3DLP

4-Chlorobenzoyl-CoA Ligase/Synthetase, Mutant D402P, bound to 4CB

Summary for 3DLP

• Entry DOI: 10.2210/pdb3dlp/pdb
• Related: 1PG4 1T5D
• Descriptor: 4-Chlorobenzoate CoA Ligase/Synthetase, 4-CHLORO-BENZOIC ACID (3 entities in total)
• Functional Keywords: adenylate-forming enzymes acyl-coa ligase domain alternation, ligase
• Biological source: Alcaligenes sp.
• Total number of polymer chains: 1
• Total formula weight: 54519.70

Authors

Wu, R.,Cao, J.,Reger, A.S.,Lu, X.,Gulick, A.M.,Dunaway-Mariano, D. (deposition date: 2008-06-28, release date: 2009-04-21, Last modification date: 2024-04-03)

Primary citation

Wu, R.,Reger, A.S.,Lu, X.,Gulick, A.M.,Dunaway-Mariano, D.
The mechanism of domain alternation in the acyl-adenylate forming ligase superfamily member 4-chlorobenzoate: coenzyme A ligase
Biochemistry, 48:4115-4125, 2009

PubMed Abstract: 4-Chlorobenzoate:CoA ligase (CBL) belongs to the adenylate-forming family of enzymes that catalyze a two-step reaction to first activate a carboxylate substrate as an adenylate and then transfer the carboxylate to the pantetheine group of either coenzyme A or an acyl-carrier protein. The active site is located at the interface of a large N-terminal domain and a smaller C-terminal domain. Crystallographic structures have been determined at multiple steps along the reaction pathway and form the basis for a proposal that the C-terminal domain rotates by approximately 140 degrees between the two states that catalyze the adenylation and thioester-forming half-reactions. The domain rotation is accompanied by a change in the main chain torsional angles of Asp402, a conserved residue located at the interdomain hinge position. We have mutated the Asp402 residue to Pro in order to test the impact of reduced main chain flexibility at the putative hinge position. The crystal structure of the D402P mutant shows that the enzyme adopts the proposed adenylate-forming conformation with very little change to the overall structure. To examine the impact of this mutation on the ability of the enzyme to catalyze the complete reaction, single turnover kinetic experiments were performed. Whereas the ability of this mutant to catalyze the adenylate-forming half-reaction is reduced by approximately 3-fold, catalysis of the second half-reaction is reduced by 4 orders of magnitude. The impact of the alanine replacement of Asp402 on the thioester-forming reaction is significant, although not as dramatic as the proline mutation, and provides evidence that the Asp402 carboxylate group, through ion pair formation with N-terminal domain residue Arg400, assists in the transition to the thioester-forming conformer. Together these results support the domain alternation hypothesis.

PubMed: 19320426
DOI: 10.1021/bi9002327

Experimental method

X-RAY DIFFRACTION (2.6 Å)