Analysis of the binding of 1,3-diacetylchloramphenicol to chloramphenicol acetyltransferase by isotope-edited proton NMR and site-directed mutagenesis

Abstract

The binary complex of diacetylchloramphenicol and chloramphenicol acetyltransferase (CAT) has been studied by a combination of isotope-edited 1H NMR spectroscopy and site-directed mutagenesis. One-dimensional HMQC spectra of the complex between 1,3-[2-13C]diacetylchloramphenicol and the type III natural variant of CAT revealed the two methyl 1H signals arising from each 13C-labeled carbon atom in the acetyl groups of the bound ligand. Slow hydrolysis of the 3-acetyl group by the enzyme precluded further analysis of this binary complex. It was possible to slow down the rate of hydrolysis by use of the catalytically defective S148A mutant of CATIII (Lewendon et al., 1990); in the complex of diacetylchloramphenicol with S148A CATIII, the chemical shifts of the acetyl groups of the bound ligand were the same as in the wild-type complex. The acetyl signals were individually assigned by repeating the experiment using 1-[2-13C],3-[2-12C]diacetylchloramphenicol, where only one signal from the bound ligand was observed. A two-dimensional 1H, 1H NOESY experiment, with 13C(omega 2) half-filter, on the 1,3-[2-13C]diacetylchloramphenicol/S148A CATIII complex showed a number of intermolecular NOEs from each methyl group in the ligand to residues in the chloramphenicol binding site. The 3-acetyl group showed strong NOEs to two aromatic signals which were selected for assignment. The possibility that the NOEs originated from the aromatic protons of diacetylchloramphenicol itself was eliminated by assignment of the signals from enzyme-bound diacetylchloramphenicol and chloramphenicol using perdeuterated CATIII. Examination of the X-ray crystal structure of the chloramphenicol/CATIII binary complex indicated four plausible candidate aromatic residues: Y25, F33, F103, and F158.(ABSTRACT TRUNCATED AT 250 WORDS)