Limits...
The ancestral activation promiscuity of ADP-glucose pyrophosphorylases from oxygenic photosynthetic organisms.

Kuhn ML, Figueroa CM, Iglesias AA, Ballicora MA - BMC Evol. Biol. (2013)

Bottom Line: 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.Its presence could constitute an efficient evolutionary mechanism to accommodate the ADP-Glc PPase regulation to different metabolic needs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemistry and Biochemistry, Loyola University Chicago, 1032 W, Sheridan Rd, Chicago, IL 60660, USA.

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.

Show MeSH
Effect of OtaL on the affinity of OtaS for 3-PGA. The OtaS homotetramer is similarly activated by 3-PGA and FBP, whereas the mutant OtaS/OtaLD171A heterotetramer has a higher affinity for 3-PGA than FBP. The plots show the activation of OtaS (A) and OtaS/OtaLD171A (B) by 3-PGA (▪) and FBP (·).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3585822&req=5

Figure 3: Effect of OtaL on the affinity of OtaS for 3-PGA. The OtaS homotetramer is similarly activated by 3-PGA and FBP, whereas the mutant OtaS/OtaLD171A heterotetramer has a higher affinity for 3-PGA than FBP. The plots show the activation of OtaS (A) and OtaS/OtaLD171A (B) by 3-PGA (▪) and FBP (·).

Mentions: Contrary to what we observed in the homotetrameric OtaS enzyme, the mutant OtaSD148A/OtaL had a specificity constant for 3-PGA three orders of magnitude higher than for FBP (2000 and 0.5 mM-1, respectively) (Table 1 and Figure 2E). It is worth noting that in this mutant the second activator was Fru6P, with a specificity constant of 5.4 mM-1, a different trend compared to OtaS/OtaL and OtaS where the second activator was FBP (Table 1). A similar result was observed for the mutant OtaS/OtaLD171A, which had a specificity constant for 3-PGA that was higher than that for FBP (61 and 3.3 mM-1, respectively) (Table 1 and Figure 2F). Results obtained with this mutant resemble those for OtaS/OtaL, where FBP is a more efficient activator than Fru6P (Table 1). The effect of the addition of OtaLD171A to OtaS on the specificity for activators is particularly striking at lower concentrations (Figure 3). Between 0.1-0.2 mM OtaS/OtaLD171A is almost fully activated by 3-PGA but only slightly by FBP. On the other hand, OtaS is poorly activated by both.


The ancestral activation promiscuity of ADP-glucose pyrophosphorylases from oxygenic photosynthetic organisms.

Kuhn ML, Figueroa CM, Iglesias AA, Ballicora MA - BMC Evol. Biol. (2013)

Effect of OtaL on the affinity of OtaS for 3-PGA. The OtaS homotetramer is similarly activated by 3-PGA and FBP, whereas the mutant OtaS/OtaLD171A heterotetramer has a higher affinity for 3-PGA than FBP. The plots show the activation of OtaS (A) and OtaS/OtaLD171A (B) by 3-PGA (▪) and FBP (·).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3585822&req=5

Figure 3: Effect of OtaL on the affinity of OtaS for 3-PGA. The OtaS homotetramer is similarly activated by 3-PGA and FBP, whereas the mutant OtaS/OtaLD171A heterotetramer has a higher affinity for 3-PGA than FBP. The plots show the activation of OtaS (A) and OtaS/OtaLD171A (B) by 3-PGA (▪) and FBP (·).
Mentions: Contrary to what we observed in the homotetrameric OtaS enzyme, the mutant OtaSD148A/OtaL had a specificity constant for 3-PGA three orders of magnitude higher than for FBP (2000 and 0.5 mM-1, respectively) (Table 1 and Figure 2E). It is worth noting that in this mutant the second activator was Fru6P, with a specificity constant of 5.4 mM-1, a different trend compared to OtaS/OtaL and OtaS where the second activator was FBP (Table 1). A similar result was observed for the mutant OtaS/OtaLD171A, which had a specificity constant for 3-PGA that was higher than that for FBP (61 and 3.3 mM-1, respectively) (Table 1 and Figure 2F). Results obtained with this mutant resemble those for OtaS/OtaL, where FBP is a more efficient activator than Fru6P (Table 1). The effect of the addition of OtaLD171A to OtaS on the specificity for activators is particularly striking at lower concentrations (Figure 3). Between 0.1-0.2 mM OtaS/OtaLD171A is almost fully activated by 3-PGA but only slightly by FBP. On the other hand, OtaS is poorly activated by both.

Bottom Line: 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.Its presence could constitute an efficient evolutionary mechanism to accommodate the ADP-Glc PPase regulation to different metabolic needs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemistry and Biochemistry, Loyola University Chicago, 1032 W, Sheridan Rd, Chicago, IL 60660, USA.

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.

Show MeSH