Kinetic and Structural Study of GalU and GalF Proteins from Escherichia coli

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

Mendoza

Congreso; XLVIII Reunión Anual de la SAIB; 2012

SAIB

UDPGlc, a key glycosyl donor for carbohydrate metabolism, isproduced from UTP and GlclP by UnPGlc pyrophosphorylase(calm. Previously, we demonstmtedthat in K cdi another prokin(GalF) can catalyze the reamnn. We compared the kinetic andnzluctural properties of both enzymes, and constructed a GalFmutant (MI 5TITI hR) which exhibiteda partla1 '"resurrection"oftheactivity. In this work we cumplemented an E. coti strain deficient inthc galU gtl~cw ith construchons that allow ovmprcssmn ofthcenzymes (pGALU, pGALF and pMl571-Il bR). In accordance within vitro r e d s , transEod cells with pCiALU were able to fermentgalactose within the Grst 24 h, cells complemented withpM 1 5TH16R &r 190 EO, whereas cells carrying pGALF neededmore ha^^ 240 11. We also produced the d o g o u s 'l'20MR2 I H GalUmutani, which had a Y, three orders of magnitude lower and a S,,hr Glc IF MI-fnld higher than the wild typc. Rmults mppnrt keymles for kinetics d critical restdues present in CalU and ahsent inCralF. In ddihon, we hypothesize that differences between enzymeactiviVes would, in part, be due to oligomeriza~ion slams of there~pectivep rotein. In fact, produdiwn of monomeric GalU gave anPnyyme with firnibr afinity for s~~lwinthwut with 2 h r l d lnwerV,, han the tetramer. Wediscuss abou t a putative in vivo function ofGalF and its possible interaction with GalU.