Characterization of the Acyl Substrate Binding Pocket of Acetyl-CoA Synthetase

Abstract

AMP-forming acetyl-CoA synthetase [ACS; acetate:CoA ligase (AMP-forming), EC 6.2.1.1] catalyzes the activation of acetate to acetyl-CoA in a two-step reaction. This enzyme is a member of the adenylate-forming enzyme superfamily that includes firefly luciferase, nonribosomal peptide synthetases, and acyl- and aryl-CoA synthetases/ligases. Although the structures of several superfamily members demonstrate that these enzymes have a similar fold and domain structure, the low sequence conservation and diversity of the substrates utilized have limited the utility of these structures in understanding substrate binding in more distantly related enzymes in this superfamily. The crystal structures of the Salmonella enterica ACS and Saccharomyces cerevisiae ACS1 have allowed a directed approach to investigating substrate binding and catalysis in ACS. In the S. enterica ACS structure, the propyl group of adenosine 5'-propylphosphate, which mimics the acyl-adenylate intermediate, lies in a hydrophobic pocket. Modeling of the Methanothermobacter thermautotrophicus Z245 ACS (MT-ACS1) on the S. cerevisiae ACS structure showed similar active site architecture, and alignment of the amino acid sequences of proven ACSs indicates that the four residues that compose the putative acetate binding pocket are well conserved. These four residues, Ile312, Thr313, Val388, and Trp416 of MT-ACS1, were targeted for alteration, and our results support that they do indeed form the acetate binding pocket and that alterations at these positions significantly alter the enzyme's affinity for acetate as well as the range of acyl substrates that can be utilized. In particular, Trp416 appears to be the primary determinant for acyl chain length that can be accommodated in the binding site.

AMP-Forming Acetyl Coenzyme A Synthetase in the Outermost Membrane of the Hyperthermophilic Crenarchaeon Ignicoccus hospitalis

“…We also checked the Ignicoccus ACS sequence for the presence of amino residues putatively involved in acetate binding. Four such residues, which were identified for the S. enterica enzyme to be important for substrate binding (27) are found in a similar arrangement see (Fig. S1 in the supplemental material, marked in blue).…”

AMP‐forming acetyl‐CoA synthetases in Archaea show unexpected diversity in substrate utilization

“…Four residues (Ile 312 , Thr 313 , Val 388 , and Trp 416 ) have been shown to form the acetate binding pocket of MT-ACS1 and have been shown to be important in acyl substrate selection (Ingram-Smith et al 2006). Alteration of any of these four residues influences substrate affinity or substrate range, or both, as well as catalysis (Ingram-Smith et al 2006).…”

“…Four residues (Ile 312 , Thr 313 , Val 388 , and Trp 416 ) have been shown to form the acetate binding pocket of MT-ACS1 and have been shown to be important in acyl substrate selection (Ingram-Smith et al 2006). Alteration of any of these four residues influences substrate affinity or substrate range, or both, as well as catalysis (Ingram-Smith et al 2006). For example, alteration of Trp 416 to Gly, the residue found in short and medium chain acyl-CoA synthetases other than acetyl-and propionyl-CoA synthetases, expands the substrate range of MT-ACS1 such that the enzyme can utilize substrates ranging from acetate to octanoate (including some branched chain acyl substrates) and changes the substrate preference from acetate to valerate.…”

“…Of the eleven residues identified by ConSurf analysis to be highly conserved in traditional ACSs but not the AF-ACS2/PA-ACS subclade (Figure 4), six of these positions are in close proximity to Val 388 and Trp 416 of MT-ACS1 ( Figure 5A), the two acetate pocket residues that were shown to have the greatest influence on substrate range and preference (Ingram-Smith et al 2006 , and Sulfolobus acidocaldarius (SA-ACS) using Clustal X (Thompson et al 1997). A partial alignment is shown here.…”

“…MT-ACS1 is a typical ACS in that acetate is the strongly preferred substrate over propionate and it cannot utilize larger substrates such as butyrate. Through modeling of MT-ACS1 on the S. enterica and yeast ACS structures, Ingram-Smith et al (2006) identified four residues that comprise at least part of the acetate binding pocket of MT-ACS1 and have shown that alterations of these residues can greatly influence acyl substrate range and preference.…”