Unveiling a putative class 3 L-asparaginase in Glutamicibacter sp.: from soil isolation to in silico characterization.

BACKGROUND: Type II L-asparaginases are important therapeutic agents for treating childhood leukemia, but numerous side effects of these enzymes, besides neutralization by antibodies due to repeated injection, prompt extensive research to identify alternatives in other classes of this enzyme.

RESULTS: Among the six asparaginase-producing strains, the best-producing isolate was identified as Glutamicibacter. The asparaginase and glutaminase activities in culture supernatant were 15 U/ml and 1.5 U/ml, respectively. The full-length asparaginase gene was sequenced, and the phylogenetic tree revealed strong clustering with recently identified class 3 asparaginases from Rhizobium etli. The presence of key residues from Class 3 ReAV and ReAIV further confirms this similarity. The AlphaFold 3 server confidently predicted the enzyme's 3D structure. Molecular docking of L-asparagine and L-glutamine using Rosetta revealed binding energies of -9.99 REU for both substrates that were higher than those of the Type IV and V enzymes. Molecular dynamics results indicated that enzyme-Asn was more stable than enzyme-Gln with average RMSD values of 0.33 nm and 0.44 nm, respectively. Enzyme-Asn reached a relatively stable plateau after 0.05 ns, whereas enzyme-Gln showed a sudden jump after 0.75 ns, indicating the separation of glutamine from the protein. A comparison of the antigenic determinants of GlAVI with those of ReAIV, ReAV, EcAII, and ErAII revealed that GlAVI exhibited lower immunogenicity than the other enzymes. Allergenic epitopes were predicted using bioinformatics, demonstrating that nonallergenic epitopes cover a significant portion of the enzyme.

CONCLUSION: The newly isolated Glutamicibacter L-asparaginase appears to be a new type of class 3 asparaginases with high potential for novel therapeutic drug development because of its less and dissimilar immunological properties.