The ANL superfamily of adenylating enzymes contains acyl- and aryl-CoA synthetases, firefly luciferase, and the adenylation domains of the modular Non-Ribosomal Peptide Synthetases (NRPSs). Members of this family catalyze two partial reactions, the initial adenylation of a carboxylate to form an acyl-AMP intermediate, followed by a second partial reaction, most commonly, the formation of a thioester. Recent biochemical and structural evidence has been presented that supports the use by this enzyme family of a remarkable catalytic strategy for the two catalytic steps. The enzymes use a 140° domain rotation to present opposing faces of the dynamic C-terminal domain to the active site for the different partial reactions. Support for this Domain Alternation strategy is presented along with an explanation of the advantage of this catalytic strategy on the reaction catalyzed by the ANL enzymes. Finally, the ramifications of this domain rotation in the catalytic cycle of the modular NRPS enzymes are discussed.

Many important natural products are produced by multidomain nonribosomal peptide synthetases (NRPSs)1–4. During synthesis, intermediates are covalently bound to integrated carrier domains and transported to neighboring catalytic domains in an assembly line fashion5. Understanding the structural basis for catalysis with NRPSs will facilitate bioengineering to create novel products. Here we describe the structures of two different holo-NRPSs modules, each revealing a distinct step in the catalytic cycle. One structure depicts the carrier domain cofactor bound to the peptide bond-forming condensation domain, whereas a second structure captures the installation of the amino acid onto the cofactor within the adenylation domain. These structures demonstrate that a conformational change within the adenylation domain guides transfer of intermediates between domains. Furthermore, one structure shows that the condensation and adenylation domains simultaneously adopt their catalytic conformations, increasing the overall efficiency in a revised structural cycle. These structures and single-particle electron microscopy analysis demonstrate a highly dynamic domain architecture and provide the foundation for understanding the structural mechanisms that could enable engineering novel NRPSs.

Acetyl-coenzyme A synthetase catalyzes the two-step synthesis of acetyl-CoA from acetate, ATP, and CoA and belongs to a family of adenylate-forming enzymes that generate an acyl-AMP intermediate. This family includes other acyl- and aryl-CoA synthetases, firefly luciferase, and the adenylation domains of the modular nonribosomal peptide synthetases. We have determined the X-ray crystal structure of acetyl-CoA synthetase complexed with adenosine-5'-propylphosphate and CoA. The structure identifies the CoA binding pocket as well as a new conformation for members of this enzyme family in which the approximately 110-residue C-terminal domain exhibits a large rotation compared to structures of peptide synthetase adenylation domains. This domain movement presents a new set of residues to the active site and removes a conserved lysine residue that was previously shown to be important for catalysis of the adenylation half-reaction. Comparison of our structure with kinetic and structural data of closely related enzymes suggests that the members of the adenylate-forming family of enzymes may adopt two different orientations to catalyze the two half-reactions. Additionally, we provide a structural explanation for the recently shown control of enzyme activity by acetylation of an active site lysine.