对一种新型L-天冬氨酸酶的结构行为进行评估，并以小角散射数据为支持证据，证明其在单体形式下具有催化活性。
Assessment of structural behaviour of a new L-asparaginase and SAXS data-based evidence for catalytic activity in its monomeric form.
发表日期：2023 Sep 07
作者：
Kanti N Mihooliya, Jitender Nandal, Nidhi Kalidas, Ashish, Subhash Chand, Dipesh K Verma, Mani S Bhattacharyya, Debendra K Sahoo
来源：
Int J Biol Macromol
摘要：
本研究报道了来自Pseudomonas resinovorans IGS-131的一种新型无谷氨酰胺酸酶的重组L-天门冬氨酸酶（PrASNase）的结构和功能特征。PrASNase对L-天门冬氨酸具有底物特异性，其动力学参数Km、Vmax和kcat分别为9.49 × 10-3 M、25.13 IUmL-1 min-1和3.01 × 103 s-1。CD光谱表明，PrASNase由18.5%的螺旋、21.5%的反平行β折叠、4.2%的平行β折叠、14%的转弯和其他结构组成。功能特征使用FTIR进行，分子对接预测底物与PrASNase的结合口袋中的丝氨酸、丙氨酸和谷氨酰胺发生相互作用。与已知的天门冬氨酸酶不同，利用小角X射线散射（SAXS）和分析超速离心（AUC）的结构特征表明，PrASNase在低温条件下以单体形式存在于溶液中，并随着温度升高以高级别寡聚化。通过SAXS研究和酶活性测定，发现PrASNase在37 °C时主要为单体，并具有催化活性。此外，这种无谷氨酰胺酸酶的PrASNase对WIL2-S和TF-1.28细胞显示抗肿瘤效果，其IC50分别为7.4 μg.mL-1和5.6 μg.mL-1。这可能是使用SAXS和AUC发现完全活性的L-天门冬氨酸酶以单体形式存在的首次报道，并证明了PrASNase在抑制癌细胞方面的潜力，使其成为潜在的治疗候选物。版权所有 © 2023年。由Elsevier B.V.出版。
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 9.49 × 10-3 M, 25.13 IUmL-1 min-1, and 3.01 × 103 s-1, respectively. The CD spectra showed that PrASNase consisted of 18.5 % helix, 21.5 % antiparallel sheets, 4.2 % parallel sheets, 14 % turns, and rest other structures. 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. Differing 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 °C. Furthermore, this glutaminase-free PrASNase showed killing effects against WIL2-S and TF-1.28 cells with IC50 of 7.4 μg.mL-1 and 5.6 μg.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 demonstrated the potential of PrASNase in inhibiting cancerous cells, making it a potential therapeutic candidate.Copyright © 2023. Published by Elsevier B.V.