Mutagenesis of rat acyl-CoA synthetase 4 indicates amino acids that contribute to fatty acid binding.
Although each of the five mammalian long-chain acyl-CoA synthetases (ACSL) can bind saturated and unsaturated fatty acids ranging from 12 to 22 carbons, ACSL4 prefers longer chain polyunsaturated fatty acids. In order to gain a better understanding of ACSL4 fatty acid binding, we based a mutagenesis approach on sequence alignments related to ttLC-FACS crystallized from Thermus thermophilus HB8. Four residues selected for mutagenesis corresponded to residues in ttLC-FACS that comprise the fatty acid binding pocket; the fifth residue aligned with a region thought to be involved in fatty acid selectivity of the Escherichia coli acyl-CoA synthetase, FadD. Changing an amino acid at the entry of the putative fatty acid binding pocket, G401L, resulted in an inactive enzyme. Mutating a residue near the pocket entry, L399M, did not significantly alter enzyme activity, but mutating a residue at the hydrophobic terminus of the pocket, S291Y, altered ACSL4's preference for 20:5 and 22:6 and increased its apparent K(m) for ATP. Mutating a site in a region previously identified as important for fatty acid binding also altered activation of 20:4 and 20:5. These studies suggested that the preference of ACSL4 for long-chain polyunsaturated fatty acids can be modified by altering specific amino acid residues.
Stinnett
Lori
L
Department of Nutrition, 2301 Michael Hooker Research Center, Pittsboro St., University of North Carolina, Chapel Hill, NC 27599, USA.
Lewin
Tal M
TM
Coleman
Rosalind A
RA
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D003066:Coenzyme A Ligases / Q000235:genetics* / Q000378:metabolism; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005231:Fatty Acids, Unsaturated / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D011485:Protein Binding / Q000235:genetics; D051381:Rats; D016415:Sequence Alignment; D016963:Thermus thermophilus / Q000201:enzymology / Q000235:genetics
D005231:Fatty Acids, Unsaturated; C500958:Acsl4 protein, rat; D003066:Coenzyme A Ligases