Acyl-coenzyme A synthetases (ACSs) catalyze the fundamental, initial reaction in fatty acid metabolism. "Activation" of fatty acids by thioesterification to CoA allows their participation in both anabolic and catabolic pathways. The availability of the sequenced human genome has facilitated the investigation of the number of ACS genes present. Using two conserved amino acid sequence motifs to probe human DNA databases, 26 ACS family genes/proteins were identified. ACS activity in either humans or rodents was demonstrated previously for 20 proteins, but 6 remain candidate ACSs. For two candidates, cDNA was cloned, protein was expressed in COS-1 cells, and ACS activity was detected. Amino acid sequence similarities were used to assign enzymes into subfamilies, and subfamily assignments were consistent with acyl chain length preference. Four of the 26 proteins did not fit into a subfamily, and bootstrap analysis of phylograms was consistent with evolutionary divergence. Three additional conserved amino acid sequence motifs were identified that likely have functional or structural roles. The existence of many ACSs suggests that each plays a unique role, directing the acyl-CoA product to a specific metabolic fate. Knowing the full complement of ACS genes in the human genome will facilitate future studies to characterize their specific biological functions.-Watkins, P. A., D. Maiguel, Z. Jia, and J. Pevsner. Evidence for 26 distinct acyl-coenzyme A synthetase genes in the human genome. J. Lipid Res.

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The family of proteins that includes very long-chain acyl-CoA synthetases (ACSVL) consists of six members. These enzymes have also been designated fatty acid transport proteins. We cloned full-length mouse Acsvl3 cDNA and characterized its protein product ACSVL3/ fatty acid transport protein 3. The predicted amino acid sequence contains two highly conserved motifs characteristic of acyl-CoA synthetases. Northern blot analysis revealed that the mouse Acsvl3 mRNA is highly expressed in adrenal gland, testis, and ovary, with lower expression in the brain of adult mice. A developmental Northern blot revealed that Acsvl3 mRNA levels were significantly higher in embryonic mouse brain (embryonic days 12-14) than in newborn or adult mice, suggesting a possible role in nervous system development. Immunohistochemistry revealed high ACSVL3 expression in adrenal cortical cells, spermatocytes and interstitial cells of the testis, theca cells of the ovary, cerebral cortical neurons, and cerebellar Purkinje cells. Endogenous ACSVL3 was found primarily in mitochondria of MA-10 and Neuro2a cells by both Western blot analysis of subcellular fractions and immunofluorescence analysis. In MA-10 cells, loss-of-function studies using RNA interference confirmed that endogenous ACSVL3 is an acyl-CoA synthetase capable of activating both longchain (C16:0) and very long-chain (C24:0) fatty acids. However, despite decreased acyl-CoA synthetase activity, initial rates of fatty acid uptake were unaffected by knockdown of Acsvl3 expression in MA-10 cells. These studies cast doubt on the designation of ACSVL3 as a fatty acid transport protein.The transport of fatty acids into cells and their subsequent "activation" by thioesterification to CoA are fundamental processes required for entry of fatty acids into the metabolic stream (1). The mechanism of fatty acids entry into cells remains controversial. Some investigators argue that specific proteins are required to transport the fatty acid across the plasma membrane (2-6). Others have provided evidence that proteins are not necessary for translocation of fatty acids through the lipid bilayer (7,8). One group of proteins proposed to mediate fatty acid entry into cells are the fatty acid transport proteins (FATPs) 1 (4). The mammalian FATP family consists of six homologous proteins (FATP1-6) that share 35-58% amino acid identity. 2 Studies with cultured cells overexpressing FATP1-6 have demonstrated increased rates of accretion of fluorescent or radiolabeled fatty acids (3, 9). However, interpretation of fatty acid transport studies is hampered by the fact that, once inside cells, fatty acids are rapidly metabolized. Metabolism will decrease the intracellular concentration of the unesterified fatty acid, shifting the concentration gradient across the plasma membrane to promote entry of additional fatty acids into the cell. The design of most transport studies does not distinguish between transport mechanisms that can occur in a protein-free phospholipid bilayer and transport plus metabolism.Indepe...