Acyl-adenylate motif of the acyl-adenylate/thioester-forming enzyme superfamily: a site-directed mutagenesis study with the Pseudomonas sp. strain CBS3 4-chlorobenzoate:coenzyme A ligase.
4-Chlorobenzoate:coenzyme A (4-CBA:CoA) ligase catalyzes 4-chlorobenzoyl-coenzyme A formation in a two-step reaction consisting of the adenylation of 4-chlorobenzoate with adenosine 5'-triphosphate followed by acyl transfer from the 4-chlorobenzoyl adenosine 5'-monophosphate diester intermediate to coenzyme A. In this study, two core motifs present in the Pseudomonas sp. strain CBS3 4-CBA:CoA ligase (motif I, 161T-S-G-T-T-G-L-P-K-G170, and motif II, 302Y-G-T-T-E306) and conserved among the sequences representing the acyl-adenylate/thioester-forming enzyme family (to which the ligase belongs) were tested for their possible role in substrate binding and/or catalysis. The site-directed mutants G163I, G166I, P168A, K169M, and E306Q were prepared and then subjected to steady-state and transient kinetic studies. The results, which indicate reduced catalysis of the adenylation of 4-chlorobenzoate in the mutant enzymes, are interpreted within the context of the three-dimensional structure of the acyl-adenylate/thioester-forming enzyme family member, firefly luciferase.
Chang
K H
KH
Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742, USA.
Xiang
H
H
Dunaway-Mariano
D
D
D000255:Adenosine Triphosphate / Q000378:metabolism; D002384:Catalysis; D002723:Chlorobenzoates / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003065:Coenzyme A / Q000378:metabolism; D003066:Coenzyme A Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017124:Conserved Sequence; D007700:Kinetics; D008156:Luciferases / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D011549:Pseudomonas / Q000201:enzymology*; D013050:Spectrometry, Fluorescence
D002723:Chlorobenzoates; D000255:Adenosine Triphosphate; D008156:Luciferases; C077846:4-halobenzoate-CoA ligase; D003066:Coenzyme A Ligases; C040768:4-chlorobenzoic acid; D003065:Coenzyme A