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3. Structural studies |
A. First bioinformatic model of CPTI
The crystal structure of CPTI has not yet been solved, therefore the use of bioinformatic tools is of primary importance to understand the mechanism of action of CPTI. Using an in silico macromolecular docking approach, our group has built a model in which malonyl-CoA could be attached near the catalytic core, indicating the site at which malonyl-CoA interacts with the substrate acyl-CoA14. Other members of the family that are not inhibited by malonyl-CoA, CPTII and carnitine acetyltransferase (CrAT), do not contain this domain.
We also proposed a 3-D (three-dimensional) structural model for CPTIA, based on the similarity of this enzyme to the crystallized mouse CrAT. The model includes 607 of the 773 amino acids of CPTIA, and the positions of carnitine, CoA and the palmitoyl group were assigned by superposition and docking analysis. Functional analysis of this 3-D model included the mutagenesis of several amino acids in order to identify putative catalytic residues15.
Additional research from our group includes the study of the substrate specificity of other carnitine acyltransferase enzymes such as CrAT and Carnitine octanoyltransferase (COT)16.
B. Identification of the amino acid involved in CPTI regulation by malonyl-CoA
By using the SequenceSpace algorithm program our group has identified the amino acid Met(593) that participates in malonyl-CoA inhibition of CPTI. Furthermore, by the mutation M593S we developed a mutant but active form of CPTIA (CPTIAM17) which is insensitive to malonyl-CoA and therefore leads to permanent accelerated fatty acid oxidation, independently of the glucose-derived malonyl-CoA levels. This has been a highly valuable tool for the study of fatty-acid metabolism.
C. Design of anti-obesity drugs
Through the use of bioinformatic modeling of CPTI, our group aims to find its relationship with obesity and type 2 diabetes by performing in silico design of anti-obesity drugs. We are currently studying derivates from the potential anti-obesity drug C75 and its interaction in vitro and in vivo with CPTI in the hypothalamus.
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14. Morillas, M., et al. Structural model of a malonyl-CoA-binding site of carnitine octanoyltransferase and carnitine palmitoyltransferase I: mutational analysis of a malonyl-CoA affinity domain. J Biol Chem 277, 11473-11480 (2002).
15. Morillas, M., et al. Structural model of carnitine palmitoyltransferase I based on the carnitine acetyltransferase crystal. Biochem J 379, 777-784 (2004).
16. Cordente, A.G., et al. Redesign of carnitine acetyltransferase specificity by protein engineering. J Biol Chem 279, 33899-33908 (2004).
17. Morillas, M., et al. Identification of conserved amino acid residues in rat liver carnitine palmitoyltransferase I critical for malonyl-CoA inhibition. Mutation of methionine 593 abolishes malonyl-CoA inhibition. J Biol Chem 278, 9058-9063 (2003). |
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