Kinetic and structural study of GalU and GalF proteins from Escherichia coli

EBRECHT AC; SASONI N; ORLOF A; FIGUEROA CM; KUHN M; BALLICORA MA; IGLESIAS AA

Mendoza

Congreso; XLVIII Reunión Anual de la SAIB; 2012

SAIB

UDPGlc, a key glycosyl donor for carbohydrate metabolism, is produced from UTP and Glc1P by UDPGlc pyrophosphorylase (GalU). Previously, we demonstrated that in E. coli another protein (GalF) can catalyze the reaction. We compared the kinetic and structural properties of both enzymes, and constructed a GalF mutant (M15TH16R) which exhibited a partial "resurrection" of the activity. In this work we complemented an E. coli strain deficient in the galU gene with constructions that allow overexpression of the enzymes (pGALU, pGALF and pM15TH16R). In accordance with in vitro results, transformed cells with pGALU were able to ferment galactose within the first 24 h, cells complemented with pM15TH16R after 190 h, whereas cells carrying pGALF needed more than 240 h. We also produced the analogous T20MR21H GalU mutant, which had a Vmax three orders of magnitude lower and a S0.5 for Glc1P 60-fold higher than the wild type. Results support key roles for kinetics of critical residues present in GalU and absent in GalF. In addition, we hypothesize that differences between enzyme activities would, in part, be due to oligomerization status of the respective protein. In fact, production of monomeric GalU gave an enzyme with similar affinity for substrates but with 20-fold lower Vmax than the tetramer. We discuss about a putative in vivo function of GalF and its possible interaction with GalU.