Kinetic Characterization and Homology Modeling of UDPglucose Pyrophosphorylase from Xanthomonas?

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

Mar del Plata, Buenos Aires, Argentina

Congreso; XLIII Reunión Anual de SAIB; 2007

SAIB

EN-P17.KINETIC CHARACTERIZATION AND HOMOLOGYMODELING OF UDPGLUCOSE PYROPHOSPHORYLASEFROMXANTHOMONASMachtey M, Bosco MB,Aleanzi MC, Iglesias AA.Lab Enzimología Molecular, FBCB, UNL, CC 242, S300ZAA SantaFé, Argentina. E-mail: matimach@gmail.comXanthomonas X. campestris X. axonopodiscitri XanthomonasEscherichia coliCorynebacterium glutamicumUDPglucose pyrophosphorylase (EC 2.7.7.9; UDPGlcPPase) is ofcentral importance for the synthesis of polysaccharides, sinceUDPGlc is the essential precursor in the pathway. We cloned,expressed in a recombinant manner and purified the enzyme fromtwo species: and pv.. Both enzymes exhibited identical properties.UDPGlcPPase was characterized as a homodimer, after native- andSDS-PAGE of oligomeric structures stabilized by cross-linkingwith bisuberate. One particular behavior, not described for otherUDPGlcPPases is that at dilutions utilized for activity assay theenzyme undergoes inactivation. The latter is prevented by UTP,which also stabilized the dimeric form of the enzyme in experimentsof subunits cross-linking. Kinetics of the enzyme inactivationperformed at different UTP concentrations allowed to calculate adissociation constant for the substrate of 0.010 mM. Studies ofhomology modeling were performed using the structures of therecently crystallized UDPGlcPPases from Eshcerichia coli andCorynebacterium glutamicum. This analysis shows the relevance ofthe C-terminal alpha-helix, and the helixloop-helix motif aroundK84 for a tight interaction between subunits. Results suggest that thedimer is the active form of the Xanthomonas UDPGlcPPase and thatits dissociation accounts for the enzyme inactivation.