Abstract
The covalent attachment of a 14-carbon aliphatic tail on a glycine residue of nascent translated peptide chains is catalyzed in human cells by two N-myristoyltransferase (NMT) enzymes using the rare myristoyl-CoA (C(14)-CoA) molecule as fatty acid donor. Although, NMT enzymes can only transfer a myristate group, they lack specificity for C(14)-CoA and can also bind the far more abundant palmitoyl-CoA (C(16)-CoA) molecule. We determined that the acyl-CoA binding protein, acyl-CoA binding domain (ACBD)6, stimulated the NMT reaction of NMT2. This stimulatory effect required interaction between ACBD6 and NMT2, and was enhanced by binding of ACBD6 to its ligand, C(18:2)-CoA. ACBD6 also interacted with the second human NMT enzyme, NMT1. The presence of ACBD6 prevented competition of the NMT reaction by C(16)-CoA. Mutants of ACBD6 that were either deficient in ligand binding to the N-terminal ACBD or unable to interact with NMT2 did not stimulate activity of NMT2, nor could they protect the enzyme from utilizing the competitor C(16)-CoA. These results indicate that ACBD6 can locally sequester C(16)-CoA and prevent its access to the enzyme binding site via interaction with NMT2. Thus, the ligand binding properties of the NMT/ACBD6 complex can explain how the NMT reaction can proceed in the presence of the very abundant competitive substrate, C(16)-CoA.
Highlights
The covalent attachment of a 14-carbon aliphatic tail on a glycine residue of nascent translated peptide chains is catalyzed in human cells by two N-myristoyltransferase (NMT) enzymes using the rare myristoyl-CoA (C14-CoA) molecule as fatty acid donor
CAMK2D and UGGT1, were identified at a lower specificity threshold, but NMT1 and NMT2 were the only proteins that reproducibly interacted with ACBD6 in all four experiments
The specificity of the association with ACBD6 was further confirmed with a mammalian two-hybrid interaction assay, which established a stronger binding of ACBD6 with NMT2 than with NMT1, as compared with the related ACBD5
Summary
The covalent attachment of a 14-carbon aliphatic tail on a glycine residue of nascent translated peptide chains is catalyzed in human cells by two N-myristoyltransferase (NMT) enzymes using the rare myristoyl-CoA (C14-CoA) molecule as fatty acid donor. We determined that the acyl-CoA binding protein, acyl-CoA binding domain (ACBD), stimulated the NMT reaction of NMT2. Mutants of ACBD6 that were either deficient in ligand binding to the N-terminal ACBD or unable to interact with NMT2 did not stimulate activity of NMT2, nor could they protect the enzyme from utilizing the competitor C16-CoA. The ligand binding properties of the NMT/ACBD6 complex can explain how the NMT reaction can proceed in the presence of the very abundant competitive substrate, C16-CoA.—Soupene, E., J. Members of the acyl-CoA binding domain (ACBD)containing protein family are involved in the maintenance of diverse cellular functions and can interact with a multitude of proteins implicated in a variety of cellular functions such as neural stem cell self-renewal, neurodegeneration, stress resistance, lipid homeostasis, intracellular vesicle trafficking, organelle formation, viral replication, and apoptotic response.
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