4cla
ALTERNATIVE BINDING MODES FOR CHLORAMPHENICOL AND 1-SUBSTITUTED CHLORAMPHENICOL ANALOGUES REVEALED BY SITE-DIRECTED MUTAGENESIS AND X-RAY CRYSTALLOGRAPHY OF CHLORAMPHENICOL ACETYLTRANSFERASE
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OverviewOverview
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)
About this StructureAbout this Structure
4CLA is a Single protein structure of sequence from Escherichia coli with CO and CLM as ligands. Active as Chloramphenicol O-acetyltransferase, with EC number 2.3.1.28 Full crystallographic information is available from OCA.
ReferenceReference
Alternative binding modes for chloramphenicol and 1-substituted chloramphenicol analogues revealed by site-directed mutagenesis and X-ray crystallography of chloramphenicol acetyltransferase., Murray IA, Lewendon A, Williams JA, Cullis PM, Shaw WV, Leslie AG, Biochemistry. 1991 Apr 16;30(15):3763-70. PMID:2015231
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