ADPGlcPPase and UDPGlcPPase: Understanding Carbon Partitioning in Mycobacterium tuberculosis

M.D. ASENCIÓN DIEZ, A.M. DEMONTE, F. BIGI, M. ROMANO, S.A. GUERRERO, A.A. IGLESIAS

Mar del Plata, Buenos Aires, Argentina

Congreso; XLIII Reunión Anual de SAIB; 2007

SAIB

EN-P15.A D P G L C P PA S E A N D U D P G L C P PA S E : UNDERSTANDING CARBON PARTITIONING IN MYCOBACTERIUMTUBERCULOSISAsención Diez MD, Demonte AM, Bigi F*, Romano M*, Guerrero SA, Iglesias AA.Lab de Enz Molecular y Bqca Microbiana, FBCB, UNL, Sta Fe & *Inst de Biotecnología CICVyA-INTA, BsAs. E-mail: md.asencion@gmail.comUDP-glucose pyrophosphorylase (EC 2.7.7.9; UDPGlcPPase)catalyzes the reaction Glc1P + UTP <=> UDPGlc + PPi, in thepresence ofMg . In bacteria, UDPGlc is the main glucosil donor forthe biosynthesis of structure polysaccharides, glycolipids,glycoproteins and UDPGlc derived nucleotide-sugars. On the otherhand, ADPGlc, synthesized via ADPGlcPPase (EC 2.7.7.27) is theglucosil donor for the pathway leading to glycogen. Characterizationof both NDPGlcPPases is critical for the better understanding of Glcpartitioning into reserve and structure carbohydrates.We have solvedthe expression of ADPGlcPPase. The enzyme wascharacterized and found to be regulated by PEPand Glc6P. The latter,increase the enzyme affinity for Glc1P and interestingly, was notdescribed as an effector for other ADPGlcPPases. The same schemewas used to obtain UDPGlcPPase. The U genewas cloned into pMIP12 and competent mc 155 weretransformed with the construct [pMIP12/ U]. The protein wasexpressed with a C-Term HisTag and purified chromatographically.Functional characterization was performed in both UDPGlcsynthesis and pyrophosphorolysis. The analysis of the kinetic andregulatory properties of both NDPGlcPPases is useful to propose aregulatory scenario for Glc1P partitioning into different metabolicpathways for carbon and energy in mycobacteria.