Ostreococcus tauri ADP-glucose Pyrophosphorylase: Insights into the activation binding site

C.M. FIGUEROA; M.L. KUHN; A.A. IGLESIAS; M.A. BALLICORA

Puerto Madryn

Congreso; XLVI Reunión Anual de SAIB; 2010

SAIB

O s t r e o c o c c u s t a u r i A D P - G L U C O S E PYROPHOSPHORYLASE: INSIGHTS INTO THE ACTIVATOR BINDING SITE1 2 1 2 Figueroa CM , Kuhn ML , Iglesias AA , Ballicora MA 1 2 IAL (UNL-CONICET), Santa Fe, Argentina, and Department of C h e m i s t r y , L U C , C h i c a g o , U S A . E - m a i l carfigue@fbcb.unl.edu.ar ADP-glucose pyrophosphorylase (ADPGlcPPase) controls the synthesis of starch in plants and algae. The enzyme from photosynthetic organisms is a heterotetramer, composed of two small (S) and two large (L) subunits, and is mainly activated by 3-phosphoglycerate (3PGA), the first intermediary of the carbon fixation pathway. To study the evolution of structure and function of subunits in the ADPGlcPPase family, we synthesized the genes encoding for the S (OtaS) and L (OtaL) subunits of the unicellular alga Ostreococcus tauri, one of the most ancient eukaryotic species in the green lineage. An interesting feature of the OtaL subunit is that a highly conserved Lys residue, present in the OtaS subunit, demonstrated to be important for 3PGA binding in the Anabaena ADPGlcPPase, is replaced by Arg. In this work, we constructed the mutants OtaS and OtaL and co-expressed them together or K443R R466K with the corresponding wild type subunits. 3PGA kinetics showed A values of 0.22, 0.58, 1.2 and 2.9 mM for OtaS/OtaL , 0.5 R466K OtaS/OtaL, OtaS /OtaL and OtaS /OtaL, respectively. K443R R466K K443R In addition, OtaS /OtaL displayed the lowest activation fold, K443R thus confirming the importance of the mutated Lys residue for 3PGA activation. Our results suggest that both subunits evolved independently and adopted divergent activation properties associated with metabolic differences present in each organism.