Crystal structure of 4-chlorobenzoate:CoA ligase/synthetase in the unliganded and aryl substrate-bound states

Biochemistry. 2004 Jul 13;43(27):8670-9. doi: 10.1021/bi049384m.


4-Chlorobenzoate:CoA ligase (CBAL) is a member of a family of adenylate-forming enzymes that catalyze two-step adenylation and thioester-forming reactions. In previous studies, we have provided structural evidence that members of this enzyme family (exemplified by acetyl-CoA synthetase) use a large domain rotation to catalyze the respective partial reactions [A. M. Gulick, V. J. Starai, A. R. Horswill, K. M. Homick, and J. C. Escalante-Semerena, (2003) Biochemistry 42, 2866-2873]. CBAL catalyzes the synthesis of 4-chlorobenzoyl-CoA, the first step in the 4-chlorobenzoate degredation pathway in PCB-degrading bacteria. We have solved the 2.0 A crystal structure of the CBAL enzyme from Alcaligenes sp. AL3007 using multiwavelength anomalous dispersion. The results demonstrate that in the absence of any ligands, or bound to the aryl substrate 4-chlorobenzoate, the enzyme adopts the conformation poised for catalysis of the adenylate-forming half-reaction. We hypothesize that coenzyme A binding is required for stabilization of the alternate conformation, which catalyzes the 4-CBA-CoA thioester-forming reaction. We have also determined the structure of the enzyme bound to the aryl substrate 4-chlorobenzoate. The aryl binding pocket is composed of Phe184, His207, Val208, Val209, Phe249, Ala280, Ile303, Gly305, Met310, and Asn311. The structure of the 4-chlorobenzoate binding site is discussed in the context of the binding sites of other family members to gain insight into substrate specificity and evolution of new function.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenine / metabolism
  • Amino Acid Sequence
  • Binding Sites
  • Catalysis
  • Chlorobenzoates / metabolism*
  • Coenzyme A Ligases / chemistry*
  • Coenzyme A Ligases / metabolism*
  • Crystallography, X-Ray
  • Ligands
  • Models, Molecular
  • Molecular Sequence Data
  • Protein Structure, Tertiary
  • Sequence Alignment
  • Sulfides / metabolism


  • Chlorobenzoates
  • Ligands
  • Sulfides
  • 4-halobenzoate-CoA ligase
  • Coenzyme A Ligases
  • 4-chlorobenzoic acid
  • Adenine

Associated data

  • PDB/1T5D
  • PDB/1T5H