{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,7]],"date-time":"2026-05-07T13:21:38Z","timestamp":1778160098421,"version":"3.51.4"},"reference-count":50,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2026,5,7]],"date-time":"2026-05-07T00:00:00Z","timestamp":1778112000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Cooperation Project of Pingyang Traditional Chinese Medicine Hospital","award":["2025-HT-669"],"award-info":[{"award-number":["2025-HT-669"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomolecules"],"abstract":"<jats:p>Aspartylglucosaminidase (AGA) is an amidohydrolase that can hydrolyze the amide bond between N-acetylglucosamine (GlcNAc) and asparagine (Asn), producing N-acetylglucosamine and aspartic acid (Asp). AGA is distributed in both eukaryotes and prokaryotes. In this study, we identified the sequence of AGA in Elizabethkingia meningoseptica (EmAGA), cloned and expressed it in Escherichia coli, and recharacterized its properties, confirming its substrates as aspartylglucosamine (Asn-GlcNAc) and Asn. Key residues affecting its enzymatic activity were predicted through molecular docking and conserved site analysis, and 10 key residues that affected enzymatic activity were verified, eight of which regulated activity by interfering with EmAGA\u2019s autoproteolysis, indicating that autoproteolytic cleavage into \u03b1\/\u03b2 subunits was essential for EmAGA maturation. Molecular dynamics simulations were performed on autohydrolysis-impaired mutants, which showed a more stable conformation and lower energy. In summary, EmAGA\u2019s functional characterization provided novel evidence for elucidating its molecular mechanism. Clinically used Asparaginase (ASNase) exerts its therapeutic effect on acute lymphoblastic leukemia (ALL) through its asparaginase activity, but it is limited by glutamine off-target side effects, while EmAGA also has asparaginase activity but no glutaminase activity, rendering its potential as a basis for novel anti-leukemic enzymatic therapeutics.<\/jats:p>","DOI":"10.3390\/biom16050690","type":"journal-article","created":{"date-parts":[[2026,5,7]],"date-time":"2026-05-07T12:15:07Z","timestamp":1778156107000},"page":"690","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Recharacterization of EmAGA, a Potential Candidate for Novel ALL Therapeutics"],"prefix":"10.3390","volume":"16","author":[{"given":"Qiange","family":"Lin","sequence":"first","affiliation":[{"name":"School of Medical Technology and Information Engineering, Zhejiang Chinese Medical University, Hangzhou 310053, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yuxin","family":"Zhang","sequence":"additional","affiliation":[{"name":"School of Medical Technology and Information Engineering, Zhejiang Chinese Medical University, Hangzhou 310053, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Junyou","family":"Lin","sequence":"additional","affiliation":[{"name":"School of Medical Technology and Information Engineering, Zhejiang Chinese Medical University, Hangzhou 310053, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yilin","family":"Ye","sequence":"additional","affiliation":[{"name":"School of Medical Technology and Information Engineering, Zhejiang Chinese Medical University, Hangzhou 310053, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xin","family":"Qian","sequence":"additional","affiliation":[{"name":"School of Medical Technology and Information Engineering, Zhejiang Chinese Medical University, Hangzhou 310053, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xinrong","family":"Lu","sequence":"additional","affiliation":[{"name":"Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shaoxian","family":"Lyu","sequence":"additional","affiliation":[{"name":"Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xinliu","family":"Geng","sequence":"additional","affiliation":[{"name":"Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Li","family":"Chen","sequence":"additional","affiliation":[{"name":"Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Guiqin","family":"Sun","sequence":"additional","affiliation":[{"name":"School of Medical Technology and Information Engineering, Zhejiang Chinese Medical University, Hangzhou 310053, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,5,7]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"629","DOI":"10.1146\/annurev.biochem.75.103004.142520","article-title":"Asparagine synthetase chemotherapy","volume":"75","author":"Richards","year":"2006","journal-title":"Annu. Rev. Biochem."},{"key":"ref_2","first-page":"217404","article-title":"A human-like glutaminase-free asparaginase is highly efficacious in ASNSlow leukemia and solid cancer mouse xenograft models","volume":"611","author":"Schalk","year":"2024","journal-title":"Cancer Lett."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"e46640","DOI":"10.1097\/MD.0000000000046640","article-title":"Pegasparaginase-induced hepatotoxicity in patients with acute lymphocytic leukemia in the Saudi population: A retrospective cohort study","volume":"105","author":"Alshehri","year":"2026","journal-title":"Medicine"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"117","DOI":"10.1039\/D1MD00344E","article-title":"Overview of the structure, side effects, and activity assays of l-asparaginase as a therapy drug of acute lymphoblastic leukemia","volume":"13","author":"Wang","year":"2022","journal-title":"RSC Med. Chem."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Freitas, M., Souza, P., Homem-de-Mello, M., Fonseca-Bazzo, Y.M., Silveira, D., Ferreira Filho, E.X., Pessoa Junior, A., Sarker, D., Timson, D., and In\u00e1cio, J. (2022). L-Asparaginase from Penicillium sizovae Produced by a Recombinant Komagataella phaffii Strain. Pharmaceuticals, 15.","DOI":"10.3390\/ph15060746"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"757","DOI":"10.1016\/j.leukres.2015.04.008","article-title":"Glutaminase activity determines cytotoxicity of L-asparaginases on most leukemia cell lines","volume":"39","author":"Parmentier","year":"2015","journal-title":"Leuk. Res."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"712","DOI":"10.1038\/s41375-024-02153-6","article-title":"Increase in peg-asparaginase clearance as a predictor for inactivation in patients with acute lymphoblastic leukemia","volume":"38","author":"Dam","year":"2024","journal-title":"Leukemia"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"130312","DOI":"10.1016\/j.ijbiomac.2024.130312","article-title":"Homology modeling and molecular docking studies to decrease glutamine affinity of Yarrowia lipolytica L-asparaginase","volume":"263","author":"Darvishi","year":"2024","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Banning, A., Laine, M., and Tikkanen, R. (2023). Validation of Aspartylglucosaminidase Activity Assay for Human Serum Samples: Establishment of a Biomarker for Diagnostics and Clinical Studies. Int. J. Mol. Sci., 24.","DOI":"10.20944\/preprints202302.0255.v1"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"2710","DOI":"10.1038\/s41564-024-01782-y","article-title":"Aspergillus fumigatus conidial surface-associated proteome reveals factors for fungal evasion and host immunity modulation","volume":"9","author":"Pinzan","year":"2024","journal-title":"Nat. Microbiol."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Banning, A., and Tikkanen, R. (2021). Towards Splicing Therapy for Lysosomal Storage Disorders: Methylxanthines and Luteolin Ameliorate Splicing Defects in Aspartylglucosaminuria and Classic Late Infantile Neuronal Ceroid Lipofuscinosis. Cells, 10.","DOI":"10.3390\/cells10112813"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"82","DOI":"10.1016\/j.jmb.2006.09.051","article-title":"Crystallographic snapshot of a productive glycosylasparaginase-substrate complex","volume":"366","author":"Wang","year":"2007","journal-title":"J. Mol. Biol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"961","DOI":"10.1016\/0006-291X(66)90344-5","article-title":"Enzymatic cleavage of glycopeptides","volume":"24","author":"Makino","year":"1966","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"399","DOI":"10.1006\/abbi.1995.1053","article-title":"Molecular cloning and sequence analysis of Flavobacterium meningosepticum glycosylasparaginase: A single gene encodes the \u03b1 and \u03b2 subunits","volume":"316","author":"Tarentino","year":"1995","journal-title":"Arch. Biochem. Biophys."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"527","DOI":"10.1093\/glycob\/6.5.527","article-title":"Purification, biochemistry and molecular cloning of an insect glycosylasparaginase from Spodoptera frugiperda","volume":"6","author":"Liu","year":"1996","journal-title":"Glycobiology"},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Coulette, Q., Lemauf, S., Colinet, D., Pr\u00e9vost, G., Anselme, C., Poiri\u00e9, M., and Gatti, J.L. (2017). Biochemical characterization and comparison of aspartylglucosaminidases secreted in venom of the parasitoid wasps Asobara tabida and Leptopilina heterotoma. PLoS ONE, 12.","DOI":"10.1371\/journal.pone.0181940"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"3400","DOI":"10.1111\/febs.16755","article-title":"X-ray crystallographic and mutational analysis of the NylC precursor: Catalytic mechanism of autocleavage and substrate hydrolysis of nylon hydrolase","volume":"290","author":"Negoro","year":"2023","journal-title":"Febs J."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1855","DOI":"10.1016\/j.str.2014.09.014","article-title":"Structural basis of a point mutation that causes the genetic disease aspartylglucosaminuria","volume":"22","author":"Sui","year":"2014","journal-title":"Structure"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"150","DOI":"10.1016\/j.ymgme.2017.04.008","article-title":"Crystal structure of a mutant glycosylasparaginase shedding light on aspartylglycosaminuria-causing mechanism as well as on hydrolysis of non-chitobiose substrate","volume":"121","author":"Pande","year":"2017","journal-title":"Mol. Genet. Metab."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1102","DOI":"10.1038\/nsb1295-1102","article-title":"Three-dimensional structure of human lysosomal aspartylglucosaminidase","volume":"2","author":"Oinonen","year":"1995","journal-title":"Nat. Struct. Mol. Biol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"179","DOI":"10.1006\/bbrc.1993.2457","article-title":"The first demonstration of a procaryotic glycosylasparaginase","volume":"197","author":"Tarentino","year":"1993","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"D161","DOI":"10.1093\/nar\/gkab1135","article-title":"GenBank","volume":"50","author":"Sayers","year":"2022","journal-title":"Nucleic Acids Res."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"W320","DOI":"10.1093\/nar\/gku316","article-title":"Deciphering key features in protein structures with the new ENDscript server","volume":"42","author":"Robert","year":"2014","journal-title":"Nucleic Acids Res."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"35","DOI":"10.1111\/1751-7915.12300","article-title":"Enhancement of the catalytic efficiency and thermostability of Stenotrophomonas sp. keratinase KerSMD by domain exchange with KerSMF","volume":"9","author":"Fang","year":"2016","journal-title":"Microb. Biotechnol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"988","DOI":"10.1042\/bj0280988","article-title":"A colorimetric method for the determination of N-acetylglucosamine and N-acetylchrondrosamine","volume":"28","author":"Morgan","year":"1934","journal-title":"Biochem. J."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"26","DOI":"10.1016\/j.abb.2015.05.013","article-title":"Roles of active site residues in LodA, a cysteine tryptophylquinone dependent \u03b5-lysine oxidase","volume":"579","author":"Sehanobish","year":"2015","journal-title":"Arch. Biochem. Biophys."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"493","DOI":"10.1038\/s41586-024-07487-w","article-title":"Accurate structure prediction of biomolecular interactions with AlphaFold 3","volume":"630","author":"Abramson","year":"2024","journal-title":"Nature"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1581","DOI":"10.1021\/acs.jctc.0c01327","article-title":"CHARMM36 Lipid Force Field with Explicit Treatment of Long-Range Dispersion: Parametrization and Validation for Phosphatidylethanolamine, Phosphatidylglycerol, and Ether Lipids","volume":"17","author":"Yu","year":"2021","journal-title":"J. Chem. Theory Comput."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1016\/j.softx.2015.06.001","article-title":"GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers","volume":"1\u20132","author":"Abraham","year":"2015","journal-title":"SoftwareX"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"7452","DOI":"10.1074\/jbc.M114.605493","article-title":"Identification and characterization of a novel prokaryotic peptide: N-glycosidase from Elizabethkingia meningoseptica","volume":"290","author":"Sun","year":"2015","journal-title":"J. Biol. Chem."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"26295","DOI":"10.1074\/jbc.273.41.26295","article-title":"Glycosylasparaginase-catalyzed synthesis and hydrolysis of \u03b2-aspartyl peptides","volume":"273","author":"Noronkoski","year":"1998","journal-title":"J. Biol. Chem."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"6855","DOI":"10.1016\/S0021-9258(19)50505-1","article-title":"Substrate specificity and reaction mechanism of human glycoasparaginase. The N-glycosidic linkage of various glycoasparagines is cleaved through a reaction mechanism similar to L-asparaginase","volume":"267","author":"Kaartinen","year":"1992","journal-title":"J. Biol. Chem."},{"key":"ref_33","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"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"319","DOI":"10.1016\/0165-022X(88)90066-8","article-title":"Microcalorimetric determination of substrate specificity of L-asparaginase in Eschericia coli and Erwinia carotovora","volume":"16","author":"Rekharsky","year":"1988","journal-title":"J. Biochem. Biophys. Methods"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"9688","DOI":"10.1074\/jbc.273.16.9688","article-title":"Site-directed mutagenesis of essential residues involved in the mechanism of bacterial glycosylasparaginase","volume":"273","author":"Liu","year":"1998","journal-title":"J. Biol. Chem."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"989","DOI":"10.1016\/j.ymthe.2020.11.012","article-title":"Pre-clinical Gene Therapy with AAV9\/AGA in Aspartylglucosaminuria Mice Provides Evidence for Clinical Translation","volume":"29","author":"Chen","year":"2021","journal-title":"Mol. Ther."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"e200161","DOI":"10.1212\/NXG.0000000000200161","article-title":"A 37-Year-Old Man with Intellectual Disability Discovered to Have Aspartylglucosaminuria: Implications for the Diagnosis of Genetic Causes","volume":"10","author":"Kouhashi","year":"2024","journal-title":"Neurol. Genet."},{"key":"ref_38","first-page":"3","article-title":"Effectiveness of early hematopoietic stem cell transplantation in preventing neurocognitive decline in aspartylglucosaminuria: A case series","volume":"61","author":"Selvanathan","year":"2021","journal-title":"JIMD Rep."},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Ruohola, A., Salli, E., Roine, T., Tokola, A., Laine, M., Tikkanen, R., Savolainen, S., and Autti, T. (2022). Detection of Aspartylglucosaminuria Patients from Magnetic Resonance Images by a Machine-Learning-Based Approach. Brain Sci., 12.","DOI":"10.3390\/brainsci12111522"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1013","DOI":"10.1002\/pro.3607","article-title":"The T99K variant of glycosylasparaginase shows a new structural mechanism of the genetic disease aspartylglucosaminuria","volume":"28","author":"Pande","year":"2019","journal-title":"Protein Sci."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"3212","DOI":"10.1074\/jbc.270.7.3212","article-title":"Localization of the disulfide bond involved in post-translational processing of glycosylasparaginase and disrupted by a mutation in the Finnish-type aspartylglycosaminuria","volume":"270","author":"McCormack","year":"1995","journal-title":"J. Biol. Chem."},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"D\u00edaz-Barriga, C., Villanueva-Flores, F., Quester, K., Z\u00e1rate-Romero, A., Cadena-Nava, R.D., and Huerta-Saquero, A. (2021). Asparaginase-Phage P22 Nanoreactors: Toward a Biobetter Development for Acute Lymphoblastic Leukemia Treatment. Pharmaceutics, 13.","DOI":"10.3390\/pharmaceutics13050604"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1002\/ajh.70103","article-title":"Optimizing Asparaginase Treatment for Adolescent and Young Adult (AYA) Patients with Acute Lymphoblastic Leukemia: US Consensus Panel Recommendations","volume":"101","author":"Aldoss","year":"2026","journal-title":"Am. J. Hematol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"3478","DOI":"10.1200\/JCO.24.00500","article-title":"Impact of Age on Pharmacogenomics and Treatment Outcomes of B-Cell Acute Lymphoblastic Leukemia","volume":"42","author":"Yoshimura","year":"2024","journal-title":"J. Clin. Oncol."},{"key":"ref_45","doi-asserted-by":"crossref","unstructured":"Ko, M., Kim, M.G., Yoon, S.S., Kim, I.W., Suh, S.Y., Cho, Y.S., and Oh, J.M. (2023). Clinical impacts of the concomitant use of L-asparaginase and total parenteral nutrition containing L-aspartic acid in patients with acute lymphoblastic leukemia. Front. Nutr., 10.","DOI":"10.3389\/fnut.2023.1122010"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"115473","DOI":"10.1016\/j.bcp.2023.115473","article-title":"The anticancer effect of PASylated calreticulin-targeting L-ASNase in solid tumor bearing mice with immunogenic cell death-inducing chemotherapy","volume":"210","author":"Zhang","year":"2023","journal-title":"Biochem. Pharmacol."},{"key":"ref_47","first-page":"55","article-title":"Asparaginase in the Treatment of Acute Lymphoblastic Leukemia in Adults: Current Evidence and Place in Therapy","volume":"12","author":"Juluri","year":"2022","journal-title":"Blood Lymphat. Cancer"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"2606","DOI":"10.3324\/haematol.2022.282324","article-title":"Back to the future: The amazing journey of the therapeutic anti-leukemia enzyme asparaginase Erwinia chrysanthemi","volume":"108","author":"Tong","year":"2023","journal-title":"Haematologica"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"66","DOI":"10.1182\/bloodadvances.2024013346","article-title":"Recombinant Erwinia asparaginase (JZP458) in ALL\/LBL: Complete follow-up of the Children\u2019s Oncology Group AALL1931 study","volume":"9","author":"Maese","year":"2025","journal-title":"Blood Adv."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"191","DOI":"10.1038\/s41408-024-01178-5","article-title":"Venous thromboembolism in adolescents and young adults with acute lymphoblastic leukemia treated on a pediatric-inspired regimen","volume":"14","author":"Shimony","year":"2024","journal-title":"Blood Cancer J."}],"container-title":["Biomolecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2218-273X\/16\/5\/690\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,5,7]],"date-time":"2026-05-07T13:07:02Z","timestamp":1778159222000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2218-273X\/16\/5\/690"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,5,7]]},"references-count":50,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2026,5]]}},"alternative-id":["biom16050690"],"URL":"https:\/\/doi.org\/10.3390\/biom16050690","relation":{},"ISSN":["2218-273X"],"issn-type":[{"value":"2218-273X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,5,7]]}}}