence":"first","affiliation":[{"name":"Unit of Immunology and General Pathology, Department of Molecular Medicine, University of Pavia, 27100 Pavia, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5790-1833","authenticated-orcid":false,"given":"Claudia","family":"Scotti","sequence":"additional","affiliation":[{"name":"Unit of Immunology and General Pathology, Department of Molecular Medicine, University of Pavia, 27100 Pavia, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2022,5,25]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1069","DOI":"10.1007\/s00253-014-6271-9","article-title":"Recent Research Progress on Microbial L-Asparaginases","volume":"99","author":"Zuo","year":"2014","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"627","DOI":"10.18388\/abp.2006_3291","article-title":"Structural Aspects of L-Asparaginases, Their Friends and Relations","volume":"53","author":"Michalska","year":"2006","journal-title":"Acta Biochim. Pol."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Maggi, M., Chiarelli, L.R., Valentini, G., and Scotti, C. (2015). Engineering of Helicobacter Pylori L-Asparaginase: Characterization of Two Functionally Distinct Groups of Mutants. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0117025"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1474","DOI":"10.1073\/pnas.90.4.1474","article-title":"Crystal Structure of Escherichia Coli L-Asparaginase, an Enzyme Used in Cancer Therapy","volume":"90","author":"Swain","year":"1993","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"2471","DOI":"10.1016\/j.jmb.2014.04.016","article-title":"Elucidation of the Specific Function of the Conserved Threonine Triad Responsible for Human L-Asparaginase Autocleavage and Substrate Hydrolysis","volume":"426","author":"Nomme","year":"2014","journal-title":"J. Mol. Biol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"306","DOI":"10.3390\/biom5020306","article-title":"Tackling Critical Catalytic Residues in Helicobacter Pylori L-Asparaginase","volume":"5","author":"Maggi","year":"2015","journal-title":"Biomolecules"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"211","DOI":"10.1016\/0014-5793(96)00660-6","article-title":"A Covalently Bound Catalytic Intermediate in Escherichia Coli Asparaginase: Crystal Structure of a Thr-89-Val Mutant","volume":"390","author":"Palm","year":"1996","journal-title":"FEBS Lett."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Maggi, M., Meli, M., Colombo, G., and Scotti, C. (2021). Revealing Escherichia Coli Type II L-Asparaginase Active Site Flexible Loop in Its Open, Ligand-Free Conformation. Sci. Rep., 11.","DOI":"10.1038\/s41598-021-98455-1"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1850","DOI":"10.1002\/pro.3709","article-title":"Geometric Considerations Support the Double-Displacement Catalytic Mechanism of l-Asparaginase","volume":"28","author":"Lubkowski","year":"2019","journal-title":"Protein Sci."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1927","DOI":"10.1021\/acs.biochem.0c00116","article-title":"Mechanism of Catalysis by l-Asparaginase","volume":"59","author":"Lubkowski","year":"2020","journal-title":"Biochemistry"},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Strzelczyk, P., Zhang, D., Dyba, M., Wlodawer, A., and Lubkowski, J. (2020). Generalized Enzymatic Mechanism of Catalysis by Tetrameric L-Asparaginases from Mesophilic Bacteria. Sci. Rep., 10.","DOI":"10.1038\/s41598-020-74480-4"},{"key":"ref_12","first-page":"418","article-title":"Expanding Targets for a Metabolic Therapy of Cancer: L-Asparaginase","volume":"3","author":"Covini","year":"2014","journal-title":"Top. Anti-Cancer Res."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"173","DOI":"10.1007\/978-981-13-7709-9_9","article-title":"Enzymes in Metabolic Anticancer Therapy","volume":"1148","author":"Maggi","year":"2019","journal-title":"Adv. Exp. Med. Biol."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Maggi, M., Mittelman, S.D., Parmentier, J.H., Colombo, G., Meli, M., Whitmire, J.M., Merrell, D.S., Whitelegge, J., and Scotti, C. (2017). A Protease-Resistant Escherichia Coli Asparaginase with Outstanding Stability and Enhanced Anti-Leukaemic Activity in Vitro. Sci. Rep., 7.","DOI":"10.1038\/s41598-017-15075-4"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"17664","DOI":"10.1074\/jbc.M116.728485","article-title":"Design and Characterization of Erwinia Chrysanthemi L-Asparaginase Variants with Diminished l-Glutaminase Activity","volume":"291","author":"Nguyen","year":"2016","journal-title":"J. Biol. Chem."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"4183","DOI":"10.1111\/febs.16042","article-title":"Structural and Biochemical Properties of L-Asparaginase","volume":"288","author":"Lubkowski","year":"2021","journal-title":"FEBS J."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"416","DOI":"10.1107\/S0907444902021200","article-title":"Structural Comparison of Escherichia coli L-Asparaginase in Two Monoclinic Space Groups","volume":"59","author":"Sanches","year":"2003","journal-title":"Acta Crystallogr. D Biol. Crystallogr."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"5655","DOI":"10.1021\/bi0029595","article-title":"Structural Basis for the Activity and Substrate Specificity of Erwinia Chrysanthemi L-Asparaginase","volume":"40","author":"Aghaiypour","year":"2001","journal-title":"Biochemistry"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/11\/5942\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T23:18:40Z","timestamp":1760138320000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/11\/5942"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,5,25]]},"references-count":18,"journal-issue":{"issue":"11","published-online":{"date-parts":[[2022,6]]}},"alternative-id":["ijms23115942"],"URL":"https:\/\/doi.org\/10.3390\/ijms23115942","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,5,25]]}}}