Alternative binding modes for chloramphenicol and 1-substituted chloramphenicol analogs revealed by site-directed mutagenesis and x-ray crystallography of chloramphenicol acetyltransferase

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Abstract
Leucine- 160 of chloramphenicol acetyltransferase (CAT) has been replaced by site-directedud mutagenesis to investigate enzyme-ligand interactions at the 1-hydroxyl substituent of the substrateud chloramphenicol. The consequences of the substitution of Leu-1 60 by glutamine and by phenylalanine wereud deduced from the steady-state kinetic parameters for acetyl transfer from acetyl-coA to the 3-hydroxylud of chloramphenicol and its analogues 1-deoxychloramphenicol and 1-acetylchloramphenicol. The acetylud group of the latter, which is a substrate both in vivo and in vitro, could potentially bind in a similar positionud to the 1-hydroxyl of chloramphenicol, in close proximity to the side chain of Leu-160. In the case of Gln-160ud CAT, large increases in K, for the three acetyl acceptors were accompanied by small decreases in k,, andud in apparent affinity for acetyl-coA. Such results are consistent with the introduction of the relativelyud hydrophilic amide in place of the &methyl groups of Leu-160. The kinetic properties of Phe-160 CAT wereud unexpected in that K, for each of the three acetyl acceptors was unchanged or reduced, compared to theud equivalent parameters for the wild-type enzyme, whereas k,,, fell significantly (44-83-fold) in each case.ud The ratios of specificity constants (kcat/Km) for the acetylation of chloramphenicol compared with theud alternative acyl acceptors were similar for wild-type and mutant enzymes. As the residue substitutions forud Leu- 160 do not result in enhanced discrimination against the binding and acetylation of l-acetylchloramphenicol,ud it appears unlikely that the 1-acetyl group binds to the CAT active site in the same positionud as that occupied by the 1-hydroxyl of chloramphenicol. The structure of the binary complex of chloramphenicolud and Phe-160 CAT was determined at 2.0-A resolution and found to be essentially isosteric withud that of wild-type CAT. However, the position of the bound chloramphenicol differs in Phe-160 CAT dueud to a 2.0-A movement of the 1-hydroxyl into a novel, more hydrophilic, environment. This results in newud van der Waals contacts between the aryl group of the substrate and the side chains of Phe-160 and Ile-172.ud As a consequence, the critical distance between the 3-hydroxyl of chloramphenicol and Nf2 of the imidazoleud of His-195 is extended from 2.8 to 4.7 A so that the observed mode of binding is almost certainly nonproductive.ud An additional binding mode for chloramphenicol, which is likely to be similar to but not identicalud with that which occurs in wild-type CAT, must be invoked to account for the observed activity of the Phe-160ud mutant.4147

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