title: Alternative binding modes for chloramphenicol and 1-substituted chloramphenicol analogues revealed by site-directed mutagenesis and X-ray crystallography of chloramphenicol acetyltransferase.
abstract: Leucine-160 of chloramphenicol acetyltransferase (CAT) has been replaced by site-directed mutagenesis to investigate enzyme-ligand interactions at the 1-hydroxyl substituent of the substrate chloramphenicol. The consequences of the substitution of Leu-160 by glutamine and by phenylalanine were deduced from the steady-state kinetic parameters for acetyl transfer from acetyl-CoA to the 3-hydroxyl of chloramphenicol and its analogues 1-deoxychloramphenicol and 1-acetylchloramphenicol. The acetyl group of the latter, which is a substrate both in vivo and in vitro, could potentially bind in a similar position to the 1-hydroxyl of chloramphenicol, in close proximity to the side chain of Leu-160. In the case of Gln-160 CAT, large increases in Km for the three acetyl acceptors were accompanied by small decreases in kcat and in apparent affinity for acetyl-CoA. Such results are consistent with the introduction of the relatively hydrophilic amide in place of the delta-methyl groups of Leu-160. The kinetic properties of Phe-160 CAT were unexpected in that Km for each of the three acetyl acceptors was unchanged or reduced, compared to the equivalent parameters for the wild-type enzyme, whereas kcat fell significantly (44-83-fold) in each case. The ratios of specificity constants (kcat/Km) for the acetylation of chloramphenicol compared with the alternative acyl acceptors were similar for wild-type and mutant enzymes. As the residue substitutions for Leu-160 do not result in enhanced discrimination against the binding and acetylation of 1-acetylchloramphenicol, it appears unlikely that the 1-acetyl group binds to the CAT active site in the same position as that occupied by the 1-hydroxyl of chloramphenicol.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Biochemistry, University of Leicester, U.K.
authors.forename: I A
authors.initials: IA
authors.lastname: Murray
authors.forename: A
authors.initials: A
authors.lastname: Lewendon
authors.forename: J A
authors.initials: JA
authors.lastname: Williams
authors.forename: P M
authors.initials: PM
authors.lastname: Cullis
authors.forename: W V
authors.initials: WV
authors.lastname: Shaw
authors.forename: A G
authors.initials: AG
authors.lastname: Leslie
mesh_terms: D000107:Acetylation; D001667:Binding, Competitive; D002701:Chloramphenicol / Q000031:analogs & derivatives / Q000378:metabolism*; D015500:Chloramphenicol O-Acetyltransferase / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006860:Hydrogen Bonding; D007700:Kinetics; D007930:Leucine / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000235:genetics; D011487:Protein Conformation; D013237:Stereoisomerism; D013379:Substrate Specificity; D014961:X-Ray Diffraction
chemical_list: D010649:Phenylalanine; D002701:Chloramphenicol; D015500:Chloramphenicol O-Acetyltransferase; D007930:Leucine