Abstract:
The bifunctional enzyme, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase/inosine monophosphate (IMP) cyclohydrolase (ATIC) is involved in catalyzing penultimate and final steps of purine de novo biosynthetic pathway crucial for the survival of organisms. The present study reports the characterization of ATIC from Candidatus Liberibacer asiaticus (CLasATIC) along with the identification of potential inhibitor molecules and evaluation of cell proliferative activity. CLasATIC showed both the AICAR Transformylase (AICAR TFase) activity for substrates, 10-f-THF (Km, 146.6 μM and Vmax, 0.95 μmol/min/mg) and AICAR (Km, 34.81 μM and Vmax, 0.56 μmol/min/mg) and IMP cyclohydrolase (IMPCHase) activitiy (Km, 1.81 μM and Vmax, 2.87 μmol/min/mg). The optimum pH and temperature were also identified for the enzyme activity. In-silico study has been conducted to identify potential inhibitor molecules through virtual screening and MD simulations. Out of many compounds, HNBSA, diosbulbin A and lepidine D emerged as lead compounds, exhibiting higher binding energy and stability for CLasATIC than AICAR. ITC study reports higher binding affinities for HNBSA and diosbulbin A (Kd, 12.3 μM and 34.2 μM, respectively) compared to AICAR (Kd, 83.4 μM). Likewise, DSC studies showed enhanced thermal stability for CLasATIC in the presence of inhibitors. CD and Fluorescence studies revealed significant conformational changes in CLasATIC upon binding of the inhibitors. CLasATIC demonstrated potent cell proliferative, wound healing and ROS scavenging properties evaluated by cell-based bioassays using CHO cells. This study highlights CLasATIC as a promising drug target with potential inhibitors for managing CLas and its unique cell protective, wound-healing properties for future biotechnological applications.
摘要:
双功能酶,5-氨基咪唑-4-甲酰胺核糖核苷酸(AICAR)转化酶/肌苷单磷酸(IMP)环水解酶(ATIC)参与催化嘌呤从头生物合成途径的倒数第二个和最后一个步骤,这对于生物体的存活至关重要。本研究报告了来自亚洲念珠菌(CLasATIC)的ATIC的表征,以及潜在抑制剂分子的鉴定和细胞增殖活性的评估。CLasATIC显示了AICAR转化酶(AICARTFase)对底物的活性,10-f-THF(Km,146.6μM和Vmax,0.95μmol/min/mg)和AICAR(Km,34.81μM和Vmax,0.56μmol/min/mg)和IMP环化水解酶(IMPCHase)活性(Km,1.81μM和Vmax,2.87μmol/min/mg)。还确定了酶活性的最佳pH和温度。已经进行了计算机模拟研究以通过虚拟筛选和MD模拟来鉴定潜在的抑制剂分子。在许多化合物中,HNBSA,薯片苷A和lepidineD作为先导化合物出现,比AICAR表现出更高的结合能和稳定性。ITC研究报告对HNBSA和薯脂素A的结合亲和力较高(Kd,12.3μM和34.2μM,分别)与AICAR(Kd,83.4μM)。同样,DSC研究显示在抑制剂存在下CLasATIC的热稳定性增强。CD和荧光研究揭示了在结合抑制剂时CLasATIC的显著构象变化。ClasATIC证明了有效的细胞增殖,使用CHO细胞通过基于细胞的生物测定法评估伤口愈合和ROS清除特性。这项研究强调了CLasATIC作为一种有前途的药物靶标,具有潜在的抑制剂来管理CLas及其独特的细胞保护作用,未来生物技术应用的伤口愈合特性。