Publications

• Promiscuous fatty acyl CoA ligases produce acyl-CoA and acyl-SNAC precursors for polyketide biosynthesis.

  Arora P., Vats A., Saxena P., Mohanty D., Gokhale R.S.

  The study of bioactive natural products has undergone rapid advancement with the cloning and sequencing of large number of gene clusters and the concurrent progress to manipulate complex biosynthetic systems in heterologous hosts. The genetic reconstitution necessitates that the heterologous hosts ... >> More

  The study of bioactive natural products has undergone rapid advancement with the cloning and sequencing of large number of gene clusters and the concurrent progress to manipulate complex biosynthetic systems in heterologous hosts. The genetic reconstitution necessitates that the heterologous hosts possess substrate pools that could be coordinately supplied for biosynthesis. Polyketide synthases (PKS) utilize acyl-coenzyme A (CoA) precursors and synthesize polyketides by repetitive decarboxylative condensations. Here we show that acyl-CoA ligases, which belong to a large family of acyl-activating enzymes, possess potential to produce varied starter CoA precursors that could be utilized in polyketide biosynthesis. Incidentally, such protein domains have been recognized in several PKS and nonribosomal peptide synthetase gene clusters. Our studies with mycobacterial fatty acyl-CoA ligases (FACLs) show remarkable tolerance to activate a variety of fatty acids that contain modifications at alpha, beta, omega, and omega-nu positions. This substrate flexibility extends further such that these proteins also efficiently utilize N-acetyl cysteamine, the shorter acceptor terminal portion of CoASH, to produce acyl-SNACs. We show that the in situ generated acyl-CoAs and acyl-SNACs could be channeled to types I and -III PKS systems to produce new metabolites. Together, the promiscuous activity of FACL and PKSs provides new opportunities to expand the repertoire of natural products. << Less

  J. Am. Chem. Soc. 127:9388-9389(2005) [PubMed] [EuropePMC]

  This publication is cited by 33 other entries.

• Enzymic activation and transfer of fatty acids as acyl-adenylates in mycobacteria.

  Trivedi O.A., Arora P., Sridharan V., Tickoo R., Mohanty D., Gokhale R.S.

  The metabolic repertoire in nature is augmented by generating hybrid metabolites from a limited set of gene products. In mycobacteria, several unique complex lipids are produced by the combined action of fatty acid synthases and polyketide synthases (PKSs), although it is not clear how the covalen ... >> More

  The metabolic repertoire in nature is augmented by generating hybrid metabolites from a limited set of gene products. In mycobacteria, several unique complex lipids are produced by the combined action of fatty acid synthases and polyketide synthases (PKSs), although it is not clear how the covalently sequestered biosynthetic intermediates are transferred from one enzymatic complex to another. Here we show that some of the 36 annotated fadD genes, located adjacent to the PKS genes in the Mycobacterium tuberculosis genome, constitute a new class of long-chain fatty acyl-AMP ligases (FAALs). These proteins activate long-chain fatty acids as acyl-adenylates, which are then transferred to the multifunctional PKSs for further chain extension. This mode of activation and transfer of fatty acids is contrary to the previously described universal mechanism involving the formation of acyl-coenzyme A thioesters. Similar mechanisms may operate in the biosynthesis of other lipid-containing metabolites and could have implications in engineering novel hybrid products. << Less

  Nature 428:441-445(2004) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• The Escherichia coli fadK (ydiD) gene encodes an anerobically regulated short chain acyl-CoA synthetase.

  Morgan-Kiss R.M., Cronan J.E.

  We recently reported a new metabolic competency for Escherichia coli, the ability to degrade and utilize fatty acids of various chain lengths as sole carbon and energy sources. This beta-oxidation pathway is distinct from the previously described aerobic fatty acid degradation pathway and requires ... >> More

  We recently reported a new metabolic competency for Escherichia coli, the ability to degrade and utilize fatty acids of various chain lengths as sole carbon and energy sources. This beta-oxidation pathway is distinct from the previously described aerobic fatty acid degradation pathway and requires enzymes encoded by two operons, yfcYX and ydiQRSTD. The yfcYX operon (renamed fadIJ) encodes enzymes required for hydration, oxidation, and thiolytic cleavage of the acyl chain. The ydiQRSTD operon encodes a putative acyl-CoA synthetase, ydiD (renamed fadK), as well as putative electron transport chain components. We report that FadK is as an acyl-CoA synthetase that has a preference for short chain length fatty acid substrates (<10 C atoms). The enzymatic mechanism of FadK is similar to other acyl-CoA synthetases in that it forms an acyl-AMP intermediate prior to the formation of the final acyl-CoA product. Expression of FadK is repressed during aerobic growth and is maximally expressed under anaerobic conditions in the presence of the terminal electron acceptor, fumarate. << Less

  J. Biol. Chem. 279:37324-37333(2004) [PubMed] [EuropePMC]

  This publication is cited by 7 other entries.

• Two long-chain acyl-CoA synthetases from Arabidopsis thaliana involved in peroxisomal fatty acid beta-oxidation.

  Fulda M., Shockey J., Werber M., Wolter F.P., Heinz E.

  Post-germinative growth of oilseeds is dependent on the breakdown of the stored lipid reserves. Long-chain acyl-CoA synthetase activities (LACS) are critically involved in this process by activating the released free fatty acids and thus feeding the beta-oxidation cycle in glyoxysomes. Here we rep ... >> More

  Post-germinative growth of oilseeds is dependent on the breakdown of the stored lipid reserves. Long-chain acyl-CoA synthetase activities (LACS) are critically involved in this process by activating the released free fatty acids and thus feeding the beta-oxidation cycle in glyoxysomes. Here we report on the identification of two LACS genes, AtLACS6 and AtLACS7 from Arabidopsis thaliana coding for peroxisomal LACS proteins. The subcellular localization was verified by co-expression studies of spectral variants of the green fluorescent protein (GFP). While AtLACS6 is targeted by a type 2 (PTS2) peroxisomal targeting sequence, for AtLACS7 a functional PTS1 as well as a PTS2 could be demonstrated. Possible explanations for this potentially redundant targeting information will be discussed. Expression studies of both genes revealed a strong induction 1 day after germination resembling the expression pattern of other genes involved in beta-oxidation. Analysis of the substrate specificities of the two LACS proteins demonstrated enzymatic activity for both enzymes with the whole spectrum of fatty acids found in stored lipid reserves. These results suggest that both LACS proteins might have overlapping functions and are able to initiate beta-oxidation in plant peroxisomes. << Less

  Plant J. 32:93-103(2002) [PubMed] [EuropePMC]

  This publication is cited by 6 other entries.

• The dual function of the Mycobacterium tuberculosis FadD32 required for mycolic acid biosynthesis.

  Leger M., Gavalda S., Guillet V., van der Rest B., Slama N., Montrozier H., Mourey L., Quemard A., Daffe M., Marrakchi H.

  Mycolic acids are major and specific lipids of Mycobacterium tuberculosis cell envelope. Their synthesis requires the condensation by Pks13 of a C(22)-C(26) fatty acid with the C(50)-C(60) meromycolic acid activated by FadD32, a fatty acyl-AMP ligase essential for mycobacterial growth. A combinati ... >> More

  Mycolic acids are major and specific lipids of Mycobacterium tuberculosis cell envelope. Their synthesis requires the condensation by Pks13 of a C(22)-C(26) fatty acid with the C(50)-C(60) meromycolic acid activated by FadD32, a fatty acyl-AMP ligase essential for mycobacterial growth. A combination of biochemical and enzymatic approaches demonstrated that FadD32 exhibits substrate specificity for relatively long-chain fatty acids. More importantly, FadD32 catalyzes the transfer of the synthesized acyl-adenylate onto specific thioester acceptors, thus revealing the protein acyl-ACP ligase function. Therefore, FadD32 might be the prototype of a group of M. tuberculosis polyketide-synthase-associated adenylation enzymes possessing such activity. A substrate analog of FadD32 inhibited not only the enzyme activity but also mycolic acid synthesis and mycobacterial growth, opening an avenue for the development of novel antimycobacterial agents. << Less

  Chem. Biol. 16:510-519(2009) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

• Jasmonates meet fatty acids: functional analysis of a new acyl-coenzyme A synthetase family from Arabidopsis thaliana.

  Kienow L., Schneider K., Bartsch M., Stuible H.-P., Weng H., Miersch O., Wasternack C., Kombrink E.

  Arabidopsis thaliana contains a large number of genes encoding carboxylic acid-activating enzymes, including long-chain fatty acyl-CoA synthetase (LACS), 4-coumarate:CoA ligases (4CL), and proteins closely related to 4CLs with unknown activities. The function of these 4CL-like proteins was systema ... >> More

  Arabidopsis thaliana contains a large number of genes encoding carboxylic acid-activating enzymes, including long-chain fatty acyl-CoA synthetase (LACS), 4-coumarate:CoA ligases (4CL), and proteins closely related to 4CLs with unknown activities. The function of these 4CL-like proteins was systematically explored by applying an extensive substrate screen, and it was uncovered that activation of fatty acids is the common feature of all active members of this protein family, thereby defining a new group of fatty acyl-CoA synthetase, which is distinct from the known LACS family. Significantly, four family members also displayed activity towards different biosynthetic precursors of jasmonic acid (JA), including 12-oxo-phytodienoic acid (OPDA), dinor-OPDA, 3-oxo-2(2'-[Z]-pentenyl)cyclopentane-1-octanoic acid (OPC-8), and OPC-6. Detailed analysis of in vitro properties uncovered significant differences in substrate specificity for individual enzymes, but only one protein (At1g20510) showed OPC-8:CoA ligase activity. Its in vivo function was analysed by transcript and jasmonate profiling of Arabidopsis insertion mutants for the gene. OPC-8:CoA ligase expression was activated in response to wounding or infection in the wild type but was undetectable in the mutants, which also exhibited OPC-8 accumulation and reduced levels of JA. In addition, the developmental, tissue- and cell-type specific expression pattern of the gene, and regulatory properties of its promoter were monitored by analysing promoter::GUS reporter lines. Collectively, the results demonstrate that OPC-8:CoA ligase catalyses an essential step in JA biosynthesis by initiating the beta-oxidative chain shortening of the carboxylic acid side chain of its precursors, and, in accordance with this function, the protein is localized in peroxisomes. << Less

  J. Exp. Bot. 59:403-419(2008) [PubMed] [EuropePMC]

  This publication is cited by 15 other entries.

• Mechanistic and functional insights into fatty acid activation in Mycobacterium tuberculosis.

  Arora P., Goyal A., Natarajan V.T., Rajakumara E., Verma P., Gupta R., Yousuf M., Trivedi O.A., Mohanty D., Tyagi A., Sankaranarayanan R., Gokhale R.S.

  The recent discovery of fatty acyl-AMP ligases (FAALs) in Mycobacterium tuberculosis (Mtb) provided a new perspective of fatty acid activation. These proteins convert fatty acids to the corresponding adenylates, which are intermediates of acyl-CoA-synthesizing fatty acyl-CoA ligases (FACLs). Prese ... >> More

  The recent discovery of fatty acyl-AMP ligases (FAALs) in Mycobacterium tuberculosis (Mtb) provided a new perspective of fatty acid activation. These proteins convert fatty acids to the corresponding adenylates, which are intermediates of acyl-CoA-synthesizing fatty acyl-CoA ligases (FACLs). Presently, it is not evident how obligate pathogens such as Mtb have evolved such new themes of functional versatility and whether the activation of fatty acids to acyladenylates could indeed be a general mechanism. Here, based on elucidation of the first structure of an FAAL protein and by generating loss-of-function and gain-of-function mutants that interconvert FAAL and FACL activities, we demonstrate that an insertion motif dictates formation of acyladenylate. Because FAALs in Mtb are crucial nodes in the biosynthetic network of virulent lipids, inhibitors directed against these proteins provide a unique multipronged approach to simultaneously disrupting several pathways. << Less

  Nat. Chem. Biol. 5:166-173(2009) [PubMed] [EuropePMC]

  This publication is cited by 7 other entries.

• 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.

• Molecular identification and characterization of two medium-chain acyl-CoA synthetases, MACS1 and the Sa gene product.

  Fujino T., Takei Y.A., Sone H., Ioka R.X., Kamataki A., Magoori K., Takahashi S., Sakai J., Yamamoto T.T.

  In this study, we identified and characterized two murine cDNAs encoding medium-chain acyl-CoA synthetase (MACS). One, designated MACS1, is a novel protein and the other the product of the Sa gene (Sa protein), which is preferentially expressed in spontaneously hypertensive rats. Based on the muri ... >> More

  In this study, we identified and characterized two murine cDNAs encoding medium-chain acyl-CoA synthetase (MACS). One, designated MACS1, is a novel protein and the other the product of the Sa gene (Sa protein), which is preferentially expressed in spontaneously hypertensive rats. Based on the murine MACS1 sequence, we also identified the location and organization of the human MACS1 gene, showing that the human MACS1 and Sa genes are located in the opposite transcriptional direction within a 150-kilobase region on chromosome 16p13.1. Murine MACS1 and Sa protein were overexpressed in COS cells, purified to homogeneity, and characterized. Among C4-C16 fatty acids, MACS1 preferentially utilizes octanoate, whereas isobutyrate is the most preferred fatty acid among C2-C6 fatty acids for Sa protein. Like Sa gene transcript, MACS1 mRNA was detected mainly in the liver and kidney. Subcellular fractionation revealed that both MACS1 and Sa protein are localized in the mitochondrial matrix. (14)C-Fatty acid incorporation studies indicated that acyl-CoAs produced by MACS1 and Sa protein are utilized mainly for oxidation. << Less

  J. Biol. Chem. 276:35961-35966(2001) [PubMed] [EuropePMC]

  This publication is cited by 10 other entries.

• O-MACS, a novel member of the medium-chain acyl-CoA synthetase family, specifically expressed in the olfactory epithelium in a zone-specific manner.

  Oka Y., Kobayakawa K., Nishizumi H., Miyamichi K., Hirose S., Tsuboi A., Sakano H.

  In rodents, the olfactory epithelium (OE) can be divided into four topographically distinct zones, and each member of the odorant receptor (OR) gene family is expressed only in one particular zone. To study the functional significance of the zonal structure of the OE, we searched for genes express ... >> More

  In rodents, the olfactory epithelium (OE) can be divided into four topographically distinct zones, and each member of the odorant receptor (OR) gene family is expressed only in one particular zone. To study the functional significance of the zonal structure of the OE, we searched for genes expressed in a zone-specific manner by using the differential display method. Among the clones isolated from the rat OE, we characterized a novel olfactory protein termed O-MACS, a member of the medium-chain acyl-CoA synthetase family. The o-macs gene encodes a protein of 580 amino acids, sharing 56-63% identity with other MACS family proteins. RT-PCR analysis demonstrated that the o-macs gene is expressed only in the OE, unlike other MACS family genes. In situ hybridization revealed that the o-macs transcripts are present in the neuronal cell layer of olfactory sensory neurons (OSNs) as well as in the supporting and basal cell layers in the most dorso-medial area (zone 1) of the OE. Developmental analysis revealed that the o-macs gene is already expressed on embryonic day 11.5, before the onset of the OR gene expression, in a restricted area within the rat olfactory placode. Recombinant O-MACS protein tagged with c-Myc and His6 demonstrated an acyl-CoA synthetase activity for fatty acid activation, and protein localization to mitochondria like other MACS family proteins. The present study indicates that this novel protein may play important roles in processing odorants in a zone-specific manner, or the zonal patterning of the OE during development. << Less

  Eur. J. Biochem. 270:1995-2004(2003) [PubMed] [EuropePMC]

  This publication is cited by 6 other entries.