title: The ancestral activation promiscuity of ADP-glucose pyrophosphorylases from oxygenic photosynthetic organisms.
abstract: BACKGROUND
  ADP-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the first committed step in the synthesis of glycogen in bacteria and starch in algae and plants. In oxygenic photosynthetic organisms, ADP-Glc PPase is mainly activated by 3-phosphoglycerate (3-PGA) and to a lesser extent by other metabolites. In this work, we analyzed the activation promiscuity of ADP-Glc PPase subunits from the cyanobacterium Anabaena PCC 7120, the green alga Ostreococcus tauri, and potato (Solanum tuberosum) tuber by comparing a specificity constant for 3-PGA, fructose-1,6-bisphosphate (FBP), fructose-6-phosphate, and glucose-6-phosphate.
  
  
  RESULTS
  The 3-PGA specificity constant for the enzymes from Anabaena (homotetramer), O. tauri, and potato tuber was considerably higher than for other activators. O. tauri and potato tuber enzymes were heterotetramers comprising homologous small and large subunits. Conversely, the O. tauri small subunit (OtaS) homotetramer was more promiscuous because its FBP specificity constant was similar to that for 3-PGA. To explore the role of both OtaS and OtaL (O. tauri large subunit) in determining the specificity of the heterotetramer, we knocked out the catalytic activity of each subunit individually by site-directed mutagenesis. Interestingly, the mutants OtaSD148A/OtaL and OtaS/OtaLD171A had higher specificity constants for 3-PGA than for FBP.
  
  
  CONCLUSIONS
  After gene duplication, OtaS seemed to have lost specificity for 3-PGA compared to FBP. This was physiologically and evolutionarily feasible because co-expression of both subunits restored the specificity for 3-PGA of the resulting heterotetrameric wild type enzyme. This widespread promiscuity seems to be ancestral and intrinsic to the enzyme family. Its presence could constitute an efficient evolutionary mechanism to accommodate the ADP-Glc PPase regulation to different metabolic needs.
authors: Department of Chemistry and Biochemistry, Loyola University Chicago, 1032 W, Sheridan Rd, Chicago, IL 60660, USA.
authors: Misty L
authors: ML
authors: Kuhn
authors: Carlos M
authors: CM
authors: Figueroa
authors: Alberto A
authors: AA
authors: Iglesias
authors: Miguel A
authors: MA
authors: Ballicora
mesh_terms: D017033:Anabaena / Q000201:enzymology* / Q000235:genetics; D000460:Chlorophyta / Q000201:enzymology* / Q000235:genetics; D005635:Fructosediphosphates / Q000378:metabolism; D005636:Fructosephosphates / Q000378:metabolism; D020440:Gene Duplication; D051599:Glucose-1-Phosphate Adenylyltransferase / Q000235:genetics / Q000378:metabolism*; D019298:Glucose-6-Phosphate / Q000378:metabolism; D005988:Glyceric Acids / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010802:Phylogeny; D035281:Plant Tubers / Q000201:enzymology; D011198:Solanum tuberosum / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D005635:Fructosediphosphates; D005636:Fructosephosphates; D005988:Glyceric Acids; D019298:Glucose-6-Phosphate; C027618:fructose-6-phosphate; C005156:3-phosphoglycerate; D051599:Glucose-1-Phosphate Adenylyltransferase; C029063:fructose-1,6-diphosphate