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)

Weak Substrate Binding to Transport Proteins Studied by NMR

“…This would facilitate a structural analysis of those ligands that can conform to the binding site geometry. The use of spectral editing via the substrate nuclei has also been used to good effect in some of the most complex proteins observed by high-resolution, solution state NMR (Derrick et al, 1992;Lian et al, 1994). The anisotropic features of membrane systems and their amenability to some solid-state NMR methods as demonstrated here, can open up many more diverse opportunities for detecting and quantifying these interactions by various structural methods (Watts et al, 1995), of which rotational resonance (Levitt et al, 1990) and rotational-echo double resonance (Gullian and Schaefer, 1989) are the best known.…”

The Conformation of Coenzyme A Bound to Chloramphenicol Acetyltransferase Determined by Transferred NOE Experiments

“…Perdeuterated enzyme was prepared by minor modifications of the method of Derrick et al (1992). CAT activity was assayed at 25°C as described by Lewendon et al (1988).…”

“…We have earlier shown that the use of 13 C-labelled ligand allows intermolecular NOEs to be identified even in a protein of this size (Derrick et al, 1992). However, generally 13 C-labelled CoA and chloramphenicol are not readily available, and in the work reported here we have taken the alternative approach of using the transferred NOE experiment.…”

NMR Studies of the Escherichia coli Trp Repressor ·trpRs Operator Complex

“…Partial deuteration has been used previously with Trp repressor [12,[29][30][31] and the effects on relaxation discussed. Full deuteration has been used in studies of ligand binding to large proteins such as chloramphenicol acetyl transferase [32] (reviewed in [33]). …”