W0058

Acetyl-CoA Synthetase uses a 140° Domain Rotation to Catalyze a Two-Step Reaction. Andrew M. Gulick^1,2, Kristen M. Homick¹, Vincent J. Starai³, and Jorge C. Escalante-Semerena³, ¹Hauptman-Woodward Medical Research Institute, Buffalo, NY, ²Dept. of Structural Biology, Univ. at Buffalo, Buffalo, NY, ³Dept. of Bacteriology, Univ. of Wisconsin, Madison, WI.

We have determined the 1.75 Å crystal structure of Acetyl-CoA Synthetase (Acs, 652 residues), a member of an adenylate-forming family of enzymes that catalyze two-step adenylation/thioesterification reactions. Comparison of our structure to the structures of related proteins at different stages of the catalytic mechanism suggests that these enzymes adopt one conformation to catalyze the adenylation half-reaction, then undergo a 140° rotation of the C-terminal domain to catalyze the second half-reaction.

The first step in the reaction catalyzed by Acs is an adenylation reaction in which acetate and ATP are used to form acetyl-AMP. This acyl-adenylate reacts in a second half-reaction to form a thioester with CoA. The structure was solved at 2.7 Å resolution by MIR using four heavy atom derivatives. The model was then refined against 1.75 Å synchrotron data obtained at CHESS Beamline F2.

Crystals of Acs were obtained in the presence of CoA and adenosine-5’-propylphosphate, a non-reactive mimic of the adenylate intermediate. Acs and related synthetases use a Bi-Uni-Uni-Bi ping pong kinetic mechanism. The CoA and inhibitor molecules were clearly identified in our structure and the model represents the protein conformation at the start of the second half-reaction. In comparison to the orientation of the C-terminal domain seen in the structures of two Non-Ribosomal Peptide Synthetases (NRPS) adenylation domains, the C-terminal domain of Acs is rotated by 140°. We propose that members of this adenylate-forming family of enzymes adopt one conformation to catalyze the adenylation reaction and a second conformation to catalyze the second half-reaction. This large domain movement that is used to catalyze the complete reaction has interesting implications for the NRPS adenylation domains, raising questions about how these modular enzymes are able to accommodate such a dramatic conformational change.