Publications

• A novel mammalian bubblegum-related acyl-CoA synthetase restricted to testes and possibly involved in spermatogenesis.

  Fraisl P., Tanaka H., Forss-Petter S., Lassmann H., Nishimune Y., Berger J.

  We have characterized a new, membrane-associated acyl-CoA synthetase (ACS), termed bubblegum-related protein (BGR), which upon functional analysis demonstrated ACS activity capable of activating long- and very long-chain fatty acids. By multiple tissue RNA array and Northern blot analyses, human B ... >> More

  We have characterized a new, membrane-associated acyl-CoA synthetase (ACS), termed bubblegum-related protein (BGR), which upon functional analysis demonstrated ACS activity capable of activating long- and very long-chain fatty acids. By multiple tissue RNA array and Northern blot analyses, human BGR mRNA was exclusively detected in testes. Murine Bgr mRNA was specifically expressed in pubertal and adult testes and was further demonstrated to be enriched in germ cells and Sertoli cells while present at a lower level in Leydig cells both by in situ hybridization and cell type fractionation. The complex 5'-end of the BGR mRNA appears to underlie translational control leading to differential utilization of alternative translation start sites. Thus, the BGR gene expands the bubblegum ACS family with a testes-specific, developmentally regulated member that may play a role in spermatogenesis. << Less

  Arch. Biochem. Biophys. 451:23-33(2006) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• Human very-long-chain acyl-CoA synthetase: cloning, topography, and relevance to branched-chain fatty acid metabolism.

  Steinberg S.J., Wang S.J., Kim D.G., Mihalik S.J., Watkins P.A.

  Very-long-chain acyl-CoA synthetases (VLCS) activate very-long-chain fatty acids (VLCFA) containing 22 or more carbons to their CoA derivatives. We cloned the human ortholog (hVLCS) of the gene encoding the rat liver enzyme (rVLCS). Both hVLCS and rVLCS contain 620 amino acids, are expressed prima ... >> More

  Very-long-chain acyl-CoA synthetases (VLCS) activate very-long-chain fatty acids (VLCFA) containing 22 or more carbons to their CoA derivatives. We cloned the human ortholog (hVLCS) of the gene encoding the rat liver enzyme (rVLCS). Both hVLCS and rVLCS contain 620 amino acids, are expressed primarily in liver and kidney, and have a potential peroxisome targeting signal 1 (-LKL) at their carboxy termini. When expressed in COS-1 cells, hVLCS activated the VLCFA lignoceric acid (C24:0), a long-chain fatty acid (C16:0), and two branched-chain fatty acids, phytanic acid and pristanic acid. Immunofluorescence and immunoblot studies localized hVLCS to both peroxisomes and endoplasmic reticulum. In peroxisomes of HepG2 cells, hVLCS was topographically oriented facing the matrix and not the cytoplasm. This orientation, coupled with the observation that hVLCS activates branched-chain fatty acids, suggests that hVLCS could play a role in the intraperoxisomal reactivation of pristanic acid produced via alpha-oxidation of phytanic acid. << Less

  Biochem. Biophys. Res. Commun. 257:615-621(1999) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Dissecting the role of critical residues and substrate preference of a fatty acyl-CoA synthetase (FadD13) of Mycobacterium tuberculosis.

  Khare G., Gupta V., Gupta R.K., Gupta R., Bhat R., Tyagi A.K.

  Newly emerging multi-drug resistant strains of Mycobacterium tuberculosis (M.tb) severely limit the treatment options for tuberculosis (TB); hence, new antitubercular drugs are urgently needed. The mymA operon is essential for the virulence and intracellular survival of M.tb and thus represents an ... >> More

  Newly emerging multi-drug resistant strains of Mycobacterium tuberculosis (M.tb) severely limit the treatment options for tuberculosis (TB); hence, new antitubercular drugs are urgently needed. The mymA operon is essential for the virulence and intracellular survival of M.tb and thus represents an attractive target for the development of new antitubercular drugs. This study is focused on the structure-function relationship of Fatty Acyl-CoA Synthetase (FadD13, Rv3089) belonging to the mymA operon. Eight site-directed mutants of FadD13 were designed, constructed and analyzed for the structural-functional integrity of the enzyme. The study revealed that mutation of Lys(487) resulted in approximately 95% loss of the activity thus demonstrating its crucial requirement for the enzymatic activity. Comparison of the kinetic parameters showed the residues Lys(172) and Ala(302) to be involved in the binding of ATP and Ser(404) in the binding of CoenzymeA. The influence of mutations of the residues Val(209) and Trp(377) emphasized their importance in maintaining the structural integrity of FadD13. Besides, we show a synergistic influence of fatty acid and ATP binding on the conformation and rigidity of FadD13. FadD13 represents the first Fatty Acyl-CoA Synthetase to display biphasic kinetics for fatty acids. FadD13 exhibits a distinct preference for C(26)/C(24) fatty acids, which in the light of earlier reported observations further substantiates the role of the mymA operon in remodeling the cell envelope of intracellular M.tb under acidic conditions. A three-dimensional model of FadD13 was generated; the docking of ATP to the active site verified its interaction with Lys(172), Ala(302) and Lys(487) and corresponded well with the results of the mutational studies. Our study provides a significant understanding of the FadD13 protein including the identification of residues important for its activity as well as in the maintenance of structural integrity. We believe that the findings of this study will provide valuable inputs in the development of inhibitors against the mymA operon, an important target for the development of antitubercular drugs. << Less

  PLoS ONE 4:E8387-E8387(2009) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Human liver-specific very-long-chain acyl-coenzyme A synthetase: cDNA cloning and characterization of a second enzymatically active protein.

  Steinberg S.J., Wang S.J., McGuinness M.C., Watkins P.A.

  Activation of fatty acids, catalyzed by acyl-coenzyme A (acyl-CoA) synthetases, is required for their subsequent metabolism. Peroxisomes and microsomes contain very-long-chain acyl-CoA synthetases (VLCSs) capable of activating fatty acids with a chain length of 22 or more carbons. Decreased peroxi ... >> More

  Activation of fatty acids, catalyzed by acyl-coenzyme A (acyl-CoA) synthetases, is required for their subsequent metabolism. Peroxisomes and microsomes contain very-long-chain acyl-CoA synthetases (VLCSs) capable of activating fatty acids with a chain length of 22 or more carbons. Decreased peroxisomal VLCS activity is, in part, responsible for the biochemical pathology in X-linked adrenoleukodystrophy (X-ALD), illustrating the importance of VLCSs in cellular fatty acid homeostasis. We previously cloned two human genes encoding proteins homologous to rat peroxisomal VLCS; one (hVLCS) is the human ortholog to the rat VLCS gene and another (hVLCS-H1) encodes a related heart-specific protein. Here, we report the cloning of a third gene (hVLCS-H2) and characterization of its protein product. The hVLCS-H2 gene is located on human chromosome 19 and encodes a 690-amino-acid protein. The amino acid sequence of hVLCS-H2 is 44-45% identical and 67-69% similar to those of both hVLCS and hVLCS-H1. COS-1 cells transiently overexpressing hVLCS-H2 activated the very-long-chain fatty acid lignocerate (C24:0) at a rate >1.5-fold higher than that of nontransfected cells (P < 0.002). The hVLCS-H2-dependent activation of long- and branched-chain fatty acids following transient transfection was less striking. However, hVLCS-H2-dependent acyl-CoA synthetase activity with long- and very-long-chain fatty acid substrates was detected in COS-1 cells stably expressing hVLCS-H2. For all substrates tested (C18:0, C20:0, C24:0, C26:0), the hVLCS-H2 catalyzed activity was significantly increased (P < 0.01 to P < 0.0001). By both Northern analysis and reverse transcription polymerase chain reaction, hVLCS-H2 is expressed primarily in liver. Indirect immunofluorescence of COS-1 cells or human hepatoma-derived HepG2 cells expressing epitope-tagged hVLCS-H2 revealed that the protein was associated with the endoplasmic reticulum but not with peroxisomes. Thus, the primary role of hVLCS-H2 is likely to be in fatty acid elongation or complex lipid synthesis rather than in degradation. << Less

  Mol. Genet. Metab. 68:32-42(1999) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Evidence for 26 distinct acyl-coenzyme A synthetase genes in the human genome.

  Watkins P.A., Maiguel D., Jia Z., Pevsner J.

  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 in ... >> More

  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. << Less

  J. Lipid Res. 48:2736-2750(2007) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• The human liver-specific homolog of very long-chain acyl-CoA synthetase is cholate:CoA ligase.

  Steinberg S.J., Mihalik S.J., Kim D.G., Cuebas D.A., Watkins P.A.

  Unconjugated bile acids must be activated to their CoA thioesters before conjugation to taurine or glycine can occur. A human homolog of very long-chain acyl-CoA synthetase, hVLCS-H2, has two requisite properties of a bile acid:CoA ligase, liver specificity and an endoplasmic reticulum subcellular ... >> More

  Unconjugated bile acids must be activated to their CoA thioesters before conjugation to taurine or glycine can occur. A human homolog of very long-chain acyl-CoA synthetase, hVLCS-H2, has two requisite properties of a bile acid:CoA ligase, liver specificity and an endoplasmic reticulum subcellular localization. We investigated the ability of this enzyme to activate the primary bile acid, cholic acid, to its CoA derivative. When expressed in COS-1 cells, hVLCS-H2 exhibited cholate:CoA ligase (choloyl-CoA synthetase) activity with both non-isotopic and radioactive assays. Other long- and very long-chain acyl-CoA synthetases were incapable of activating cholate. Endogenous choloyl-CoA synthetase activity was also detected in liver-derived HepG2 cells but not in kidney-derived COS-1 cells. Our results are consistent with a role for hVLCS-H2 in the re-activation and re-conjugation of bile acids entering liver from the enterohepatic circulation rather than in de novo bile acid synthesis. << Less

  J. Biol. Chem. 275:15605-15608(2000) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Mouse very long-chain acyl-CoA synthetase 3/fatty acid transport protein 3 catalyzes fatty acid activation but not fatty acid transport in MA-10 cells.

  Pei Z., Fraisl P., Berger J., Jia Z., Forss-Petter S., Watkins P.A.

  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 ... >> More

  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 long-chain (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. << Less

  J. Biol. Chem. 279:54454-54462(2004) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Biochemical studies of three Saccharomyces cerevisiae acyl-CoA synthetases, Faa1p, Faa2p, and Faa3p.

  Knoll L.J., Johnson D.R., Gordon J.I.

  The efficiency and specificity of protein N-myristoylation appear to be influenced by the availability of myristoyl-CoA and other potential acyl-CoA substrates of myristoyl-CoA:protein N-myristoyltransferase. Recent studies have revealed that Saccharomyces cerevisiae contains at least three acyl-C ... >> More

  The efficiency and specificity of protein N-myristoylation appear to be influenced by the availability of myristoyl-CoA and other potential acyl-CoA substrates of myristoyl-CoA:protein N-myristoyltransferase. Recent studies have revealed that Saccharomyces cerevisiae contains at least three acyl-CoA synthetase genes (FAA for fatty acid activation). We have expressed Faa1p, Faa2p, and Faa3p in a strain of Escherichia coli that lacks its own endogenous acyl-CoA synthetase (FadD). Each S. cerevisiae acyl-CoA synthetase contained a carboxyl-terminal His tag so that it could be purified to homogeneity in a single step using nickel chelate affinity chromatography. In vitro assays of C3:0-C24:0 fatty acids indicate that Faa1p prefers C12:0-C16:0, with myristic and pentadecanoic acid (C15:0) having the highest activities. Faa2p can accommodate a wider range of acyl chain lengths: C9:0-C13:0 are preferred and have equivalent activities, although C7:0-C17:0 fatty acids are tolerated as substrates with no greater than a 2-fold variation in specific activity. The myristoyl-CoA synthetase activities of Faa1p and Faa2p are 2 orders of magnitude greater than that of Faa3p in vitro. Faa3p has a preference for C16 and C18 fatty acids with a cis-double bond at C-9-C-10. The temperature optimum for Faa1p is 30 degrees C, while Faa2p and Faa3p have the greatest activities at 25 degrees C. These in vitro observations were confirmed using two in vivo assays: (i) measurement of the ability of each S. cerevisiae acyl-CoA synthetase to direct the incorporation of exogenously derived tritiated myristate, palmitate, or oleate into cellular phospholipids produced in a fadD-strain of E. coli during exponential growth at 24 or 37 degrees C and (ii) measurement of the incorporation of [3H]myristate into a yeast N-myristoylprotein coexpressed with Nmt1p and Faa1p, Faa2p, or Faa3p in the fadD-strain. << Less

  J. Biol. Chem. 269:16348-16356(1994) [PubMed] [EuropePMC]

  This publication is cited by 19 other entries.

• Comparative biochemical studies of the murine fatty acid transport proteins (FATP) expressed in yeast.

  DiRusso C.C., Li H., Darwis D., Watkins P.A., Berger J., Black P.N.

  The fatty acid transport protein (FATP) family is a group of proteins that are predicted to be components of specific fatty acid trafficking pathways. In mammalian systems, six different isoforms have been identified, which function in the import of exogenous fatty acids or in the activation of ve ... >> More

  The fatty acid transport protein (FATP) family is a group of proteins that are predicted to be components of specific fatty acid trafficking pathways. In mammalian systems, six different isoforms have been identified, which function in the import of exogenous fatty acids or in the activation of very long-chain fatty acids. This has led to controversy as to whether these proteins function as membrane-bound fatty acid transporters or as acyl-CoA synthetases, which activate long-chain fatty acids concomitant with transport. The yeast FATP orthologue, Fat1p, is a dual functional protein and is required for both the import of long-chain fatty acids and the activation of very long-chain fatty acids; these activities intrinsic to Fat1p are separable functions. To more precisely define the roles of the different mammalian isoforms in fatty acid trafficking, the six murine proteins (mmFATP1-6) were expressed and characterized in a genetically defined yeast strain, which cannot transport long-chain fatty acids and has reduced long-chain acyl-CoA synthetase activity (fat1Delta faa1Delta). Each isoform was evaluated for fatty acid transport, fatty acid activation (using C18:1, C20:4, and C24:0 as substrates), and accumulation of very long-chain fatty acids. Murine FATP1, -2, and -4 complemented the defects in fatty acid transport and very long-chain fatty acid activation associated with a deletion of the yeast FAT1 gene; mmFATP3, -5, and -6 did not complement the transport function even though each was localized to the yeast plasma membrane. Both mmFATP3 and -6 activated C20:4 and C20:4, while the expression of mmFATP5 did not substantially increase acyl-CoA synthetases activities using the substrates tested. These data support the conclusion that the different mmFATP isoforms play unique roles in fatty acid trafficking, including the transport of exogenous long-chain fatty acids. << Less

  J. Biol. Chem. 280:16829-16837(2005) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

• Functional domains of the fatty acid transport proteins: studies using protein chimeras.

  DiRusso C.C., Darwis D., Obermeyer T., Black P.N.

  Fatty acid transport proteins (FATP) function in fatty acid trafficking pathways, several of which have been shown to participate in the transport of exogenous fatty acids into the cell. Members of this protein family also function as acyl CoA synthetases with specificity towards very long chain f ... >> More

  Fatty acid transport proteins (FATP) function in fatty acid trafficking pathways, several of which have been shown to participate in the transport of exogenous fatty acids into the cell. Members of this protein family also function as acyl CoA synthetases with specificity towards very long chain fatty acids or bile acids. These proteins have two identifying sequence motifs: The ATP/AMP motif, an approximately 100 amino acid segment required for ATP binding and common to members of the adenylate-forming super family of proteins, and the FATP/VLACS motif that consists of approximately 50 amino acid residues and is restricted to members of the FATP family. This latter motif has been implicated in fatty acid transport in the yeast FATP orthologue Fat1p. In the present studies using a yeast strain containing deletions in FAT1 (encoding Fat1p) and FAA1 (encoding the major acyl CoA synthetase (Acsl) Faa1p) as an experimental platform, the phenotypic and functional properties of specific murine FATP1-FATP4 and FATP6-FATP4 protein chimeras were evaluated in order to define elements within these proteins that further distinguish the fatty acid transport and activation functions. As expected from previous work FATP1 and FATP4 were functional in the fatty acid transport pathway, while and FATP6 was not. All three isoforms were able to activate the very long chain fatty acids arachidonate (C(20:4)) and lignocerate (C(24:0)), but with distinguishing activities between saturated and highly unsaturated ligands. A 73 amino acid segment common to FATP1 and FATP4 and between the ATP/AMP and FATP/VLACS motifs was identified by studying the chimeras, which is hypothesized to contribute to the transport function. << Less

  Biochim Biophys Acta 1781:135-143(2008) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Human fatty acid transport protein 2a/very long chain acyl-CoA synthetase 1 (FATP2a/Acsvl1) has a preference in mediating the channeling of exogenous n-3 fatty acids into phosphatidylinositol.

  Melton E.M., Cerny R.L., Watkins P.A., DiRusso C.C., Black P.N.

  The trafficking of fatty acids across the membrane and into downstream metabolic pathways requires their activation to CoA thioesters. Members of the fatty acid transport protein/very long chain acyl-CoA synthetase (FATP/Acsvl) family are emerging as key players in the trafficking of exogenous fat ... >> More

  The trafficking of fatty acids across the membrane and into downstream metabolic pathways requires their activation to CoA thioesters. Members of the fatty acid transport protein/very long chain acyl-CoA synthetase (FATP/Acsvl) family are emerging as key players in the trafficking of exogenous fatty acids into the cell and in intracellular fatty acid homeostasis. We have expressed two naturally occurring splice variants of human FATP2 (Acsvl1) in yeast and 293T-REx cells and addressed their roles in fatty acid transport, activation, and intracellular trafficking. Although both forms (FATP2a (M(r) 70,000) and FATP2b (M(r) 65,000 and lacking exon3, which encodes part of the ATP binding site)) were functional in fatty acid import, only FATP2a had acyl-CoA synthetase activity, with an apparent preference toward very long chain fatty acids. To further address the roles of FATP2a or FATP2b in fatty acid uptake and activation, LC-MS/MS was used to separate and quantify different acyl-CoA species (C14-C24) and to monitor the trafficking of different classes of exogenous fatty acids into intracellular acyl-CoA pools in 293T-REx cells expressing either isoform. The use of stable isotopically labeled fatty acids demonstrated FATP2a is involved in the uptake and activation of exogenous fatty acids, with a preference toward n-3 fatty acids (C18:3 and C22:6). Using the same cells expressing FATP2a or FATP2b, electrospray ionization/MS was used to follow the trafficking of stable isotopically labeled n-3 fatty acids into phosphatidylcholine and phosphatidylinositol. The expression of FATP2a resulted in the trafficking of C18:3-CoA and C22:6-CoA into both phosphatidylcholine and phosphatidylinositol but with a distinct preference for phosphatidylinositol. Collectively these data demonstrate FATP2a functions in fatty acid transport and activation and provides specificity toward n-3 fatty acids in which the corresponding n-3 acyl-CoAs are preferentially trafficked into acyl-CoA pools destined for phosphatidylinositol incorporation. << Less

  J. Biol. Chem. 286:30670-30679(2011) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.