title: Crystal structure of 4-chlorobenzoate:CoA ligase/synthetase in the unliganded and aryl substrate-bound states.
abstract: 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.
authors.affiliation: Hauptman-Woodward Medical Research Institute, State University of New York at Buffalo, Buffalo, New York 14203-1196, USA. gulick@hwi.buffalo.edu
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Gulick
authors.forename: Xuefeng
authors.initials: X
authors.lastname: Lu
authors.forename: Debra
authors.initials: D
authors.lastname: Dunaway-Mariano
mesh_terms: D000225:Adenine / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D002723:Chlorobenzoates / Q000378:metabolism*; D003066:Coenzyme A Ligases / Q000737:chemistry* / Q000378:metabolism*; D018360:Crystallography, X-Ray; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D013440:Sulfides / Q000378:metabolism
chemical_list: D002723:Chlorobenzoates; D008024:Ligands; D013440:Sulfides; C077846:4-halobenzoate-CoA ligase; D003066:Coenzyme A Ligases; C040768:4-chlorobenzoic acid; D000225:Adenine