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Title: Alternative binding modes for chloramphenicol and 1-substituted chloramphenicol analogs revealed by site-directed mutagenesis and x-ray crystallography of chloramphenicol acetyltransferase
Authors: Murray, Iain A.
Lewendon, Ann
Williams, John A.
Cullis, Paul M.
Shaw, William V.
Leslie, A. G. W.
First Published: 16-Apr-1991
Publisher: American Chemical Society
Citation: Biochemistry, 1991, 30 (15), pp. 3763-3770
Abstract: ABSTRACT: Leucine- 160 of chloramphenicol acetyltransferase (CAT) has been replaced by site-directed mutagenesis to investigate enzyme-ligand interactions at the 1-hydroxyl substituent of the substrate chloramphenicol.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-1 60 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 K, for the three acetyl acceptors were accompanied by small decreases in k,, and in apparent affinity for acetyl-coA. Such results are consistent with the introduction of the relatively hydrophilic amide in place of the &methyl groups of Leu-160. The kinetic properties of Phe-160 CAT were unexpected in that K, for each of the three acetyl acceptors was unchanged or reduced, compared to the equivalent parameters for the wild-type enzyme, whereas k,,, 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 l-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. The structure of the binary complex of chloramphenicol and Phe-160 CAT was determined at 2.0-A resolution and found to be essentially isosteric with that of wild-type CAT. However, the position of the bound chloramphenicol differs in Phe-160 CAT due to a 2.0-A movement of the 1-hydroxyl into a novel, more hydrophilic, environment. This results in new van der Waals contacts between the aryl group of the substrate and the side chains of Phe-160 and Ile-172. As a consequence, the critical distance between the 3-hydroxyl of chloramphenicol and Nf2 of the imidazole of His-195 is extended from 2.8 to 4.7 A so that the observed mode of binding is almost certainly nonproductive. An additional binding mode for chloramphenicol, which is likely to be similar to but not identical with that which occurs in wild-type CAT, must be invoked to account for the observed activity of the Phe-160 mutant.
DOI Link: 10.1021/bi00229a025
ISSN: 0006-2960
Type: Journal Article
Appears in Collections:Published Articles, Dept. of Chemistry

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