Kinetic and structural chacacterization of enzymes involved in UDP-glucose metabolism in Escherichia coli

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

Chicago

Conferencia; The 33rd Midwest Enzyme Chemistry Conference; 2013

In bacteria, UDP-glucose (UDP-Glc) has a number of important functions, being a central intermediate in carbohydrates metabolism. This key metabolite is produced from UTP and Glc-1-phosphate (Glc-1P), in a Mg2+-dependent reaction catalyzed by UDP-Glc pyrophosphorylase (UDP-Glc PPase). The enzyme is encoded by the galU gene, which deletion generates cells unable to ferment galactose. In some bacteria, it is found a second gene, galF, encoding for a protein with high sequence identity to GalU, but which functionality is not understood. We cloned the genes encoding for GalU and GalF in Escherichia coli. After recombinant expression and purification we determined that GalF is an active enzyme. However, its activity and affinity for Glc-1P were considerably lower than those of GalU. We hypothesized that differences are mainly due to the absence of key catalytic residues and to the quaternary structure of the proteins. In silico analysis showed that GalU residues Thr20 and Arg21 are substituted in GalF by Met and His, respectively. Thus, we constructed the mutant GalF M15TH16R, which exhibited a partial ?resurrection? of the activity. Interestingly, over-expression of GalF in an E. coli strain deficient in galU allowed cells to grow in galactose medium. Also, expression of GalF M15TH16R was more efficient in compensating the absence of GalU, which is in good agreement with in vitro results obtained. We also produced the analogous mutant of GalU T20MR21H, which exhibited a Vmax three orders of magnitude lower and an S0.5 for Glc-1P 60-fold higher than GalU. Results support key role for kinetics of critical residues present in GalU but absent in GalF. In addition, we hypothesize that differences between enzyme activities would, in part, be due to oligomerization status of the respective protein. According to this, production of monomeric GalU resulted in an enzyme with 20-fold lower Vmax than the tetramer. To further analyze the catalytic site of E. coli UDP-Glc PPase, we generated, in both enzymes, a mutant of a lysine residue that presumable interacts with Glc-1P. The GalU mutant K202A exhibited a 30-fold increase in the S0.5 value for this substrate compared to the wild type; whereas GalF mutant K198A and wild type had similar affinity for Glc-1P. Results suggest that the galF gene would be a duplication of galU, after which it evolved to code for a less active, but hypothetically regulatory subunit.