The 2.1 A crystal structure of an acyl-CoA synthetase from Methanosarcina acetivorans reveals an alternate acyl-binding pocket for small branched acyl substrates.

Enzyme ligases use the chemical energy derived from hydrolysis of the phosphate esters of ATP to generate chemical bonds. We have been studying a large family of acyl-AMP forming ligases that activate a substrate carboxylate with ATP to form an acyl- or aryladenylate. In a second enzymatic step, these enzymes catalyze the formation of a thioester with CoA or the CoA derived pantetheine cofactor of an acyl carrier protein. Work in our lab has recently demonstrated a novel mechanism that these enzymes use to segregate kinetically and conformationally the activities of these two half-reactions 1–4. In this model, the ping-pong catalytic mechanism uses domain rearrangements to isolate the two partial reactions. The acyl-AMP forming superfamily of adenylating enzymes can be divided into three sub-families, acyl- and aryl-CoA synthetases, the adenylation domains of non-ribosomal peptide synthetases (NRPSs), and firefly luciferase enzymes 5–7. The first two sub-families are closely related and catalyze the formation of a thioester between the original carboxylate substrate and either CoA or the pantetheine cofactor of an NRPS acyl carrier protein (Scheme 1). The acyl- and aryl-CoA synthetase subfamily is broadly reactive with a diverse array of organic acids, ranging from small molecules like acetate and propionate to larger fatty acids and aromatic compounds. In the reaction catalyzed by firefly luciferase, the activated luciferyl-adenylate reacts in an oxidative decarboxylation reaction to produce a high energy intermediate that decomposes to yield a photon of light 8. Strengthening the ties between the different sub-families, the luciferase enzyme has been reported to catalyze the formation of luciferyl-CoA9. Scheme 1 A number of crystal structures have been determined for members of the adenylate-forming family of enzymes 1,2,10–22. These structures have demonstrated that the enzymes are composed of two domains, a larger N-terminal domain and a smaller C-terminal domain. Interestingly, these studies identified two catalytically relevant conformations, one for the first adenylation reaction, and a second conformation for the thioester-forming reaction. The two conformations differ by a ~140° domain rotation that occurs between the first and second half-reactions. A conserved residue, either an aspartic acid or less commonly a lysine, is present at the pivot point between the two domains. This hinge residue adopts alternate main chain torsion angles to accommodate the large conformational change. Our structural and functional studies of 4-chlorobenzoyl-CoA ligase have demonstrated that the rotation of the C-terminal domain serves to remove from the active site several residues that play catalytic roles in the initial adenylation reaction to thereby avoid steric hindrance of the thioester-forming reaction3. Accompanying this removal of these residues, the rotation of the C-terminal domain creates a binding tunnel for the CoA pantetheine moiety. The structures of these enzymes have allowed the characterization and prediction of the binding pocket of different family members, most notably, the NRPS adenylation domains23,24. Using several well-conserved sequences within the N-terminal domain as “anchor residues”, it is possible to identify the regions of the protein that form the amino acyl-binding pocket in different family members. A more elaborate computational approach using support vector machines correlates well with these earlier methods and extends the predictions to additional NRPS adenylation domains25. These studies have allowed the prediction of enzyme specificity in certain instances26 yet the ultimate goal of engineering the complete synthesis of a novel peptide by altering the adenylation domain specificity remains elusive. An important determinant in predictions of NRPS adenylation domain activity is the presence of a conserved aspartic acid residue that interacts with the amino group of the substrate amino acids. Because of the fixed position of the carboxylate and the α-amino group of the amino acid substrate, the side chains of the L-amino acids are directed into a single pocket composed of enzyme residues that are tailored for the specific amino acid substrate. Less well characterized are the binding pockets of acyl- and aryl-CoA synthetases and thus a protein sequence is not sufficient to predict with certainty the specific activity. To date, structures have been determined for short chain acyl-CoA synthetases2,15,20, several aryl-CoA synthetases (often called ligases)1,10, a medium chain acyl-CoA synthetase 16, and a longer chain fatty acyl-CoA synthetase14. These structures have provided insights for initial attempts at modulating substrate specificity27. To provide further insight into this enzyme family, and to allow a better understanding of the relationship between acyl substrate specificity and the acyl binding pocket residues, Ingram-Smith et al. have recently cloned a putative acyl-CoA synthetase from the methanoarchaeon Methanosarcina acetivorans and characterized the catalytic preferences of this enzyme. This enzyme was analyzed for the formation of acyl-adenylates and acyl-CoA thioesters with a panel of substrates. This analysis showed that the enzyme is most active with propionate, butyrate, and the branched analogs, 2-methyl-propionate, butyrate, and pentanoate. The specific activity is weaker for smaller or larger acids. Interestingly, with some substrates, the enzyme appears to release the acyl-adenylate intermediate in the presence of CoA while the affinity of the enzyme for some substrates causes the acyl-adenylate to be retained until CoA binds. The full functional characterization of this protein will be presented elsewhere (C. Ingram-Smith, Y. Meng, L. L. Cooper, and K.S. Smith, submitted). We present here the three-dimensional structure of this enzyme, solved by multi-wavelength anomalous dispersion phasing methods and refined against data to 2.1 A resolution. This work identifies the active site residues of this newly identified family member and demonstrates the architecture for the acyl-binding pocket. The pocket of this acyl-CoA synthetase is shown to have a cavity volume comparable to the enzyme 4-chlorobenzoyl-CoA ligase. The pocket, however, is much more shallow in the current structure than previously observed acyl binding pockets within this family. The pocket of this enzyme is compared to the acyl binding pockets of previously characterized enzymes. The structural diversity of these active site residues are described and emphasize the need for structural information to enable the prediction of substrate specificity with the acyl-CoA synthetases.