UDPglucose pyrophosphorylase from Xanthomonas spp. Characterization of the enzyme kinetics, structure and inactivation related to oligomeric dissociation

M.B. BOSCO, M. MACHTEY, A.A. IGLESIAS, M. ALEANZI

BIOCHIMIE

Elsevier

Lugar: París; Año: 2009 vol. 91 p. 204 - 213

0300-9084

The genes encoding for UDPglucose pyrophosphorylase in two Xanthomonas spp. were cloned and overexpressed in Escherichia coli. After purification to electrophoretic homogeneity, the recombinant proteins were characterized, and both exhibited similar structural and kineticproperties. They were identified as dimeric proteins of molecular mass 60 kDa, exhibiting relatively high specific activity (w80 Units/mg) for UDPglucose synthesis. Both enzymes utilized UTP or TTP as substrate with similar affinity. The purified Xanthomonas enzyme was inactivatedafter dilution into the assay medium. Studies of crosslinking with the bifunctional lysyl reagent bisuberate suggest that inactivation occurs by enzyme dissociation to monomers. UTP effectively protects the enzyme against inactivation, from which a dissociation constant of 15 mM wascalculated for the interaction substrate-enzyme. The UTP binding to the enzyme would induce conformational changes in the protein, favoring the subunits interaction to form an active dimer. This view was reinforced by protein modeling of the Xanthomonas enzyme on the basis of theprokaryotic UDPglucose pyrophosphorylase crystallographic structure. The in silico approach pointed out two main critical regions in the enzyme involved in subunit-subunit interaction: the region surrounding the catalytic-substrate binding site and the C-term.