Assessment of structural behaviour of new L-asparaginase and SAXS data-based evidence for catalytic activity in its monomeric form
The present study reports the structural and functional characterization of a new glutaminase-free recombinant L-asparaginase (PrASNase) from Pseudomonas resinovorans IGS-131. PrASNase showed substrate specificity to L-asparagine, and its kinetic parameters, Km, Vmax, and kcat were 9.49x10-3 M, 25.13 IUmL-1min-1, and 3.01x103 s-1, respectively. The CD spectra showed that PrASNase consists of 30.9% -helix and 69.1% other structures in its native form. FTIR was used for the functional characterization, and molecular docking predicted that the substrate interacts with serine, alanine, and glutamine in the binding pocket of PrASNase. Different from known asparaginases, structural characterization by small-angle X-ray scattering (SAXS) and analytical ultracentrifugation (AUC) unambiguously revealed PrASNase to exist as a monomer in solution at low temperatures and oligomerized to a higher state with temperature rise. Through SAXS studies and enzyme assay, PrASNase was found to be mostly monomer and catalytically active at 37{degrees}C. Furthermore, this glutaminase-free PrASNase showed killing effects against WIL2-S and TF-1.28 cells with IC50 of 7.4 {micro}g.mL-1 and 5.6 {micro}g.mL-1, respectively. This is probably the first report with significant findings of fully active L-asparaginase in monomeric form using SAXS and AUC and demonstrates the potential of PrASNase in inhibiting cancerous cells, making it a potential therapeutic candidate. HIGHLIGHTSO_LIA new L-asparaginase (PrASNase) was structurally and functionally characterized. C_LIO_LISAXS revealed PrASNase is functionally active in monomeric form and oligomerizes with temperature rise. C_LIO_LIMonomeric PrASNase showed an IC50 value of 7.4 and 5.6 {micro}g mL-1 against WIL2-S and TF-1.28 cells. C_LIO_LICytotoxicity of PrASNase against leukemic cell lines showed its potential as a biotherapeutic. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/522448v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@ece494org.highwire.dtl.DTLVardef@92cdbeorg.highwire.dtl.DTLVardef@19a2borg.highwire.dtl.DTLVardef@1308c70_HPS_FORMAT_FIGEXP M_FIG C_FIG