RRC ID 79460
Author Kato S, Tamura K, Masuda Y, Konishi M, Yamanaka K, Oikawa T.
Title A novel type IIb L-asparaginase from Latilactobacillus sakei LK-145: characterization and application.
Journal Arch Microbiol
Abstract We succeeded in homogeneously expressing and purifying L-asparaginase from Latilactobacillus sakei LK-145 (Ls-Asn1) and its mutated enzymes C196S, C264S, C290S, C196S/C264S, C196S/C290S, C264S/C290S, and C196S/C264S/C290S-Ls-Asn1. Enzymological studies using purified enzymes revealed that all cysteine residues of Ls-Asn1 were found to affect the catalytic activity of Ls-Asn1 to varying degrees. The mutation of Cys196 did not affect the specific activity, but the mutation of Cys264, even a single mutation, significantly decreased the specific activity. Furthermore, C264S/C290S- and C196S/C264S/C290S-Ls-Asn1 almost completely lost their activity, suggesting that C290 cooperates with C264 to influence the catalytic activity of Ls-Asn1. The detailed enzymatic properties of three single-mutated enzymes (C196S, C264S, and C290S-Ls-Asn1) were investigated for comparison with Ls-Asn1. We found that only C196S-Ls-Asn1 has almost the same enzymatic properties as that of Ls-Asn1 except for its increased stability for thermal, pH, and the metals NaCl, KCl, CaCl2, and FeCl2. We measured the growth inhibitory effect of Ls-Asn1 and C196S-Ls-Asn1 on Jurkat cells, a human T-cell acute lymphoblastic leukemia cell line, using L-asparaginase from Escherichia coli K-12 as a reference. Only C196S-Ls-Asn1 effectively and selectively inhibited the growth of Jurkat T-cell leukemia, which suggested that it exhibited antileukemic activity. Furthermore, based on alignment, phylogenetic tree analysis, and structural modeling, we also proposed that Ls-Asn1 is a so-called "Type IIb" novel type of asparaginase that is distinct from previously reported type I or type II asparaginases. Based on the above results, Ls-Asn1 is expected to be useful as a new leukemia therapeutic agent.
Volume 206(6)
Pages 266
Published 2024-5-18
DOI 10.1007/s00203-024-03979-5
PII 10.1007/s00203-024-03979-5
PMID 38761213
MeSH Amino Acid Sequence Antineoplastic Agents / chemistry Antineoplastic Agents / metabolism Antineoplastic Agents / pharmacology Asparaginase* / chemistry Asparaginase* / genetics Asparaginase* / isolation & purification Asparaginase* / metabolism Asparaginase* / pharmacology Bacillaceae / enzymology Bacillaceae / genetics Bacterial Proteins / chemistry Bacterial Proteins / genetics Bacterial Proteins / metabolism Humans Hydrogen-Ion Concentration Jurkat Cells Kinetics Mutation
Resource
Human and Animal Cells Jurkat(RCB0806) 293(RCB1637)