Structural insights into GDP-mediated regulation of a bacterial acyl-CoA thioesterase

Abstract

Thioesterases catalyze the cleavage of thioester bonds within many activated fatty acids and acyl-CoA substrates. They are expressed ubiquitously in both prokaryotes and eukaryotes and are subdivided into 25 thioesterase families according to their catalytic active site, protein oligomerization, and substrate specificity. Although many of these enzyme families are well-characterized in terms of function and substrate specificity, regulation across most thioesterase families is poorly understood. Here, we characterized a TE6 thioesterase from the bacterium Neisseria meningitidis Structural analysis with X-ray crystallographic diffraction data to 2.0-Å revealed that each protein subunit harbors a hot dog-fold and that the TE6 enzyme forms a hexamer with D3 symmetry. An assessment of thioesterase activity against a range of acyl-CoA substrates revealed the greatest activity against acetyl-CoA, and structure-guided mutagenesis of putative active site residues identified Asn²⁴ and Asp³⁹ as being essential for activity. Our structural analysis revealed that six GDP nucleotides bound the enzyme in close proximity to an intersubunit disulfide bond interactions that covalently link thioesterase domains in a double hot dog dimer. Structure-guided mutagenesis of residues within the GDP-binding pocket identified Arg⁹³ as playing a key role in the nucleotide interaction and revealed that GDP is required for activity. All mutations were confirmed to be specific and not to have resulted from structural perturbations by X-ray crystallography. This is the first report of a bacterial GDP-regulated thioesterase and of covalent linkage of thioesterase domains through a disulfide bond, revealing structural similarities with ADP regulation in the human ACOT12 thioesterase.

Keywords: Neisseria meningitidis; acetyl coenzyme A (acetyl-CoA); coenzyme A (CoA); enzyme kinetics; enzyme regulation; enzyme structure; hotdog-fold; hydrolase; thioesterase.

Figure 1.

Structure of NmACT. A, each NmACT protomer contains a five-stranded β-sheet (pink) that wraps a central α-helix (cyan). An additional C-terminal α-helix packs against the β-sheet on the opposite side of the central α-helix. B, topology of NmACT and associated primary sequence.

Figure 2.

Quaternary structure of NmACT. A, the asymmetric unit is composed of four thioesterase arranged as double hot dog dimers. B, the biological unit, which is composed of a timer-of-double hot dog thioesterases, is formed from three asymmetric units.

Figure 3.

Small angle scattering profile of experimental data (small gray dots) and overlaid predicted scattering profile (dark line) of a hexameric thioesterase arranged with D3 symmetry as determined in the crystal structures.

Figure 4.

The biological unit of the NmACT is composed of a hexamer of thioesterase domain protomers exhibiting D3 symmetry (left). The two interfaces that mediate arrangement of the biological assembly are depicted middle and right panels.

Figure 5.

Each biological unit contained six GDP molecules, six CoA molecules, and six disulfide bonds. Both the molecules and disulfide bonds are from interactions across thioesterase protomers in a double hot dog configuration.

Figure 6.

Surface view of a double hot dog domain and bound GDP. Left, residues that contribute the binding pocket are depicted. Right, detailed bonding interactions of GDP using Ligplot. Hydrogen bonds are depicted as green dashes, and hydrophobic interactions as red fans.

Figure 7.

Activity of NmACT and mutants against acetyl-CoA substrate. Upper panel, screening of substrates (330 μm of each substrate per reaction, acetyl-CoA (C2-CoA), malonyl-CoA (C3-CoA), butyryl-CoA (C4-CoA), hexanoyl-CoA (C6-CoA), octanoyl-CoA (C8-CoA), decanoyl-CoA (C10-CoA), lauroyl-CoA (C12-CoA), myristoyl-CoA (C14-CoA), palmitoyl-CoA (C16-CoA), stearoyl-CoA (C18-CoA), and arachidonoyl-CoA (C20-CoA)) against NmACT. Lower panel, specific activity comparison of NmACT and mutants including NmACT-Cys¹⁵⁸-X, NmACT-N24A, NmACT-D39A, and NmACT-Cys¹⁵⁸-X:R93E.

Figure 8.

Structural alignment of thioesterases deposited to the PDB. The Asn²⁴ and Asp³⁹ residues, identified as catalytically important, are highly conserved.

Figure 9.

Domain organization of TE6 thioesterases. Prokaryotic thioesterases harbor single thioesterase domains, whereas eukaryotic ones contain fused thioesterase domains. The linkage of thioesterase domains by a disulfide bond may be analogous to a double hot dog domain fusion observed in eukaryotes.

Figure 10.

Dimer of (A) NmACT-WT and (B) truncated version NmACT-Cys¹⁵⁸-X showing the presence of CoA and GDP supported by a 2F_o − F_c annealed omit map contoured at 2σ (green mesh), whereas (C) Cys¹⁵⁸-X:R93E has no GDP supported, by an absence of density from a 2F_o − F_c annealed omit map contoured at 2σ (green mesh).

Figure 11.

Quaternary structures of NmACT wild-type, truncated mutant (Cys¹⁵⁸-X), and mutants targeting the active site (N24A and D39A) and GDP binding (Cys¹⁵⁸-X:R39E) confirming similar structures and mutations do not disrupt the structure.

Figure 12.

Structural comparison of NmACT and hACOT12. The overall arrangement of thioesterase domains is highly similar. NmACT binds six GDP and six CoA molecules, whereas hACOT12 binds only half the number due to non-identical hot dog domains. Fusion of the thioesterase domains have arisen through domain duplication, whereas NmACT has fused double hot dog dimers through a disulfide linkage.

Figure 13.

Superimposition of a double hot dog dimer of NmACT with a protomer of hACOT12. Shown are the GDP and ADP nucleotides superimposed and positioned in highly similar orientations.

Figure 14.

The nucleotide-binding residues in ACOT12 are highly similar in NmACT. NmACT contains single thioesterase domains fused as a double hot dog dimer through a disulfide interaction, whereas ACOT12 is fused through a 26-amino acid linker.