Abstract

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’s autoproteolysis, indicating that autoproteolytic cleavage into α/β 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’s 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.

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