Construction and Characterization of a Chimeric NDPglucose Pyrophosphorylase

L.I. MARTÍNEZ, S.A. GUERRERO, J. PREISS, A.A. IGLESIAS

Mar del Plata, Buenos Aires, Argentina

Congreso; XLIII Reunión Anual de SAIB; 2007

SAIB

EN-P20.CONSTRUCTION AND CHARACTERIZATION OF ACHIMERICNDPGLUCOSEPYROPHOSPHORYLASEMartínez LI, Guerrero SA, Preiss J, Iglesias AA.Lab Enzimología Molecular, FBCB, UNL-CONICET, Santa Fe,Argentina and *Dept Biochem Mol Biol, MSU, USA. E-mail:lucilama@fbcb.unl.edu.arStreptococcus mutansEscherichia coliE. coliN u c l e o s i d e - d i p h o s p h o - s u g a r p y r o p h o s p h o r y l a s e s(NDPSugPPases) are enzymes involved in carbohydratemetabolism, catalyzing the synthesis of different NDPSugderivatives. All NDPSugPPases from bacteria have a similar 3Dstructure, except for ADPGlcPPases that are longer proteins mainlybecause of an additional C-term domain. Most of theADPGlcPPasesare allosterically regulated; with the C-term domain playing a keyrole in this characteristic, distinctive respect to otherNDPSugPPases. We constructed a chimeric gene codifying for ahybrid combining the whole UDPGlcPPase (EC 2.7.7.9) fromplus the C-term (amino acids 295 to 432) ofthe ADPGlcPPase (EC 2.7.7.27) from . The formeris an unregulated enzyme exhibiting specificity toward UTP,whereas the ADPGlcPPase is specific for ATP and is allostericallyregulated by fructose-1,6-bisP (activator) andAMP (inhibitor). Thehybrid gene was expressed in as a recombinant-His-tagprotein and then purified. The chimeric protein was active asUDPGlcPPase and it was sensitive to activation by 3P-glycerate.Thus, the hybrid enzyme exhibited regulatory properties similar tothe ADPGlcPPase from cyanobacteria. Results agree with the viewthat the C-term domain is critical for allosteric regulation of PPases,being its interaction with the N-term determinant for the specificityto the regulatory molecule.