Characterization of enzymes involved in UDP-glucose metabolism in Escherichia coli

A.C. EBRECHT; N. SASONI; C. ORLOF; C.M. FIGUEROA; M.L. KUHN; M.A. BALLICORA; A.A. IGLESIAS

Potrero de los Funes

Congreso; XLVII Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2011

UDP-Glc is a key glycosyl donor for carbohydrate metabolism in bacteria. It is produced from UTP and Glc-1P by UDP-Glc pyrophosphorylase (GalU). Another protein (GalF), with high sequence identity to GalU, was reported in Escherichia coli. We compared the kinetic and structural properties of both recombinant proteins. Values of S0.5 for GalU were determined to be 0.16 mM (UTP) and 0.035 mM (Glc-1P), whereas those for GalF were 0.91 mM and 0.32 mM, respectively. GalU displayed a four order of magnitude higher Vmax than GalF. In silico analysis showed that GalF lacks two key catalytic residues; thus, we constructed the mutant GalF M15T/H16R, which exhibited similar affinity for both of the substrates, but had a 10-fold higher Vmax than GalF. Although the latter Vmax is substantially lower than that of GalU, mutations effectively enhanced the catalytic ability of GalF.  Size exclusion chromatography revealed that GalU is tetrameric, whereas GalF and its mutant are monomers; which could account for the only partial "resurrection" observed for mutated GalF. We hypothesize that, after gene duplication, GalU retained the catalytic function and GalF acquired new roles, including the modulation of GalU activity. Funded by ANPCyT (PICT?08 1754), CONICET (PIP 2519), UNL (CAI+D?09), and NSF (MCB 1024945).