Identification of local carboxy-terminal hydrophobic interactions essential for folding or stability of chloramphenicol acetyltransferase.
The role of the carboxy terminal in folding and stabilization of type I chloramphenicol acetyltransferase (CAT1) has been studied by mutagenesis and Fourier transform infrared analysis. We have shown that a CAT mutant truncated by seven amino acid residues folds into active protein. In this study, the last three residues of this truncated CAT mutant were randomized to detect structural information required for achieving a native enzyme conformation. Statistical analysis of sequencing data from randomly chosen mutants revealed that the amino-terminal CAT fragment of 212 amino acid residues is the shortest deletion mutant able to adopt a soluble, enzymatically active structure. This minimal length corresponds to a protein with full-length alpha5-helix in the three-dimensional crystal structure of CAT type III. The amino acid preferences at the carboxy terminal in the randomization experiments suggest that this helix also forms completely in the shortened CAT mutants. In addition correct folding and/or stabilization requires the formation of a hydrophobic + microdomain at the end of the alpha5-helix. The role of this hydrophobic interaction in CAT folding and structure stabilization is discussed.
Van der Schueren
J
J
Laboratory of Gene Technology, Katholieke Universiteit Leuven, Belgium.
Robben
J
J
Goossens
K
K
Heremans
K
K
Volckaert
G
G
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D015500:Chloramphenicol O-Acetyltransferase / Q000737:chemistry* / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017384:Sequence Deletion; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature
D004269:DNA, Bacterial; D015500:Chloramphenicol O-Acetyltransferase