Alternative binding modes for chloramphenicol and 1-substituted chloramphenicol analogues revealed by site-directed mutagenesis and X-ray crystallography of chloramphenicol acetyltransferase.
Leucine-160 of chloramphenicol acetyltransferase (CAT) has been replaced by site-directed mutagenesis to investigate enzyme-ligand interactions at the 1-hydroxyl substituent of the substrate chloramphenicol.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)
Murray
I A
IA
Department of Biochemistry, University of Leicester, U.K.
Lewendon
A
A
Williams
J A
JA
Cullis
P M
PM
Shaw
W V
WV
Leslie
A G
AG
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
D010649:Phenylalanine; D002701:Chloramphenicol; D015500:Chloramphenicol O-Acetyltransferase; D007930:Leucine