Complex formation and regulation of Escherichia coli acetyl-CoA carboxylase

Acetyl-CoA carboxylase is a biotin-dependent, multifunctional enzyme that catalyzes the first committed step in fatty acid synthesis. The Escherichia coli enzyme is composed of a homodimeric biotin carboxylase, biotinylated biotin carboxyl carrier protein (BCCP), and an α2β2 heterotetrameric carboxyltransferase. Catalysis by acetyl-CoA carboxylase proceeds via two half-reactions. In the first half-reaction, biotin carboxylase catalyzes the ATP-dependent carboxylation of biotin, which is covalently attached to BCCP, to form carboxybiotin. In the second half-reaction, carboxyltransferase transfers the carboxyl group from carboxybiotin to acetyl-CoA to form malonyl-CoA. All biotin-dependent carboxylases are proposed to have a two-site ping-pong mechanism where the carboxylase and transferase activities are separate and do not interact. This posits two hypotheses: either biotin carboxylase and BCCP undergo the first half-reaction, BCCP dissociates, and then BCCP interacts with carboxyltransferase to complete the second-half reaction, or all three components form a macromolecular enzyme complex. To determine which hypothesis is correct, a steady-state enzyme kinetic analysis of E. coli acetyl-CoA carboxylase was carried out. The results indicated the two active sites of acetyl-CoA carboxylase interact. Both in vitro and in vivo pull-down assays demonstrated that the three components of E. coli acetyl-CoA carboxylase form a multimeric complex and that complex formation is unaffected by acetyl-CoA, AMPPNP, and mRNA coding for carboxyltransferase. The second study focuses on the crystallization of part of the acetyl-CoA carboxylase complex. To date, the three-dimensional structures of the individual subunits, except the N-terminal domain of BCCP, have been solved; however, the structural basis for how BCCP reacts with biotin carboxylase or carboxyltransferase is unknown. Therefore, we report here the first crystal structure of E. coli BCCP complexed with biotin carboxylase to a resolution of 2.49 Å. The protein-protein complex shows unique quaternary structure and two distinct interfaces for each BCCP monomer. The structure supports a model by which biotin protein ligase can biotinylate the complex. The BCCP binding sites are unique compared to phylogenetically related biotin-dependent carboxylases and therefore, provide novel targets for developing antibiotics against bacterial acetyl-CoA carboxylase. Taken together, these findings provide structural and functional insight into the regulation and complex formation of E. coli acetyl-CoA carboxylase.