In its typical biochemical role, CoA is covalently linked to metabolic intermediates through a high-energy bond (ΔGO°’ = -31.5 kJ/mol) between the pantetheine moiety of CoA and a carboxyl group. The thioester bond may be formed using the free energy of a metabolic reaction, as in the production of acetyl-CoA by pyruvate dehydrogenase or by the 3-ketoacyl thiolase reaction in the final step of each b-oxidation cycle. However, many carboxylate compounds are activated to the corresponding CoA derivative by the action of CoA ligase enzymes. The CoA ligase reaction proceeds through a two-step mechanism involving the conversion of the carboxylate and ATP to an enzyme-bound carboxyl-AMP intermediate with the release of PPi. Then, the activated carbonyl carbon of the adenylate is coupled to the thiol of CoA before AMP and the thioester are released from the enzyme. Hydrolysis of PPi in vivo makes the reaction irreversible:
CoA + R-COO- + ATP –> R-COSCoA + AMP + PPi
Closely related acyl carrier protein (ACP) ligases act in the same way since ACP shares the same phosphopantetheine prosthetic group, and a few CoA ligases are capable of using ACP as an alternative substrate. It is possible that a small number of the genes we are studying encode carboxy-ACP ligases. Also, one subset of the Arabidopsis AMP-binding proteins contains enzymes that activate carboxyl groups for conjugation to amino acids. The genes encoding these functions will not be studied under this grant. More broadly, the CoA ligases are related to firefly luciferase and to activation domains of proteins involved in non-ribosomal peptide synthesis. Crystal structures for luciferases and the PheA domain of gramicidin S synthetase are available, and these have been used as the basis for modeling the structures of several plant AAEs.