Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, India.
FEBS J. 2017 Dec;284(24):4233-4261. doi: 10.1111/febs.14303. Epub 2017 Nov 13.
The 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase/inosine monophosphate (IMP) cyclohydrolase (ATIC) catalyzes final two steps of purine nucleotide de novo biosynthetic pathway. This study reports the characterization of ATIC from Staphylococcus lugdunensis (SlugATIC). Apart from kinetic analysis and a detailed biophysical characterization of SlugATIC, the role of ATIC in cell proliferation has been demonstrated for the first time. The purified recombinant SlugATIC and its truncated domains exist mainly in dimeric form was revealed in gel-filtration and glutaraldehyde cross-linking studies. The two activities reside on separate domains was demonstrated in kinetic analysis of SlugATIC and reconstituted truncated N-terminal IMP cyclohydrolase (IMPCHase) and C-terminal AICAR transformylase (AICAR TFase) domains. Site-directed mutagenesis showed that Lys255 and His256 are the key catalytic residues, while Asn415 substantially contributes to AICAR TFase activity in SlugATIC. The differential scanning calorimetry (DSC) analysis revealed a molten globule-like structure for independent N-terminal domain as compared with a relatively stable conformational state in full-length SlugATIC signifying the importance of covalently linked domains. Unlike reported crystal structures, the DSC studies revealed significant conformational changes on binding of leading ligand to AICAR TFase domain in SlugATIC. The cell proliferation activity of SlugATIC was observed where it promoted proliferation and viability of NIH 3T3 and RIN-5F cells, exhibited in vitro wound healing in NIH 3T3 fibroblast cells, and rescued RIN-5F cells from the cytotoxic effects of palmitic acid and high glucose. The results suggest that ATIC, an important drug target, can also be exploited for its cell proliferative properties.
5-氨基咪唑-4-甲酰胺核苷酸（AICAR）转化酶/次黄嘌呤核苷酸（IMP）环化水解酶（ATIC）催化嘌呤核苷酸从头生物合成途径的最后两步。本研究报道了来自金黄色葡萄球菌（SlugATIC）的 ATIC 的特性。除了对 SlugATIC 的动力学分析和详细的生物物理特性进行表征外，还首次证明了 ATIC 在细胞增殖中的作用。凝胶过滤和戊二醛交联研究表明，纯化的重组 SlugATIC 及其截断结构域主要以二聚体形式存在。动力学分析表明，SlugATIC 的两个活性位于不同的结构域上，并重新构建了截断的 N 端 IMP 环化水解酶（IMPCHase）和 C 端 AICAR 转化酶（AICAR TFase）结构域。定点突变显示 Lys255 和 His256 是关键的催化残基，而 Asn415 在 SlugATIC 的 AICAR TFase 活性中起着重要作用。差示扫描量热法（DSC）分析表明，与全长 SlugATIC 相比，独立的 N 端结构域具有类似无规卷曲的结构，表明共价连接的结构域的重要性。与报道的晶体结构不同，DSC 研究显示，在 SlugATIC 的 AICAR TFase 结构域结合先导配体时，会发生显著的构象变化。SlugATIC 的细胞增殖活性表现为它促进 NIH 3T3 和 RIN-5F 细胞的增殖和活力，在 NIH 3T3 成纤维细胞中表现出体外伤口愈合，并从棕榈酸和高葡萄糖的细胞毒性作用中拯救 RIN-5F 细胞。结果表明，ATIC 作为一个重要的药物靶点，也可以利用其细胞增殖特性。
