Abstract
Acyl-coenzyme A (acyl-CoA) thioesterases play a crucial role in the metabolism of activated fatty acids, coenzyme A, and other metabolic precursor molecules including arachidonic acid and palmitic acid. These enzymes hydrolyze coenzyme A from acyl-CoA esters to mediate a range of cellular functions including β-oxidation, lipid biosynthesis, and signal transduction. Here, we present the crystal structure of a hexameric hot-dog domain-containing acyl-CoA thioesterase from Bacillus halodurans in the apo-form and provide structural and comparative analyses to the coenzyme A-bound form to identify key conformational changes induced upon ligand binding. We observed dramatic ligand-induced changes at both the hot-dog dimer and the trimer-of-dimer interfaces; the dimer interfaces in the apo-structure differ by over 20% and decrease to about half the size in the ligand-bound state. We also assessed the specificity of the enzyme against a range of fatty acyl-CoA substrates and have identified a preference for short-chain fatty acyl-CoAs. Coenzyme A was shown both to negatively regulate enzyme activity, representing a direct inhibitory feedback, and consistent with the structural data, to destabilize the quaternary structure of the enzyme. Coenzyme A-induced conformational changes in the C-terminal helices of enzyme were assessed through mutational analysis and shown to play a role in regulating enzyme activity. The conformational changes are likely to be conserved from bacteria through to humans and provide a greater understanding, particularly at a structural level, of thioesterase function and regulation.
Highlights
IntroductionSignificant variation is observed at the C-terminal ␣-helices to accommodate the ligand (Fig. 5)
Upon ligand binding, significant variation is observed at the C-terminal ␣-helices to accommodate the ligand (Fig. 5)
We found that CoA dramatically inhibited the enzyme (Fig. 8)
Summary
Significant variation is observed at the C-terminal ␣-helices to accommodate the ligand (Fig. 5). In the apo-form, both helices within the double hot-dog are bound through interactions involving Asn[149] and Arg[153]; Ala[156] and Ala[145]; and Arg[157] and Glu[135]. In the ligand-bound form, these interactions are disrupted due to favorable interactions with the ligand: in particular, Lys[147] interaction with the 3Ј-phosphoryl group and Arg[150] interaction with the adenine bas
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