Abstract
Carnitine octanoyltransferase (COT) and carnitine palmitoyltransferase (CPT) I, which facilitate the transport of medium- and long-chain fatty acids through the peroxisomal and mitochondrial membranes, are physiologically inhibited by malonyl-CoA. Using an "in silico" macromolecular docking approach, we built a model in which malonyl-CoA could be attached near the catalytic core. This disrupts the positioning of the acyl-CoA substrate in the channel in the model reported for both proteins (Morillas, M., Gómez-Puertas, P., Roca, R., Serra, D., Asins, G., Valencia, A., and Hegardt, F. G. (2001) J. Biol. Chem. 276, 45001-45008). The putative malonyl-CoA domain contained His(340), implicated together with His(131) in COT malonyl-CoA sensitivity (Morillas, M., Clotet, J., Rubi, B., Serra, D., Asins, G., Ariño, J., and Hegardt F. G. (2000) FEBS Lett. 466, 183-186). When we mutated COT His(131) the IC(50) increased, and malonyl-CoA competed with the substrate decanoyl-CoA. Mutation of COT Ala(332), present in the domain 8 amino acids away from His(340), decreased the malonyl-CoA sensitivity of COT. The homologous histidine and alanine residues of L-CPT I, His(277), His(483), and Ala(478) were also mutated, which decreased malonyl-CoA sensitivity. Natural mutation of Pro(479), which is also located in the malonyl-CoA predicted site, to Leu in a patient with human L-CPT I hereditary deficiency, modified malonyl-CoA sensitivity. We conclude that this malonyl-CoA domain is present in both COT and L-CPT I proteins and might be the site at which malonyl-CoA interacts with the substrate acyl-CoA. Other malonyl-CoA non-inhibitable members of the family, CPT II and carnitine acetyltransferase, do not contain this domain.
Highlights
Carnitine octanoyltransferase (COT) and carnitine palmitoyltransferase (CPT) I, which facilitate the transport of medium- and long-chain fatty acids through the peroxisomal and mitochondrial membranes, are physiologically inhibited by malonyl-CoA
Positioning of Malonyl-CoA in the COT Structural Model— Once the structural models of COT and L-CPT I catalytic core, including the putative substrate-binding site, active site, were established [31], we performed exhaustive in silico molecular docking analysis to find clues to the putative binding site of malonyl-CoA
After analysis of the best solutions given by the program for the interaction between the malonyl-CoA molecule and the structural model for the catalytic core of rat COT, a common position was observed for the five best predicted solutions in terms of low energy of the complex and lowest macromolecular distance
Summary
Structural Model Building—Structural models of rat CPT I and COT were obtained as previously described [31]. Using the three-dimensioinal structure of rat enoyl-CoA hydratase (PDB entry 2dub chain E), as selected previously for the homologous proteins CPT I and COT [31], the program Swiss-Pdb Viewer and the SWISS-MODEL server facilities [42,43,44,45] were used to build the coordinates for the three-dimensional model of the active site-surrounding regions of CAT and CPT II. Macromolecular Docking—Docking calculations to obtain a molecular model of the interaction between the inhibitor (ligand) malonyl-CoA and the putative receptor proteins CPT I, COT, CPT II, and CAT were performed using the spherical polar Fourier correlations based program “Hex” [51]. Mutant L-CPT I A478G was constructed using the “QuikChange” polymerase chain reaction-based mutagenesis procedure (Stratagene) with pYESLCPTIwt plasmid as template and the primer 5Ј-CACTCCTGCGCGGACGGGCCCATCGTGGGCCATTTG-3Ј(mutated nucleotide is underlined). Mitochondria were dispersed in 250 mM sucrose, 10 mM Tris-HCl, pH 7.4, and 1 mM EDTA to a final concentration of 40 mg/ml and stored at Ϫ70 °C
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