Cloning and molecular modeling of a thermostable carboxylesterase from the chicken uropygial glands

• The chicken uropygial carboxylesterase (cuCES) cDNA was synthesized by RT-PCR.
• cuCES shows a high degree of homology with human liver carboxylesterase 1 (hCES1).
• A 3D structure model of the cuCES was built using the structure of hCES1 as template.
• Thermostability of cuCES was explained based on molecular dynamics (MD) simulations.
• Chicken CES could not accommodate large substrate molecule in its active site pocket.

Starting from total uropygial glands mRNAs, chicken uropygial carboxylesterase (cuCES) cDNA was synthesized by RT-PCR and cloned into the PGEM-T vector. Amino acid sequence of the cuCES is compared to that of human liver carboxylesterase 1 (hCES1). Given the high amino acid sequence homology between the two enzymes, a 3-D structure model of the chicken carboxylesterase was built using the structure of hCES1 as template. By following this model and utilizing molecular dynamics (MD) simulations, the resistance of the chicken carboxylesterase at high temperatures could be explained. The docking of substrate analogs into the cuCES active site was used to explain the fact that the chicken carboxylesterase cannot hydrolyze efficiently large substrate molecules.

The helices α1 (residues 72–82) and α9 (residues 340–346) which are located on the top of the central β-sheet were proposed as potential lid and second lid, respectively. These two structural units (α1 and α9) may act as a double door controlling the access to the active site of the chicken uropygial carboxylesterase.Figure optionsDownload high-quality image (254 K)Download as PowerPoint slide