Structural model of a malonyl-CoA-binding site of carnitine octanoyltransferase and carnitine palmitoyltransferase I. Mutational analysis of a malonyl-CoA affinity domain

Montserrat Morillas, Paulino Gómez-Puertas, Blanca Rubí, Josep Clotet, Joaquín Ariño, Alfonso Valencia, Fausto G. Hegardt, Dolors Serra, Guillermina Asins

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35 Citations (Scopus)

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 His340, implicated together with His131 in COT malonyl-CoA sensitivity (Morillas, M., Clotet, J., Rubí, B., Serra, D., Asins, G., Ariño, J., and Hegardt F. G. (2000) FEBS Lett. 466, 183-186). When we mutated COT His131 the IC50 increased, and malonyl-CoA competed with the substrate decanoyl-CoA. Mutation of COT Ala332, present in the domain 8 amino acids away from His340, decreased the malonyl-CoA sensitivity of COT. The homologous histidine and alanine residues of L-CPT I, His277, His483, and Ala478 were also mutated, which decreased malonyl-CoA sensitivity. Natural mutation of Pro479, 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 noninhibitable members of the family, CPT II and carnitine acetyltransferase, do not contain this domain.
Original languageEnglish
Pages (from-to)11473-11480
JournalJournal of Biological Chemistry
Volume277
DOIs
Publication statusPublished - 29 Mar 2002

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