Structural Characterization of a 140° Domain Movement in the Two-Step Reaction Catalyzed by 4-Chlorobenzoate:CoA Ligase

Abstract

Members of the adenylate-forming family of enzymes play a role in the metabolism of halogenated aromatics and of short, medium, and long chain fatty acids, as well as in the biosynthesis of menaquinone, peptide antibiotics, and peptide siderophores. This family includes a subfamily of acyl- and aryl-CoA ligases that catalyze thioester synthesis through two half-reactions. A carboxylate substrate first reacts with ATP to form an acyl-adenylate. Subsequent to the release of the product PPi, the enzyme binds CoA, which attacks the activated acyl group to displace AMP. Structural and functional studies on different family members suggest that these enzymes alternate between two conformations during catalysis of the two half-reactions. Specifically, after the initial adenylation step, the C-terminal domain rotates by ~140° to adopt a second conformation for thioester formation. Previously, we determined the structure of 4-chlorobenzoate:CoA ligase (CBL) in the adenylate forming conformation bound to 4-chlorobenzoate. We have determined two new crystal structures. We have determined the structure of CBL in the original adenylate-forming conformation, bound to the adenylate intermediate. Additionally, we have used a novel product analog, 4-chlorophenacyl-CoA, to trap the enzyme in the thioester-forming conformation and determined this structure in a new crystal form. This work identifies a novel binding pocket for the CoA nucleotide. The structures presented herein provide the foundation for biochemical analyses presented in the accompanying manuscript (Wu et al.). The complete characterization of this enzyme allows us to provide an explanation for the use of the domain alternation strategy by these enzymes.

The Crystal Structure of the Adenylation Enzyme VinN Reveals a Unique β-Amino Acid Recognition Mechanism

“…However, we could not obtain any VinN crystals. Adenylation enzymes have been reported to change conformation at the C-terminal region during reaction (37). After the initial adenylation step, the C-terminal domain region is rotated by ϳ140°for the following thioester bond formation step.…”

Structural Insights into Anthranilate Priming during Type II Polyketide Biosynthesis

“…In conformation 2, the C-terminal domain is rotated ∼140°r elative to conformation 1, resulting in the movement of the A10 core motif away from the N-terminal active site. 17 In the AuaEII crystal structure, the C-terminal domain is in conformation 1, and the conserved lysine Lys506 of the A10 core interacts with the phosphate of the anthraniloyl-AMP intermediate ( Figure 2C). The charge−charge interaction between Lys506 and the anthraniloyl-AMP phosphate may be important for stabilizing conformation 1 of AuaEII and promoting crystallization.…”

Defining a Structural and Kinetic Rationale for Paralogous Copies of Phenylacetate-CoA Ligases from the Cystic Fibrosis Pathogen Burkholderia cenocepacia J2315

“…PaaK1 and PaaK2 are members of the adenylate-forming enzyme (AFE) family PFAM00501, which incorporates enzymes involved in metabolism of short-to-long chain fatty acids, aromatic compounds, biosynthesis of siderophores and peptide antibiotics, and luciferases (9). The family maintains a general architecture of two ␣/␤ domains with the active site formed at the interface of the N-and C-terminal domains (10 -13).…”

“…Sequences reveal noteworthy divergent features of PaaK1 and PaaK2 with respect to the superfamily, including a novel N-terminal sequence spanning ϳ70 residues. The general mechanism of the AFE superfamily indicates that catalysis relies on a large conformational change (9,14), yet it is rare that a single enzyme is captured in multiple conformations. Furthermore, there are very few ATP-bound structures (15) mak-ing it difficult to refine the general catalytic mechanism of these metabolically important enzymes.…”