AbstractGlycoconjugates play a central role in several cellular processes and alteration in their composition is associated to human pathologies. The hexosamine biosynthetic pathway is a route through which cells obtain substrates for cellular glycosylation, and is controlled by the glutamine: fructose-6-phosphate amidotransferase (GFAT). Human isoform 2 GFAT (hGFAT2) has been implicated in diabetes and cancer, however, there is no information about structural and enzymatic properties of this enzyme. Here, we report a successful expression and purification of a catalytically active recombinant hGFAT2 (rhGFAT2) in E. coli cells fused or not to a HisTag at the C-terminal end. Our enzyme kinetics data suggest that hGFAT2 does not follow the ordered bi-bi mechanism, and performs the glucosamine-6-phosphate synthesis much slowly than previously reported for other GFATs. In addition, hGFAT2 is able to isomerase fructose-6-phosphate into glucose-6-phosphate even in presence of equimolar amounts of glutamine, in an unproductive glutamine hydrolysis. Structural analysis of the generated three-dimensional model rhGFAT2, corroborated by circular dichroism data, indicated the presence of a partially structured loop in glutaminase domain, whose sequence is present in eukaryotic enzymes but absent in the E. coli homolog. Molecular dynamics simulations show such loop as the most flexible portion of the protein, which interacts with the protein mainly through the interdomain region, and plays a key role on conformational states of hGFAT2. Altogether, our study provides the first comprehensive set of data on the structure, kinetics and mechanics of hGFAT2, which will certainly contribute for further studies focusing on drug development targeting hGFAT2.
High level of soluble expression and purification of catalytically active native UDP-galactose 4-epimerase of <i>Aeromonas hydrophila</i> in <i>E. coli</i>
