Mishra Saurabh, Bhagavat Raghu, Chandra Nagasuma, Vijayarangan Namperumalsamy, Rajeswari Haryadi, Ajitkumar Parthasarathi
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, Karnataka, India.
Protein Expr Purif. 2012 Nov;86(1):58-67. doi: 10.1016/j.pep.2012.08.020. Epub 2012 Sep 13.
The role of FIC (Filamentation induced by cAMP)(2) domain containing proteins in the regulation of many vital pathways, mostly through the transfer of NMPs from NTPs to specific target proteins (NMPylation), in microorganisms, higher eukaryotes, and plants is emerging. The identity and function of FIC domain containing protein of the human pathogen, Mycobacterium tuberculosis, remains unknown. In this regard, M. tuberculosis fic gene (Mtfic) was cloned, overexpressed, and purified to homogeneity for its biochemical characterisation. It has the characteristic FIC motif, HPFREGNGRSTR (HPFxxGNGRxxR), spanning 144th to 155th residue. Neither the His-tagged nor the GST-tagged MtFic protein, overexpressed in Escherichia coli, nor expression of Mtfic in Mycobacterium smegmatis, yielded the protein in the soluble fraction. However, the maltose binding protein (MBP) tagged MtFic (MBP-MtFic) could be obtained partly in the soluble fraction. The cloned, overexpressed, and purified recombinant MBP-MtFic showed conversion of ATP, GTP, CTP, and UTP into AMP, GMP, CMP, and UMP, respectively. Sequence alignment with several FIC motif containing proteins, complemented with homology modeling on the FIC motif containing protein, VbhT of Bartonella schoenbuchensis as the template, showed conservation and interaction of residues constituting the FIC domain. Site-specific mutagenesis of the His144, or Glu148, or Asn150 of the FIC motif, or of Arg87 residue that constitutes the FIC domain, or complete deletion of the FIC motif, abolished the NTP to NMP conversion activity. The design of NMP formation assay using the recombinant, soluble MtFic would enable identification of its target substrate for NMPylation.
含FIC（cAMP诱导的丝状化）(2)结构域的蛋白质在调节许多重要途径中的作用正在显现，这些途径主要通过将NMP从NTP转移到特定靶蛋白（NMP化）来实现，涉及微生物、高等真核生物和植物。人类病原体结核分枝杆菌中含FIC结构域蛋白质的身份和功能仍然未知。在这方面，结核分枝杆菌fic基因（Mtfic）被克隆、过表达并纯化至同质，以进行其生化特性分析。它具有特征性的FIC基序，HPFREGNGRSTR（HPFxxGNGRxxR），跨越第144至155个残基。在大肠杆菌中过表达的His标签或GST标签的MtFic蛋白，以及耻垢分枝杆菌中Mtfic的表达，均未在可溶部分产生该蛋白。然而，麦芽糖结合蛋白（MBP）标签的MtFic（MBP-MtFic）可以部分地在可溶部分获得。克隆、过表达并纯化的重组MBP-MtFic分别显示ATP、GTP、CTP和UTP转化为AMP、GMP、CMP和UMP。与几种含FIC基序的蛋白质进行序列比对，并以含FIC基序的蛋白质、以Bartonella schoenbuchensis的VbhT为模板进行同源建模，显示了构成FIC结构域的残基的保守性和相互作用。FIC基序的His144、Glu148、Asn150或构成FIC结构域的Arg87残基的位点特异性诱变，或FIC基序的完全缺失，均消除了NTP向NMP的转化活性。使用重组可溶MtFic设计NMP形成测定法将能够鉴定其NMP化的靶底物。
