Crystal structure of a thiolase fromEscherichia coliat 1.8 Å resolution

Abstract

Thiolases catalyze the Claisen condensation of two acetyl-CoA molecules to give acetoacetyl-CoA, as well as the reverse degradative reaction. Four genes coding for thiolases or thiolase-like proteins are found in theEscherichia coligenome. In this communication, the successful cloning, purification, crystallization and structure determination at 1.8 Å resolution of a homotetramericE. colithiolase are reported. The structure ofE. colithiolase co-crystallized with acetyl-CoA at 1.9 Å resolution is also reported. As observed in other tetrameric thiolases, the presentE. colithiolase is a dimer of two tight dimers and probably functions as a biodegradative enzyme. Comparison of the structure and biochemical properties of theE. colienzyme with those of other well studied thiolases reveals certain novel features of this enzyme, such as the modification of a lysine in the dimeric interface, the possible oxidation of the catalytic Cys88 in the structure of the enzyme obtained in the presence of CoA and active-site hydration. The tetrameric enzyme also displays an interesting departure from exact 222 symmetry, which is probably related to the deformation of the tetramerization domain that stabilizes the oligomeric structure of the protein. The current study allows the identification of substrate-binding amino-acid residues and water networks at the active site and provides the structural framework required for understanding the biochemical properties as well as the physiological function of thisE. colithiolase.