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Characterization of AICAR transformylase/IMP cyclohydrolase (ATIC) from Staphylococcus lugdunensis.

The FEBS journal (2017-10-25)
Preeti Verma, Bibekananda Kar, Ritu Varshney, Partha Roy, Ashwani K Sharma
ABSTRACT

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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
AICAR, ≥98% (HPLC), powder
Sigma-Aldrich
Phenylmethanesulfonyl chloride, 98%

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