Phylogenetic and experimental characterization of an acyl-ACP thioesterase family reveals significant diversity in enzymatic specificity and activity

PubMed Central

2011-01-01

Background Acyl-acyl carrier protein thioesterases (acyl-ACP TEs) catalyze the hydrolysis of the thioester bond that links the acyl chain to the sulfhydryl group of the phosphopantetheine prosthetic group of ACP. This reaction terminates acyl chain elongation of fatty acid biosynthesis, and in plant seeds it is the biochemical determinant of the fatty acid compositions of storage lipids. Results To explore acyl-ACP TE diversity and to identify novel acyl ACP-TEs, 31 acyl-ACP TEs from wide-ranging phylogenetic sources were characterized to ascertain their in vivo activities and substrate specificities. These acyl-ACP TEs were chosen by two different approaches: 1) 24 TEs were selected from public databases on the basis of phylogenetic analysis and fatty acid profile knowledge of their source organisms; and 2) seven TEs were molecularly cloned from oil palm (Elaeis guineensis), coconut (Cocos nucifera) and Cuphea viscosissima, organisms that produce medium-chain and short-chain fatty acids in their seeds. The in vivo substrate specificities of the acyl-ACP TEs were determined in E. coli. Based on their specificities, these enzymes were clustered into three classes: 1) Class I acyl-ACP TEs act primarily on 14- and 16-carbon acyl-ACP substrates; 2) Class II acyl-ACP TEs have broad substrate specificities, with major activities toward 8- and 14-carbon acyl-ACP substrates; and 3) Class III acyl-ACP TEs act predominantly on 8-carbon acyl-ACPs. Several novel acyl-ACP TEs act on short-chain and unsaturated acyl-ACP or 3-ketoacyl-ACP substrates, indicating the diversity of enzymatic specificity in this enzyme family. Conclusion These acyl-ACP TEs can potentially be used to diversify the fatty acid biosynthesis pathway to produce novel fatty acids. PMID:21831316

Knockout of the regulatory site of 3-ketoacyl-ACP synthase III enhances short- and medium-chain acyl-ACP synthesis.

PubMed

Abbadi, A; Brummel, M; Spener, F

2000-10-01

3-ketoacyl-acyl carrier protein synthase (KAS) III catalyses the first condensing step of the fatty acid synthase (FAS) type II reaction in plants and bacteria, using acetyl CoA and malonyl-acyl carrier protein (ACP) as substrates. Enzymatic characterization of recombinant KAS III from Cuphea wrightii embryo shows that this enzyme is strongly inhibited by medium-chain acyl-ACP end products of the FAS reaction, i.e. inhibition by lauroyl-ACP was uncompetitive towards acetyl CoA and non-competitive with regard to malonyl-ACP. This indicated a distinct attachment site for regulatory acyl-ACPs. Based on alignment of primary structures of various KAS IIIs and 3-ketoacyl CoA synthases, we suspected the motif G290NTSAAS296 to be responsible for binding of regulatory acyl-ACPs. Deletion of the tetrapeptide G290NTS293 led to a change of secondary structure and complete loss of KAS III condensing activity. Exchange of asparagine291 to aspartate, alanine294 to serine and alanine295 to proline, however, produced mutant enzymes with slightly reduced condensing activity, yet with insensitivity towards acyl-ACPs. To assess the potential of unregulated KAS III as tool in oil production, we designed in vitro experiments employing FAS preparations from medium-chain fatty acid-producing Cuphea lanceolata seeds and long-chain fatty acid-producing rape seeds, each supplemented with a fivefold excess of the N291D KAS III mutant. High amounts of short-chain acyl-ACPs in the case of C. lanceolata, and of medium-chain acyl-ACPs in the case of rape seed preparations, were obtained. This approach targets regulation and offers new possibilities to derive transgenic or non-transgenic plants for production of seed oils with new qualities.

Characterization of Lipid A Variants by Energy-Resolved Mass Spectrometry: Impact of Acyl Chains

NASA Astrophysics Data System (ADS)

Crittenden, Christopher M.; Akin, Lucas D.; Morrison, Lindsay J.; Trent, M. Stephen; Brodbelt, Jennifer S.

2017-06-01

Lipid A molecules consist of a diglucosamine sugar core with a number of appended acyl chains that vary in their length and connectivity. Because of the challenging nature of characterizing these molecules and differentiating between isomeric species, an energy-resolved MS/MS strategy was undertaken to track the fragmentation trends and map genealogies of product ions originating from consecutive cleavages of acyl chains. Generalizations were developed based on the number and locations of the primary and secondary acyl chains as well as variations in preferential cleavages arising from the location of the phosphate groups. Secondary acyl chain cleavage occurs most readily for lipid A species at the 3' position, followed by primary acyl chain fragmentation at both the 3' and 3 positions. In the instances of bisphosphorylated lipid A variants, phosphate loss occurs readily in conjunction with the most favorable primary and secondary acyl chain cleavages. [Figure not available: see fulltext.

Elucidating the substrate specificities of acyl-lipid thioesterases from diverse plant taxa.

PubMed

Kalinger, Rebecca S; Pulsifer, Ian P; Rowland, Owen

2018-06-01

Acyl-ACP thioesterase enzymes, which cleave fatty acyl thioester bonds to release free fatty acids, contribute to much of the fatty acid diversity in plants. In Arabidopsis thaliana, a family of four single hot-dog fold domain, plastid-localized acyl-lipid thioesterases (AtALT1-4) generate medium-chain (C6-C14) fatty and β-keto fatty acids as secondary metabolites. These volatile products may serve to attract insect pollinators or deter predatory insects. Homologs of AtALT1-4 are present in all plant taxa, but are nearly all uncharacterized. Despite high sequence identity, AtALT1-4 generate different lipid products, suggesting that ALT homologs in other plants also have highly varied activities. We investigated the catalytic diversity of ALT-like thioesterases by screening the substrate specificities of 15 ALT homologs from monocots, eudicots, a lycophyte, a green microalga, and the ancient gymnosperm Gingko biloba, via expression in Escherichia coli. Overall, these enzymes had highly varied substrate preferences compared to one another and to AtALT1-4, and could be classified into four catalytic groups comprising members from diverse taxa. Group 1 ALTs primarily generated 14:1 β-keto fatty acids, Group 2 ALTs produced 6-10 carbon fatty/β-keto fatty acids, Group 3 ALTs predominantly produced 12-14 carbon fatty acids, and Group 4 ALTs mainly generated 16 carbon fatty acids. Enzymes in each group differed significantly in the quantities of lipids and types of minor products they generated in E. coli. Medium-chain fatty acids are used to manufacture insecticides, pharmaceuticals, and biofuels, and ALT-like proteins are ideal candidates for metabolic engineering to produce specific fatty acids in significant quantities. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Two Predicted Transmembrane Domains Exclude Very Long Chain Fatty acyl-CoAs from the Active Site of Mouse Wax Synthase

PubMed Central

Kawelke, Steffen; Feussner, Ivo

2015-01-01

Wax esters are used as coatings or storage lipids in all kingdoms of life. They are synthesized from a fatty alcohol and an acyl-CoA by wax synthases. In order to get insights into the structure-function relationships of a wax synthase from Mus musculus, a domain swap experiment between the mouse acyl-CoA:wax alcohol acyltransferase (AWAT2) and the homologous mouse acyl-CoA:diacylglycerol O-acyltransferase 2 (DGAT2) was performed. This showed that the substrate specificity of AWAT2 is partially determined by two predicted transmembrane domains near the amino terminus of AWAT2. Upon exchange of the two domains for the respective part of DGAT2, the resulting chimeric enzyme was capable of incorporating up to 20% of very long acyl chains in the wax esters upon expression in S. cerevisiae strain H1246. The amount of very long acyl chains in wax esters synthesized by wild type AWAT2 was negligible. The effect was narrowed down to a single amino acid position within one of the predicted membrane domains, the AWAT2 N36R variant. Taken together, we provide first evidence that two predicted transmembrane domains in AWAT2 are involved in determining its acyl chain length specificity. PMID:26714272

Sirtuin 3 (SIRT3) Protein Regulates Long-chain Acyl-CoA Dehydrogenase by Deacetylating Conserved Lysines Near the Active Site

PubMed Central

Bharathi, Sivakama S.; Zhang, Yuxun; Mohsen, Al-Walid; Uppala, Radha; Balasubramani, Manimalha; Schreiber, Emanuel; Uechi, Guy; Beck, Megan E.; Rardin, Matthew J.; Vockley, Jerry; Verdin, Eric; Gibson, Bradford W.; Hirschey, Matthew D.; Goetzman, Eric S.

2013-01-01

Long-chain acyl-CoA dehydrogenase (LCAD) is a key mitochondrial fatty acid oxidation enzyme. We previously demonstrated increased LCAD lysine acetylation in SIRT3 knockout mice concomitant with reduced LCAD activity and reduced fatty acid oxidation. To study the effects of acetylation on LCAD and determine sirtuin 3 (SIRT3) target sites, we chemically acetylated recombinant LCAD. Acetylation impeded substrate binding and reduced catalytic efficiency. Deacetylation with recombinant SIRT3 partially restored activity. Residues Lys-318 and Lys-322 were identified as SIRT3-targeted lysines. Arginine substitutions at Lys-318 and Lys-322 prevented the acetylation-induced activity loss. Lys-318 and Lys-322 flank residues Arg-317 and Phe-320, which are conserved among all acyl-CoA dehydrogenases and coordinate the enzyme-bound FAD cofactor in the active site. We propose that acetylation at Lys-318/Lys-322 causes a conformational change which reduces hydride transfer from substrate to FAD. Medium-chain acyl-CoA dehydrogenase and acyl-CoA dehydrogenase 9, two related enzymes with lysines at positions equivalent to Lys-318/Lys-322, were also efficiently deacetylated by SIRT3 following chemical acetylation. These results suggest that acetylation/deacetylation at Lys-318/Lys-322 is a mode of regulating fatty acid oxidation. The same mechanism may regulate other acyl-CoA dehydrogenases. PMID:24121500

The level of circulating octanoate does not predict ghrelin O-acyl transferase (GOAT)-mediated acylation of ghrelin during fasting.

PubMed

Nass, Ralf; Nikolayev, Alexander; Liu, Jianhua; Pezzoli, Suzan S; Farhy, Leon S; Patrie, James; Gaylinn, Bruce D; Heiman, Mark; Thorner, Michael O

2015-01-01

Acyl-ghrelin is a 28-amino acid peptide released from the stomach. Ghrelin O-acyl transferase (GOAT) attaches an 8-carbon medium-chain fatty acid (MCFA) (octanoate) to serine 3 of ghrelin. This acylation is necessary for the activity of ghrelin. Animal data suggest that MCFAs provide substrate for GOAT and an increase in nutritional octanoate increases acyl-ghrelin. To address the question of the source of substrate for acylation, we studied whether the decline in ghrelin acylation during fasting is associated with a decline in circulating MCFAs. Eight healthy young men (aged 18-28 years, body mass index range, 20.6-26.2 kg/m(2)) had blood drawn every 10 minutes for acyl- and desacyl-ghrelin and every hour for free fatty acids (FFAs) during the last 24 hours of a 61.5-hour fast and during a fed day. FFAs were measured by a highly sensitive liquid chromatography-mass spectroscopy method. Acyl- and desacyl-ghrelin were measured in an in-house assay; the results were published previously. Ghrelin acylation was assessed by the ratio of acyl-ghrelin to total ghrelin. With the exception of MCFAs C8 and C10, all other FFAs, the MCFAs (C6 and C12), and the long-chain fatty acids (C14-C18) significantly increased with fasting (P < .05). There was no significant association between the fold change in ghrelin acylation and circulating FFAs. These results suggest that changes in circulating MCFAs are not linked to the decline in ghrelin acylation during fasting and support the hypothesis that acylation of ghrelin depends at least partially on the availability of gastroluminal MCFAs or the regulation of GOAT activity.

Computational Redesign of Acyl-ACP Thioesterase with Improved Selectivity toward Medium-Chain-Length Fatty Acids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Grisewood, Matthew J.; Hernández-Lozada, Néstor J.; Thoden, James B.

Enzyme and metabolic engineering offer the potential to develop biocatalysts for converting natural resources to a wide range of chemicals. To broaden the scope of potential products beyond natural metabolites, methods of engineering enzymes to accept alternative substrates and/or perform novel chemistries must be developed. DNA synthesis can create large libraries of enzyme-coding sequences, but most biochemistries lack a simple assay to screen for promising enzyme variants. Our solution to this challenge is structure-guided mutagenesis, in which optimization algorithms select the best sequences from libraries based on specified criteria (i.e., binding selectivity). We demonstrate this approach by identifying medium-chain (C8–C12)more » acyl-ACP thioesterases through structure-guided mutagenesis. Medium-chain fatty acids, which are products of thioesterase-catalyzed hydrolysis, are limited in natural abundance, compared to long-chain fatty acids; the limited supply leads to high costs of C6–C10 oleochemicals such as fatty alcohols, amines, and esters. Here, we applied computational tools to tune substrate binding of the highly active ‘TesA thioesterase in Escherichia coli. We used the IPRO algorithm to design thioesterase variants with enhanced C12 or C8 specificity, while maintaining high activity. After four rounds of structure-guided mutagenesis, we identified 3 variants with enhanced production of dodecanoic acid (C12) and 27 variants with enhanced production of octanoic acid (C8). The top variants reached up to 49% C12 and 50% C8 while exceeding native levels of total free fatty acids. A comparably sized library created by random mutagenesis failed to identify promising mutants. The chain length-preference of ‘TesA and the best mutant were confirmed in vitro using acyl-CoA substrates. Molecular dynamics simulations, confirmed by resolved crystal structures, of ‘TesA variants suggest that hydrophobic forces govern ‘TesA substrate specificity

Computational Redesign of Acyl-ACP Thioesterase with Improved Selectivity toward Medium-Chain-Length Fatty Acids

DOE PAGES

Grisewood, Matthew J.; Hernández-Lozada, Néstor J.; Thoden, James B.; ...

2017-04-20

Enzyme and metabolic engineering offer the potential to develop biocatalysts for converting natural resources to a wide range of chemicals. To broaden the scope of potential products beyond natural metabolites, methods of engineering enzymes to accept alternative substrates and/or perform novel chemistries must be developed. DNA synthesis can create large libraries of enzyme-coding sequences, but most biochemistries lack a simple assay to screen for promising enzyme variants. Our solution to this challenge is structure-guided mutagenesis, in which optimization algorithms select the best sequences from libraries based on specified criteria (i.e., binding selectivity). We demonstrate this approach by identifying medium-chain (C8–C12)more » acyl-ACP thioesterases through structure-guided mutagenesis. Medium-chain fatty acids, which are products of thioesterase-catalyzed hydrolysis, are limited in natural abundance, compared to long-chain fatty acids; the limited supply leads to high costs of C6–C10 oleochemicals such as fatty alcohols, amines, and esters. Here, we applied computational tools to tune substrate binding of the highly active ‘TesA thioesterase in Escherichia coli. We used the IPRO algorithm to design thioesterase variants with enhanced C12 or C8 specificity, while maintaining high activity. After four rounds of structure-guided mutagenesis, we identified 3 variants with enhanced production of dodecanoic acid (C12) and 27 variants with enhanced production of octanoic acid (C8). The top variants reached up to 49% C12 and 50% C8 while exceeding native levels of total free fatty acids. A comparably sized library created by random mutagenesis failed to identify promising mutants. The chain length-preference of ‘TesA and the best mutant were confirmed in vitro using acyl-CoA substrates. Molecular dynamics simulations, confirmed by resolved crystal structures, of ‘TesA variants suggest that hydrophobic forces govern ‘TesA substrate specificity

Evidence for involvement of medium chain acyl-CoA dehydrogenase in the metabolism of phenylbutyrate

PubMed Central

Kormanik, Kaitlyn; Kang, Heejung; Cuebas, Dean; Vockley, Jerry; Mohsen, Al-Walid

2012-01-01

Sodium phenylbutyrate is used for treating urea cycle disorders, providing an alternative for ammonia excretion. Following conversion to its CoA ester, phenylbutyryl-CoA is postulated to undergo one round of β-oxidation to phenylacetyl-CoA, the active metabolite. Molecular modeling suggests that medium chain acyl-CoA dehydrogenase (MCAD; EC 1.3.99.3), a key enzyme in straight chain fatty acid β-oxidation, could utilize phenylbutyryl-CoA as substrate. Moreover, phenylpropionyl-CoA has been shown to be a substrate for MCAD and its intermediates accumulate in patients with MCAD deficiency. We have examined the involvement of MCAD and other acyl-CoA dehydrogenases (ACADs) in the metabolism of phenylbutyryl-CoA. Anaerobic titration of purified recombinant human MCAD with phenylbutyryl-CoA caused changes in the MCAD spectrum that are similar to those induced by octanoyl-CoA, its bona fide substrate, and unique to the development of the charge transfer ternary complex. The calculated apparent dissociation constant (KD app) for these substrates was 2.16 μM and 0.12 μM, respectively. The MCAD reductive and oxidative half reactions were monitored using the electron transfer flavoprotein (ETF) fluorescence reduction assay. The catalytic efficiency and the Km for phenylbutyryl-CoA were 0.2 mM−1· sec−1 and 5.3 μM compared to 4.0 mM−1· sec−1 and 2.8 μM for octanoyl-CoA. Extracts of wild type and MCAD-deficient lymphoblast cells were tested for the ability to reduce ETF using phenylbutyryl-CoA as substrate. While ETF reduction activity was detected in extracts of wild type cells, it was undetectable in extracts of cells deficient in MCAD. The results are consistent with MCAD playing a key role in phenylbutyrate metabolism. PMID:23141465

Evidence for involvement of medium chain acyl-CoA dehydrogenase in the metabolism of phenylbutyrate.

PubMed

Kormanik, Kaitlyn; Kang, Heejung; Cuebas, Dean; Vockley, Jerry; Mohsen, Al-Walid

2012-12-01

Sodium phenylbutyrate is used for treating urea cycle disorders, providing an alternative for ammonia excretion. Following conversion to its CoA ester, phenylbutyryl-CoA is postulated to undergo one round of β-oxidation to phenylacetyl-CoA, the active metabolite. Molecular modeling suggests that medium chain acyl-CoA dehydrogenase (MCAD; EC 1.3.99.3), a key enzyme in straight chain fatty acid β-oxidation, could utilize phenylbutyryl-CoA as substrate. Moreover, phenylpropionyl-CoA has been shown to be a substrate for MCAD and its intermediates accumulate in patients with MCAD deficiency. We have examined the involvement of MCAD and other acyl-CoA dehydrogenases (ACADs) in the metabolism of phenylbutyryl-CoA. Anaerobic titration of purified recombinant human MCAD with phenylbutyryl-CoA caused changes in the MCAD spectrum that are similar to those induced by octanoyl-CoA, its bona fide substrate, and unique to the development of the charge transfer ternary complex. The calculated apparent dissociation constant (K(D app)) for these substrates was 2.16 μM and 0.12 μM, respectively. The MCAD reductive and oxidative half reactions were monitored using the electron transfer flavoprotein (ETF) fluorescence reduction assay. The catalytic efficiency and the K(m) for phenylbutyryl-CoA were 0.2 mM 34(-1)·sec(-1) and 5.3 μM compared to 4.0 mM(-1)·sec(-1) and 2.8 μM for octanoyl-CoA. Extracts of wild type and MCAD-deficient lymphoblast cells were tested for the ability to reduce ETF using phenylbutyryl-CoA as substrate. While ETF reduction activity was detected in extracts of wild type cells, it was undetectable in extracts of cells deficient in MCAD. The results are consistent with MCAD playing a key role in phenylbutyrate metabolism. Copyright © 2012 Elsevier Inc. All rights reserved.

Disruption of plastid acyl:acyl carrier protein synthetases increases medium chain fatty acid accumulation in seeds of transgenic Arabidopsis.

PubMed

Tjellström, Henrik; Strawsine, Merissa; Silva, Jillian; Cahoon, Edgar B; Ohlrogge, John B

2013-04-02

Engineering transgenic plants that accumulate high levels of medium-chain fatty acids (MCFA) has been least successful for shorter chain lengths (e.g., C8). We demonstrate that one limitation is the activity of acyl-ACP synthetase (AAE) that re-activates fatty acids released by acyl-ACP thioesterases. Seed expression of Cuphea pulcherrima FATB acyl-ACP thioesterase in a double mutant lacking AAE15/16 increased 8:0 accumulation almost 2-fold compared to expression in wild type. These results also provide an in planta demonstration that AAE enzymes participate not only in activation of exogenously added MCFA but also in activation of MCFA synthesized in plastids. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Sites of intermolecular crosslinking of fatty acyl chains in phospholipids carrying a photoactivable carbene precursor

PubMed Central

Gupta, Chhitar M.; Costello, Catherine E.; Khorana, H. Gobind

1979-01-01

Sonicated vesicles of 1-fatty acyl-2-ω-(2-diazo-3,3,3-trifluoropropionoxy) fatty acyl sn-glycero-3-phosphoryl-cholines were shown recently to form intermolecular crosslinks by insertion of the photogenerated carbene into a C—H bond of a neighboring hydrocarbon chain. We now report that photolysis of multilamellar dispersions gives a second series of products in which carbene insertion is accompanied by elimination of a molecule of hydrogen fluoride. The sites of crosslinking in the latter compounds have been studied by mass spectrometry using phospholipids with varying chain lengths of the fatty acyl groups carrying the carbene precursor. The patterns observed show that the point of maximum crosslinking is consistent with the recent conclusion that in phospholipids the sn-2 fatty acyl chain trails the sn-1 chain by 2-4 atoms. Images PMID:16592675

The 2.1Å Crystal Structure of an Acyl-CoA Synthetase from Methanosarcina acetivorans reveals an alternate acyl binding pocket for small branched acyl substrates†,‡

PubMed Central

Shah, Manish B.; Ingram-Smith, Cheryl; Cooper, Leroy L.; Qu, Jun; Meng, Yu; Smith, Kerry S.; Gulick, Andrew M.

2009-01-01

The acyl-AMP forming family of adenylating enzymes catalyze two-step reactions to activate a carboxylate with the chemical energy derived from ATP hydrolysis. X-ray crystal structures have been determined for multiple members of this family and, together with biochemical studies, provide insights into the active site and catalytic mechanisms used by these enzymes. These studies have shown that the enzymes use a domain rotation of 140° to reconfigure a single active site to catalyze the two partial reactions. We present here the crystal structure of a new medium chain acyl-CoA synthetase from Methanosarcina acetivorans. The binding pocket for the three substrates is analyzed, with many conserved residues present in the AMP binding pocket. The CoA binding pocket is compared to the pockets of both acetyl-CoA synthetase and 4-chlorobenzoate:CoA ligase. Most interestingly, the acyl binding pocket of the new structure is compared with other acyl- and aryl-CoA synthetases. A comparison of the acyl-binding pocket of the acyl-CoA synthetase from M. acetivorans with other structures identifies a shallow pocket that is used to bind the medium chain carboxylates. These insights emphasize the high sequence and structural diversity among this family in the area of the acyl binding pocket. PMID:19544569

Genetics Home Reference: short-chain acyl-CoA dehydrogenase deficiency

MedlinePlus

... Orphanet: Short chain acyl-CoA dehydrogenase deficiency Screening, Technology and Research in Genetics Patient Support and Advocacy Resources (5 links) Children Living with Inherited Metabolic Disease (CLIMB) Children's Mitochondrial ...

Modified Acyl-ACP desaturase

DOEpatents

Cahoon, Edgar B.; Shanklin, John; Lindqvist, Ylva; Schneider, Gunter

1999-03-30

Disclosed is a method for modifying the chain length and double bond positional specificities of a soluble plant fatty acid desaturase. More specifically, the method involves modifying amino acid contact residues in the substrate binding channel of the soluble fatty acid desaturase which contact the fatty acid. Specifically disclosed is the modification of an acyl-ACP desaturase. Amino acid contact residues which lie within the substrate binding channel are identified, and subsequently replaced with different residues to effect the modification of activity.

Modified acyl-ACP desaturase

DOEpatents

Cahoon, Edgar B.; Shanklin, John; Lindgvist, Ylva; Schneider, Gunter

1998-01-06

Disclosed is a methods for modifying the chain length and double bond positional specificities of a soluble plant fatty acid desaturase. More specifically, the method involves modifying amino acid contact residues in the substrate binding channel of the soluble fatty acid desaturase which contact the fatty acid. Specifically disclosed is the modification of an acyl-ACP desaturase. Amino acid contact residues which lie within the substrate binding channel are identified, and subsequently replaced with different residues to effect the modification of activity.

Identification of amino acids conferring chain length substrate specificities on fatty alcohol-forming reductases FAR5 and FAR8 from Arabidopsis thaliana.

PubMed

Chacón, Micaëla G; Fournier, Ashley E; Tran, Frances; Dittrich-Domergue, Franziska; Pulsifer, Ian P; Domergue, Frédéric; Rowland, Owen

2013-10-18

Fatty alcohols play a variety of biological roles in all kingdoms of life. Fatty acyl reductase (FAR) enzymes catalyze the reduction of fatty acyl-coenzyme A (CoA) or fatty acyl-acyl carrier protein substrates to primary fatty alcohols. FAR enzymes have distinct substrate specificities with regard to chain length and degree of saturation. FAR5 (At3g44550) and FAR8 (At3g44560) from Arabidopsis thaliana are 85% identical at the amino acid level and are of equal length, but they possess distinct specificities for 18:0 or 16:0 acyl chain length, respectively. We used Saccharomyces cerevisiae as a heterologous expression system to assess FAR substrate specificity determinants. We identified individual amino acids that affect protein levels or 16:0-CoA versus 18:0-CoA specificity by expressing in yeast FAR5 and FAR8 domain-swap chimeras and site-specific mutants. We found that a threonine at position 347 and a serine at position 363 were important for high FAR5 and FAR8 protein accumulation in yeast and thus are likely important for protein folding and stability. Amino acids at positions 355 and 377 were important for dictating 16:0-CoA versus 18:0-CoA chain length specificity. Simultaneously converting alanine 355 and valine 377 of FAR5 to the corresponding FAR8 residues, leucine and methionine, respectively, almost fully converted FAR5 specificity from 18:0-CoA to 16:0-CoA. The reciprocal amino acid conversions, L355A and M377V, made in the active FAR8-S363P mutant background converted its specificity from 16:0-CoA to 18:0-CoA. This study is an important advancement in the engineering of highly active FAR proteins with desired specificities for the production of fatty alcohols with industrial value.

Modified acyl-ACP desaturase

DOEpatents

Cahoon, E.B.; Shanklin, J.; Lindgvist, Y.; Schneider, G.

1998-01-06

Disclosed is a method for modifying the chain length and double bond positional specificities of a soluble plant fatty acid desaturase. More specifically, the method involves modifying amino acid contact residues in the substrate binding channel of the soluble fatty acid desaturase which contact the fatty acid. Specifically disclosed is the modification of an acyl-ACP desaturase. Amino acid contact residues which lie within the substrate binding channel are identified, and subsequently replaced with different residues to effect the modification of activity. 1 fig.

Modified Acyl-ACP desaturase

DOEpatents

Cahoon, E.B.; Shanklin, J.; Lindqvist, Y.; Schneider, G.

1999-03-30

Disclosed is a method for modifying the chain length and double bond positional specificities of a soluble plant fatty acid desaturase. More specifically, the method involves modifying amino acid contact residues in the substrate binding channel of the soluble fatty acid desaturase which contact the fatty acid. Specifically disclosed is the modification of an acyl-ACP desaturase. Amino acid contact residues which lie within the substrate binding channel are identified, and subsequently replaced with different residues to effect the modification of activity. 2 figs.

Effect of acyl chain length on selective biocatalytic deacylation on O-aryl glycosides and separation of anomers.

PubMed

Aggarwal, Neha; Arya, Anu; Mathur, Divya; Singh, Sukhdev; Tyagi, Abhilash; Kumar, Rajesh; Rana, Neha; Singh, Rajendra; Prasad, Ashok K

2014-04-01

It has been demonstrated that Lipozyme® TL IM (Thermomyces lanuginosus lipase immobilised on silica) can selectively deacylate the ester function involving the C-5' hydroxyl group of α-anomers over the other acyl functions of anomeric mixture of peracylated O-aryl α,β-D-ribofuranoside. The analysis of results of biocatalytic deacylation reaction revealed that the reaction time decreases with the increase in the acyl chain length from C1 to C4. The unique selectivity of Lipozyme® TL IM has been harnessed for the separation of anomeric mixture of peracylated O-aryl α,β-D-ribofuranosides, The lipase mediated selective deacylation methodology has been used for the synthesis of O-aryl α-D-ribofuranosides and O-aryl β-D-ribofuranosides in pure forms, which can be used as chromogenic substrate for the detection of pathogenic microbial parasites containing glycosidases. Copyright © 2014. Published by Elsevier Inc.

Genetics Home Reference: very long-chain acyl-CoA dehydrogenase deficiency

MedlinePlus

... Very long chain acyl-CoA dehydrogenase deficiency Screening, Technology, and Research in Genetics Virginia Department of Health (PDF) Patient Support and Advocacy Resources (4 links) Children's Mitochondrial Disease Network (UK) FOD (Fatty Oxidation Disorders) ...

Distinct membrane properties are differentially influenced by cardiolipin content and acyl chain composition in biomimetic membranes.

PubMed

Pennington, Edward Ross; Fix, Amy; Sullivan, E Madison; Brown, David A; Kennedy, Anthony; Shaikh, Saame Raza

2017-02-01

Cardiolipin (CL) has a critical role in maintaining mitochondrial inner membrane structure. In several conditions such as heart failure and aging, there is loss of CL content and remodeling of CL acyl chains, which are hypothesized to impair mitochondrial inner membrane biophysical organization. Therefore, this study discriminated how CL content and acyl chain composition influenced select properties of simple and complex mitochondrial mimicking model membranes. We focused on monolayer excess area/molecule (a measure of lipid miscibility), bilayer phase transitions, and microdomain organization. In monolayer compression studies, loss of tetralinoleoyl [(18:2) 4 ] CL content decreased the excess area/molecule. Replacement of (18:2) 4 CL acyl chains with tetraoleoyl [(18:1) 4 ] CL or tetradocosahexaenoyl [(22:6) 4 ] CL generally had little influence on monolayer excess area/molecule; in contrast, replacement of (18:2) 4 CL acyl chains with tetramyristoyl [(14:0) 4 ] CL increased monolayer excess area/molecule. In bilayers, calorimetric studies showed that substitution of (18:2) 4 CL with (18:1) 4 CL or (22:6) 4 CL lowered the phase transition temperature of phosphatidylcholine vesicles whereas (14:0) 4 CL had no effect. Finally, quantitative imaging of giant unilamellar vesicles revealed differential effects of CL content and acyl chain composition on microdomain organization, visualized with the fluorescent probe Texas Red DHPE. Notably, microdomain areas were decreased by differing magnitudes upon lowering of (18:2) 4 CL content and substitution of (18:2) 4 CL with (14:0) 4 CL or (22:6) 4 CL. Conversely, exchanging (18:2) 4 CL with (18:1) 4 CL increased microdomain area. Altogether, these data demonstrate that CL content and fatty acyl composition differentially target membrane physical properties, which has implications for understanding how CL regulates mitochondrial activity and the design of CL-specific therapeutics. Copyright © 2016 Elsevier B.V. All rights

Long Chain N-acyl Homoserine Lactone Production by Enterobacter sp. Isolated from Human Tongue Surfaces

PubMed Central

Yin, Wai-Fong; Purmal, Kathiravan; Chin, Shenyang; Chan, Xin-Yue; Chan, Kok-Gan

2012-01-01

We report the isolation of N-acyl homoserine lactone-producing Enterobacter sp. isolate T1-1 from the posterior dorsal surfaces of the tongue of a healthy individual. Spent supernatants extract from Enterobacter sp. isolate T1-1 activated the biosensor Agrobacterium tumefaciens NTL4(pZLR4), suggesting production of long chain AHLs by these isolates. High resolution mass spectrometry analysis of these extracts confirmed that Enterobacter sp. isolate T1-1 produced a long chain N-acyl homoserine lactone, namely N-dodecanoyl-homoserine lactone (C12-HSL). To the best of our knowledge, this is the first isolation of Enterobacter sp., strain T1-1 from the posterior dorsal surface of the human tongue and N-acyl homoserine lactones production by this bacterium. PMID:23202161

Endothelial cell palmitoylproteomics identifies novel lipid modified targets and potential substrates for protein acyl transferases

PubMed Central

Marin, Ethan P.; Derakhshan, Behrad; Lam, TuKiet T.; Davalos, Alberto; Sessa, William C.

2012-01-01

Rationale Protein S-palmitoylation is the post-translational attachment of a saturated 16-carbon palmitic acid to a cysteine side chain via a thioester bond. Palmitoylation can affect protein localization, trafficking, stability, and function. The extent and roles of palmitoylation in endothelial cell (EC) biology is not well understood, in part due to technological limits on palmitoylprotein detection. Objective To develop a method using acyl-biotinyl exchange (ABE) technology coupled with mass spectrometry to globally isolate and identify palmitoylproteins in EC. Methods and Results More than 150 putative palmitoyl proteins were identified in EC using ABE and mass spectrometry. Among the novel palmitoylproteins identified is superoxide dismutase 1 (SOD1), an intensively studied enzyme that protects all cells from oxidative damage. Mutation of cysteine 6 prevents palmitoylation, leads to reduction in SOD1 activity in vivo and in vitro, and inhibits nuclear localization, thereby supporting a functional role for SOD1 palmitoylation. Moreover, we used ABE to search for substrates of particular protein acyl transferases in EC. We found that palmitoylation of the cell adhesion protein PECAM1 is dependent on the protein acyl transferase ZDHHC21. We show that knockdown of ZDHHC21 leads to reduced levels of PECAM1 at the cell surface. Conclusions Our data demonstrate the utility of EC palmitoylproteomics to reveal new insights into the role of this important post-translational lipid modification in EC biology. PMID:22496122

Functional role of a distal (3'-phosphate) group of CoA in the recombinant human liver medium-chain acyl-CoA dehydrogenase-catalysed reaction.

PubMed Central

Peterson, K L; Srivastava, D K

1997-01-01

The X-ray crystallographic structure of medium-chain acyl-CoA dehydrogenase (MCAD)-octenoyl-CoA complex reveals that the 3'-phosphate group of CoA is confined to the exterior of the protein structure [approx. 15 A (1.5 nm) away from the enzyme active site], and is fully exposed to the outside solvent environment. To ascertain whether such a distal (3'-phosphate) fragment of CoA plays any significant role in the enzyme catalysis, we investigated the recombinant human liver MCAD (HMCAD)-catalysed reaction by using normal (phospho) and 3'-phosphate-truncated (dephospho) forms of octanoyl-CoA and butyryl-CoA substrates. The steady-state kinetic data revealed that deletion of the 3'-phosphate group from octanoyl-CoA substrate increased the turnover rate of the enzyme to about one-quarter, whereas that from butyryl-CoA substrate decreased the turnover rate of the enzyme to about one-fifth; the Km values of both these substrates were increased by 5-10-fold on deletion of the 3'-phosphate group from the corresponding acyl-CoA substrates. The transient kinetics for the reductive half-reaction, oxidative half-reaction and the dissociation 'off-rate' (of the reaction product from the oxidized enzyme site) were all found to be affected by deletions of the 3'-phosphate group from octanoyl-CoA and butyryl-CoA substrates. A cumulative account of these results reveals that, although the 3'-phosphate group of acyl-CoA substrates might seem 'useless' on the basis of the structural data, it has an essential functional role during HMCAD catalysis. PMID:9271097

Plant Microsomal Phospholipid Acyl Hydrolases Have Selectivities for Uncommon Fatty Acids.

PubMed

Stahl, U.; Banas, A.; Stymne, S.

1995-03-01

Developing endosperms and embryos accumulating triacylglycerols rich in caproyl (decanoyl) groups (i.e. developing embryos of Cuphea procumbens and Ulmus glabra) had microsomal acyl hydrolases with high selectivities toward phosphatidylcholine with this acyl group. Similarly, membranes from Euphorbia lagascae and Ricinus communis endosperms, which accumulate triacylglycerols with vernoleate (12-epoxy-octadeca-9-enoate) and ricinoleate (12-hydroxy-octadeca-9-enoate), respectively, had acyl hydrolases that selectively removed their respective oxygenated acyl group from the phospholipids. The activities toward phospholipid substrates with epoxy, hydroxy, and medium-chain acyl groups varied greatly between microsomal preparations from different plant species. Epoxidated and hydroxylated acyl groups in sn-1 and sn-2 positions of phosphatidylcholine and in sn-1-lysophosphatidylcholine were hydrolyzed to a similar extent, whereas the hydrolysis of caproyl groups was highly dependent on the positional localization.

Progress toward Understanding Protein S-acylation: Prospective in Plants

PubMed Central

Li, Yaxiao; Qi, Baoxiu

2017-01-01

S-acylation, also known as S-palmitoylation or palmitoylation, is a reversible post-translational lipid modification in which long chain fatty acid, usually the 16-carbon palmitate, covalently attaches to a cysteine residue(s) throughout the protein via a thioester bond. It is involved in an array of important biological processes during growth and development, reproduction and stress responses in plant. S-acylation is a ubiquitous mechanism in eukaryotes catalyzed by a family of enzymes called Protein S-Acyl Transferases (PATs). Since the discovery of the first PAT in yeast in 2002 research in S-acylation has accelerated in the mammalian system and followed by in plant. However, it is still a difficult field to study due to the large number of PATs and even larger number of putative S-acylated substrate proteins they modify in each genome. This is coupled with drawbacks in the techniques used to study S-acylation, leading to the slower progress in this field compared to protein phosphorylation, for example. In this review we will summarize the discoveries made so far based on knowledge learnt from the characterization of protein S-acyltransferases and the S-acylated proteins, the interaction mechanisms between PAT and its specific substrate protein(s) in yeast and mammals. Research in protein S-acylation and PATs in plants will also be covered although this area is currently less well studied in yeast and mammalian systems. PMID:28392791

Acyl-chain remodeling of dioctanoyl-phosphatidylcholine in Saccharomyces cerevisiae mutant defective in de novo and salvage phosphatidylcholine synthesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kishino, Hideyuki; Eguchi, Hiroki; Takagi, Keiko

2014-03-07

Highlights: • Dioctanoyl-PC (diC8PC) supported growth of a yeast mutant defective in PC synthesis. • diC8PC was converted to PC species containing longer acyl residues in the mutant. • Both acyl residues of diC8PC were replaced by longer fatty acids in vitro. • This system will contribute to the elucidation of the acyl chain remodeling of PC. - Abstract: A yeast strain, in which endogenous phosphatidylcholine (PC) synthesis is controllable, was constructed by the replacement of the promoter of PCT1, encoding CTP:phosphocholine cytidylyltransferase, with GAL1 promoter in a double deletion mutant of PEM1 and PEM2, encoding phosphatidylethanolamine methyltransferase and phospholipidmore » methyltransferase, respectively. This mutant did not grow in the glucose-containing medium, but the addition of dioctanoyl-phosphatidylcholine (diC8PC) supported its growth. Analyses of the metabolism of {sup 13}C-labeled diC8PC ((methyl-{sup 13}C){sub 3}-diC8PC) in this strain using electrospray ionization tandem mass spectrometry revealed that it was converted to PC species containing acyl residues of 16 or 18 carbons at both sn-1 and sn-2 positions. In addition, both acyl residues of (methyl-{sup 13}C){sub 3}-diC8PC were replaced with 16:1 acyl chains in the in vitro reaction using the yeast cell extract in the presence of palmitoleoyl-CoA. These results indicate that PC containing short acyl residues was remodeled to those with acyl chains of physiological length in yeast.« less

Interaction of cholesterol with sphingomyelins and acyl-chain-matched phosphatidylcholines: a comparative study of the effect of the chain length.

PubMed Central

Ramstedt, B; Slotte, J P

1999-01-01

In this study we have synthesized sphingomyelins (SM) and phosphatidylcholines (PC) with amide-linked or sn-2 linked acyl chains with lengths from 14 to 24 carbons. The purpose was to examine how the chain length and degree of unsaturation affected the interaction of cholesterol with these phospholipids in model membrane systems. Monolayers of saturated SMs and PCs with acyl chain lengths above 14 carbons were condensed and displayed a high collapse pressure ( approximately 70 mN/m). Monolayers of N-14:0-SM and 1(16:0)-2(14:0)-PC had a much lower collapse pressure (58-60 mN/m) and monounsaturated SMs collapsed at approximately 50 mN/m. The relative interaction of cholesterol with these phospholipids was determined at 22 degreesC by measuring the rate of cholesterol desorption from mixed monolayers (50 mol % cholesterol; 20 mN/m) to beta-cyclodextrin in the subphase (1.7 mM). The rate of cholesterol desorption was lower from saturated SM monolayers than from chain-matched PC monolayers. In SM monolayers, the rate of cholesterol desorption was very slow for all N-linked chains, whereas for PC monolayers we could observe higher desorption rates from monolayers of longer PCs. These results show that cholesterol interacts favorably with SMs (low rate of desorption), whereas its interaction (or miscibility) with long chain PCs is weaker. Introduction of a single cis-unsaturation in the N-linked acyl chain of SMs led to faster rates of cholesterol desorption as compared with saturated SMs. The exception was monolayers of N-22:1-SM and N-24:1-SM from which cholesterol desorbed almost as slowly as from the corresponding saturated SM monolayers. The results of this study suggest that cholesterol is most likely capable of interacting with all physiologically relevant (including long-chain) SMs present in the plasma membrane of cells. PMID:9929492

Plant Microsomal Phospholipid Acyl Hydrolases Have Selectivities for Uncommon Fatty Acids.

PubMed Central

Stahl, U.; Banas, A.; Stymne, S.

1995-01-01

Developing endosperms and embryos accumulating triacylglycerols rich in caproyl (decanoyl) groups (i.e. developing embryos of Cuphea procumbens and Ulmus glabra) had microsomal acyl hydrolases with high selectivities toward phosphatidylcholine with this acyl group. Similarly, membranes from Euphorbia lagascae and Ricinus communis endosperms, which accumulate triacylglycerols with vernoleate (12-epoxy-octadeca-9-enoate) and ricinoleate (12-hydroxy-octadeca-9-enoate), respectively, had acyl hydrolases that selectively removed their respective oxygenated acyl group from the phospholipids. The activities toward phospholipid substrates with epoxy, hydroxy, and medium-chain acyl groups varied greatly between microsomal preparations from different plant species. Epoxidated and hydroxylated acyl groups in sn-1 and sn-2 positions of phosphatidylcholine and in sn-1-lysophosphatidylcholine were hydrolyzed to a similar extent, whereas the hydrolysis of caproyl groups was highly dependent on the positional localization. PMID:12228415

Degree of fatty acyl chain unsaturation in biliary lecithin dictates cholesterol nucleation and crystal growth.

PubMed

Tazuma, S; Ochi, H; Teramen, K; Yamashita, Y; Horikawa, K; Miura, H; Hirano, N; Sasaki, M; Aihara, N; Hatsushika, S

1994-11-17

To clarify factors involved in the formation of cholesterol gallstones, we studied the relationship between the degree of fatty acyl chain unsaturation of biliary lecithin and bile metastability. We used supersaturated model bile solutions (molar taurocholate/lecithin/cholesterol ratio (73:19.5:7.5), total lipid concentration 9 g/dl) that contained equimolar egg yolk or soybean lecithins or a sn-1 palmitoyl, sn-2 linoleoyl phosphatidylcholine. Gel permeation chromatographic studies showed that the vesicular cholesterol distribution and dimension were inversely related to the degree of unsaturation of the lecithin species, estimated by reverse phase, high-performance liquid chromatography. Differential interference contrast microscopy and assay of cholesterol crystal growth showed that a higher degree of fatty acyl chain unsaturation of the lecithin species was associated with a faster nucleation time and rate of crystal growth. Our results suggest that vesicular lecithins containing more unsaturated fatty acyl chains bind less tightly to cholesterol than lecithins containing predominantly saturated fatty acids, and that the biliary lecithin species dictates, in part, the nucleation and growth of cholesterol crystals in bile.

An Unusual Fatty Acyl:Adenylate Ligase (FAAL)-Acyl Carrier Protein (ACP) Didomain in Ambruticin Biosynthesis.

PubMed

Hemmerling, Franziska; Lebe, Karen E; Wunderlich, Johannes; Hahn, Frank

2018-03-08

The divinylcyclopropane (DVC) fragment of the ambruticins is proposed to be formed by a unique polyene cyclisation mechanism, in which the unusual didomain AmbG plays a key role. It is proposed to activate the branched thioester carboxylic acid resulting from polyene cyclisation and to transfer it to its associated acyl carrier protein (ACP). After oxidative decarboxylation, the intermediate is channelled back into polyketide synthase (PKS) processing. AmbG was previously annotated as an adenylation-thiolation didomain with a very unusual substrate selectivity code but has not yet been biochemically studied. On the basis of sequence and homology model analysis, we reannotate AmbG as a fatty acyl:adenylate ligase (FAAL)-acyl carrier protein didomain with unusual substrate specificity. The expected adenylate-forming activity on fatty acids was confirmed by in vitro studies. AmbG also adenylates a number of structurally diverse carboxylic acids, including functionalised fatty acids and unsaturated and aromatic carboxylic acids. HPLC-MS analysis and competition experiments show that AmbG preferentially acylates its ACP with long-chain hydrophobic acids and tolerates a π system and a branch near the carboxylic acid. AmbG is the first characterised example of a FAAL-ACP didomain that is centrally located in a PKS and apparently activates a polyketidic intermediate. This is an important step towards deeper biosynthetic studies such as partial reconstitution of the ambruticin pathway to elucidate DVC formation. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Role of long-chain fatty acyl-CoA esters in the regulation of metabolism and in cell signalling.

PubMed Central

Faergeman, N J; Knudsen, J

1997-01-01

The intracellular concentration of free unbound acyl-CoA esters is tightly controlled by feedback inhibition of the acyl-CoA synthetase and is buffered by specific acyl-CoA binding proteins. Excessive increases in the concentration are expected to be prevented by conversion into acylcarnitines or by hydrolysis by acyl-CoA hydrolases. Under normal physiological conditions the free cytosolic concentration of acyl-CoA esters will be in the low nanomolar range, and it is unlikely to exceed 200 nM under the most extreme conditions. The fact that acetyl-CoA carboxylase is active during fatty acid synthesis (Ki for acyl-CoA is 5 nM) indicates strongly that the free cytosolic acyl-CoA concentration is below 5 nM under these conditions. Only a limited number of the reported experiments on the effects of acyl-CoA on cellular functions and enzymes have been carried out at low physiological concentrations in the presence of the appropriate acyl-CoA-buffering binding proteins. Re-evaluation of many of the reported effects is therefore urgently required. However, the observations that the ryanodine-senstitive Ca2+-release channel is regulated by long-chain acyl-CoA esters in the presence of a molar excess of acyl-CoA binding protein and that acetyl-CoA carboxylase, the AMP kinase kinase and the Escherichia coli transcription factor FadR are affected by low nanomolar concentrations of acyl-CoA indicate that long-chain acyl-CoA esters can act as regulatory molecules in vivo. This view is further supported by the observation that fatty acids do not repress expression of acetyl-CoA carboxylase or Delta9-desaturase in yeast deficient in acyl-CoA synthetase. PMID:9173866

Differential utilization of enzyme-substrate interactions for acylation but not deacylation during the catalytic cycle of Kex2 protease.

PubMed

Rockwell, N C; Fuller, R S

2001-10-19

Kex2 protease from Saccharomyces cerevisiae is the prototype for a family of eukaryotic proprotein processing proteases belonging to the subtilase superfamily of serine proteases. Kex2 can be distinguished from degradative subtilisins on the basis of stringent substrate specificity and distinct pre-steady-state behavior. To better understand these mechanistic differences, we have examined the effects of substrate residues at P(1) and P(4) on individual steps in the Kex2 catalytic cycle with a systematic series of isosteric peptidyl amide and ester substrates. The results demonstrate that substrates based on known, physiological cleavage sites exhibit high acylation rates (> or =550 s(-1)) with Kex2. Substitution of Lys for the physiologically correct Arg at P(1) resulted in a > or =200-fold drop in acylation rate with almost no apparent effect on binding or deacylation. In contrast, substitution of the physiologically incorrect Ala for Nle at P(4) resulted in a much smaller defect in acylation and a modest but significant effect on binding with Lys at P(1). This substitution also had no effect on deacylation. These results demonstrate that Kex2 utilizes enzyme-substrate interactions in different ways at different steps in the catalytic cycle, with the S(1)-P(1) contact providing a key specificity determinant at the acylation step.

Insight into Coenzyme A cofactor binding and the mechanism of acyl-transfer in an acylating aldehyde dehydrogenase from Clostridium phytofermentans

PubMed Central

Tuck, Laura R.; Altenbach, Kirsten; Ang, Thiau Fu; Crawshaw, Adam D.; Campopiano, Dominic J.; Clarke, David J.; Marles-Wright, Jon

2016-01-01

The breakdown of fucose and rhamnose released from plant cell walls by the cellulolytic soil bacterium Clostridium phytofermentans produces toxic aldehyde intermediates. To enable growth on these carbon sources, the pathway for the breakdown of fucose and rhamnose is encapsulated within a bacterial microcompartment (BMC). These proteinaceous organelles sequester the toxic aldehyde intermediates and allow the efficient action of acylating aldehyde dehydrogenase enzymes to produce an acyl-CoA that is ultimately used in substrate-level phosphorylation to produce ATP. Here we analyse the kinetics of the aldehyde dehydrogenase enzyme from the fucose/rhamnose utilisation BMC with different short-chain fatty aldehydes and show that it has activity against substrates with up to six carbon atoms, with optimal activity against propionaldehyde. We have also determined the X-ray crystal structure of this enzyme in complex with CoA and show that the adenine nucleotide of this cofactor is bound in a distinct pocket to the same group in NAD+. This work is the first report of the structure of CoA bound to an aldehyde dehydrogenase enzyme and our crystallographic model provides important insight into the differences within the active site that distinguish the acylating from non-acylating aldehyde dehydrogenase enzymes. PMID:26899032

Insight into Coenzyme A cofactor binding and the mechanism of acyl-transfer in an acylating aldehyde dehydrogenase from Clostridium phytofermentans.

PubMed

Tuck, Laura R; Altenbach, Kirsten; Ang, Thiau Fu; Crawshaw, Adam D; Campopiano, Dominic J; Clarke, David J; Marles-Wright, Jon

2016-02-22

The breakdown of fucose and rhamnose released from plant cell walls by the cellulolytic soil bacterium Clostridium phytofermentans produces toxic aldehyde intermediates. To enable growth on these carbon sources, the pathway for the breakdown of fucose and rhamnose is encapsulated within a bacterial microcompartment (BMC). These proteinaceous organelles sequester the toxic aldehyde intermediates and allow the efficient action of acylating aldehyde dehydrogenase enzymes to produce an acyl-CoA that is ultimately used in substrate-level phosphorylation to produce ATP. Here we analyse the kinetics of the aldehyde dehydrogenase enzyme from the fucose/rhamnose utilisation BMC with different short-chain fatty aldehydes and show that it has activity against substrates with up to six carbon atoms, with optimal activity against propionaldehyde. We have also determined the X-ray crystal structure of this enzyme in complex with CoA and show that the adenine nucleotide of this cofactor is bound in a distinct pocket to the same group in NAD(+). This work is the first report of the structure of CoA bound to an aldehyde dehydrogenase enzyme and our crystallographic model provides important insight into the differences within the active site that distinguish the acylating from non-acylating aldehyde dehydrogenase enzymes.

Structural characterization of acyl-CoA oxidases reveals a direct link between pheromone biosynthesis and metabolic state in Caenorhabditis elegans

PubMed Central

Zhang, Xinxing; Jones, Rachel A.; Bruner, Steven D.; Butcher, Rebecca A.

2016-01-01

Caenorhabditis elegans secretes ascarosides as pheromones to communicate with other worms and to coordinate the development and behavior of the population. Peroxisomal β-oxidation cycles shorten the side chains of ascaroside precursors to produce the short-chain ascaroside pheromones. Acyl-CoA oxidases, which catalyze the first step in these β-oxidation cycles, have different side chain-length specificities and enable C. elegans to regulate the production of specific ascaroside pheromones. Here, we determine the crystal structure of the acyl-CoA oxidase 1 (ACOX-1) homodimer and the ACOX-2 homodimer bound to its substrate. Our results provide a molecular basis for the substrate specificities of the acyl-CoA oxidases and reveal why some of these enzymes have a very broad substrate range, whereas others are quite specific. Our results also enable predictions to be made for the roles of uncharacterized acyl-CoA oxidases in C. elegans and in other nematode species. Remarkably, we show that most of the C. elegans acyl-CoA oxidases that participate in ascaroside biosynthesis contain a conserved ATP-binding pocket that lies at the dimer interface, and we identify key residues in this binding pocket. ATP binding induces a structural change that is associated with tighter binding of the FAD cofactor. Mutations that disrupt ATP binding reduce FAD binding and reduce enzyme activity. Thus, ATP may serve as a regulator of acyl-CoA oxidase activity, thereby directly linking ascaroside biosynthesis to ATP concentration and metabolic state. PMID:27551084

Remote control of regioselectivity in acyl-acyl carrier protein-desaturases.

PubMed

Guy, Jodie E; Whittle, Edward; Moche, Martin; Lengqvist, Johan; Lindqvist, Ylva; Shanklin, John

2011-10-04

Regiospecific desaturation of long-chain saturated fatty acids has been described as approaching the limits of the discriminatory power of enzymes because the substrate entirely lacks distinguishing features close to the site of dehydrogenation. To identify the elusive mechanism underlying regioselectivity, we have determined two crystal structures of the archetypal Δ9 desaturase from castor in complex with acyl carrier protein (ACP), which show the bound ACP ideally situated to position C9 and C10 of the acyl chain adjacent to the diiron active site for Δ9 desaturation. Analysis of the structures and modeling of the complex between the highly homologous ivy Δ4 desaturase and ACP, identified a residue located at the entrance to the binding cavity, Asp280 in the castor desaturase (Lys275 in the ivy desaturase), which is strictly conserved within Δ9 and Δ4 enzymes but differs between them. We hypothesized that interaction between Lys275 and the phosphate of the pantetheine, seen in the ivy model, is key to positioning C4 and C5 adjacent to the diiron center for Δ4 desaturation. Mutating castor Asp280 to Lys resulted in a major shift from Δ9 to Δ4 desaturation. Thus, interaction between desaturase side-chain 280 and phospho-serine 38 of ACP, approximately 27 Å from the site of double-bond formation, predisposes ACP binding that favors either Δ9 or Δ4 desaturation via repulsion (acidic side chain) or attraction (positively charged side chain), respectively. Understanding the mechanism underlying remote control of regioselectivity provides the foundation for reengineering desaturase enzymes to create designer chemical feedstocks that would provide alternatives to those currently obtained from petrochemicals.

Regioselective self-acylating cyclodextrins in organic solvent

NASA Astrophysics Data System (ADS)

Cho, Eunae; Yun, Deokgyu; Jeong, Daham; Im, Jieun; Kim, Hyunki; Dindulkar, Someshwar D.; Choi, Youngjin; Jung, Seunho

2016-03-01

Amphiphilic cyclodextrins have been synthesized with self-acylating reaction using vinyl esters in dimethylformamide. In the present study no base, catalyst, or enzyme was used, and the structural analyses using thin layer chromatography, nuclear magnetic resonance spectroscopy and mass spectrometry show that the cyclodextrin is substituted preferentially by one acyl moiety at the C2 position of the glucose unit, suggesting that cyclodextrin functions as a regioselective catalytic carbohydrate in organic solvent. In the self-acylation, the most acidic OH group at the 2-position and the inclusion complexing ability of cyclodextrin were considered to be significant. The substrate preference was also observed in favor of the long-chain acyl group, which could be attributed to the inclusion ability of cyclodextrin cavity. Furthermore, using the model amphiphilic building block, 2-O-mono-lauryl β-cyclodextrin, the self-organized supramolecular architecture with nano-vesicular morphology in water was investigated by fluorescence spectroscopy, dynamic light scattering and transmission electron microscopy. The cavity-type nano-assembled vesicle and the novel synthetic methods for the preparation of mono-acylated cyclodextrin should be of great interest with regard to drug/gene delivery systems, functional surfactants, and carbohydrate derivatization methods.

Acyl chain length and charge effect on Tamoxifen-lipid model membrane interactions

NASA Astrophysics Data System (ADS)

Bilge, Duygu; Kazanci, Nadide; Severcan, Feride

2013-05-01

Tamoxifen (TAM), which is an antiestrogenic agent, is widely used during chemotherapy of breast, pancreas, brain and liver cancers. In this study, TAM and model membrane interactions in the form of multilamellar vesicles (MLVs) were studied for lipids containing different acyl chain length and different charge status as a function of different TAM (1, 6, 9 and 15 mol%) concentrations. Zwitterionic lipids namely dipalmitoyl phosphatidylcholine (DPPC), and dimyristoylphosphatidylcholine (DMPC) lipids were used to see the acyl chain length effect and anionic dipalmitoyl phosphtidylglycerol (DPPG) lipid was used to see the charge effect. For this purpose Fourier transform-infrared (FTIR) spectroscopic and differential scanning calorimetric (DSC) techniques have been conducted. For zwitterionic lipid, concentration dependent different action of TAM was observed both in the gel and liquid crystalline phases by significantly increasing the lipid order and decreasing the dynamics for 1 mol% TAM, while decreasing the lipid order and increasing the dynamics of the lipids for higher concentrations (6, 9 and 15 mol%). However, different than neutral lipids, the dynamics and disorder of DPPG liposome increased for all TAM concentrations. The interactions between TAM and head group of multilamellar liposomes was monitored by analyzing the Cdbnd O stretching and PO2- antisymmetric double bond stretching bands. Increasing Tamoxifen concentrations led to a dehydration around these functional groups in the polar part of the lipids. DSC studies showed that for all types of lipids, TAM eliminates the pre-transition, shifts the main phase transition to lower temperatures and broadened the phase transition curve. The results indicate that not the acyl chain length but the charge status of the polar head group induces different effects on lipid membranes order and dynamics.

Remote control of regioselectivity in acyl-acyl carrier protein-desaturases

PubMed Central

Guy, Jodie E.; Whittle, Edward; Moche, Martin; Lengqvist, Johan; Lindqvist, Ylva; Shanklin, John

2011-01-01

Regiospecific desaturation of long-chain saturated fatty acids has been described as approaching the limits of the discriminatory power of enzymes because the substrate entirely lacks distinguishing features close to the site of dehydrogenation. To identify the elusive mechanism underlying regioselectivity, we have determined two crystal structures of the archetypal Δ9 desaturase from castor in complex with acyl carrier protein (ACP), which show the bound ACP ideally situated to position C9 and C10 of the acyl chain adjacent to the diiron active site for Δ9 desaturation. Analysis of the structures and modeling of the complex between the highly homologous ivy Δ4 desaturase and ACP, identified a residue located at the entrance to the binding cavity, Asp280 in the castor desaturase (Lys275 in the ivy desaturase), which is strictly conserved within Δ9 and Δ4 enzymes but differs between them. We hypothesized that interaction between Lys275 and the phosphate of the pantetheine, seen in the ivy model, is key to positioning C4 and C5 adjacent to the diiron center for Δ4 desaturation. Mutating castor Asp280 to Lys resulted in a major shift from Δ9 to Δ4 desaturation. Thus, interaction between desaturase side-chain 280 and phospho-serine 38 of ACP, approximately 27 Å from the site of double-bond formation, predisposes ACP binding that favors either Δ9 or Δ4 desaturation via repulsion (acidic side chain) or attraction (positively charged side chain), respectively. Understanding the mechanism underlying remote control of regioselectivity provides the foundation for reengineering desaturase enzymes to create designer chemical feedstocks that would provide alternatives to those currently obtained from petrochemicals. PMID:21930947

Effects of Nanoparticle Morphology and Acyl Chain Length on Spontaneous Lipid Transfer Rates

DOE PAGES

Xia, Yan; Li, Ming; Charubin, Kamil; ...

2015-11-05

In this paper, we report on studies of lipid transfer rates between different morphology nanoparticles and lipids with different length acyl chains. The lipid transfer rate of dimyristoylphosphatidylcholine (di-C 14, DMPC) in discoidal “bicelles” (0.156 h –1) is 2 orders of magnitude greater than that of DMPC vesicles (ULVs) (1.1 × 10 –3 h –1). For both bicellar and ULV morphologies, increasing the acyl chain length by two carbons [going from di-C 14 DMPC to di-C 16, dipalmitoylphosphatidylcholine (DPPC)] causes lipid transfer rates to decrease by more than 2 orders of magnitude. Results from small angle neutron scattering (SANS), differentialmore » scanning calorimetry (DSC), and fluorescence correlation spectroscopy (FCS) are in good agreement. Finally, the present studies highlight the importance of lipid dynamic processes taking place in different morphology biomimetic membranes.« less

Methylobacterium extorquens AM1 produces a novel type of acyl-homoserine lactone with a double unsaturated side chain under methylotrophic growth conditions.

PubMed

Nieto Penalver, Carlos G; Morin, Danièle; Cantet, Franck; Saurel, Olivier; Milon, Alain; Vorholt, Julia A

2006-01-23

Acyl-homoserine lactones (acyl-HSLs) have emerged as important regulatory molecules for many gram-negative bacteria. We have found that Methylobacterium extorquens AM1, a member of the pink-pigmented facultative methylotrophs commonly present on plant surfaces, produces several acyl-HSLs depending upon the carbon source. A novel HSL was discovered with a double unsaturated carbon chain (N-(tetradecenoyl)) (C14:2) and characterized by MS and proton NMR. This long-chain acyl-HSL is synthesized by MlaI that also directs synthesis of C14:1-HSL. The Alphaproteobacterium also produces N-hexanoyl-HSL (C6-HSL) and N-octanoyl-HSL (C8-HSL) via MsaI.

Acyl-CoA:cholesterol acyltransferases (ACATs/SOATs): Enzymes with multiple sterols as substrates and as activators.

PubMed

Rogers, Maximillian A; Liu, Jay; Song, Bao-Liang; Li, Bo-Liang; Chang, Catherine C Y; Chang, Ta-Yuan

2015-07-01

Cholesterol is essential to the growth and viability of cells. The metabolites of cholesterol include: steroids, oxysterols, and bile acids, all of which play important physiological functions. Cholesterol and its metabolites have been implicated in the pathogenesis of multiple human diseases, including: atherosclerosis, cancer, neurodegenerative diseases, and diabetes. Thus, understanding how cells maintain the homeostasis of cholesterol and its metabolites is an important area of study. Acyl-coenzyme A:cholesterol acyltransferases (ACATs, also abbreviated as SOATs) converts cholesterol to cholesteryl esters and play key roles in the regulation of cellular cholesterol homeostasis. ACATs are most unusual enzymes because (i) they metabolize diverse substrates including both sterols and certain steroids; (ii) they contain two different binding sites for steroidal molecules. In mammals, there are two ACAT genes that encode two different enzymes, ACAT1 and ACAT2. Both are allosteric enzymes that can be activated by a variety of sterols. In addition to cholesterol, other sterols that possess the 3-beta OH at C-3, including PREG, oxysterols (such as 24(S)-hydroxycholesterol and 27-hydroxycholesterol, etc.), and various plant sterols, could all be ACAT substrates. All sterols that possess the iso-octyl side chain including cholesterol, oxysterols, various plant sterols could all be activators of ACAT. PREG can only be an ACAT substrate because it lacks the iso-octyl side chain required to be an ACAT activator. The unnatural cholesterol analogs epi-cholesterol (with 3-alpha OH in steroid ring B) and ent-cholesterol (the mirror image of cholesterol) contain the iso-octyl side chain but do not have the 3-beta OH at C-3. Thus, they can only serve as activators and cannot serve as substrates. Thus, within the ACAT holoenzyme, there are site(s) that bind sterol as substrate and site(s) that bind sterol as activator; these sites are distinct from each other. These features form

Accumulation of medium-chain, saturated fatty acyl moieties in seed oils of transgenic Camelina sativa.

PubMed

Hu, Zhaohui; Wu, Qian; Dalal, Jyoti; Vasani, Naresh; Lopez, Harry O; Sederoff, Heike W; Qu, Rongda

2017-01-01

With its high seed oil content, the mustard family plant Camelina sativa has gained attention as a potential biofuel source. As a bioenergy crop, camelina has many advantages. It grows on marginal land with low demand for water and fertilizer, has a relatively short life cycle, and is stress tolerant. As most other crop seed oils, camelina seed triacylglycerols (TAGs) consist of mostly long, unsaturated fatty acyl moieties, which is not desirable for biofuel processing. In our efforts to produce shorter, saturated chain fatty acyl moieties in camelina seed oil for conversion to jet fuel, a 12:0-acyl-carrier thioesterase gene, UcFATB1, from California bay (Umbellularia californica Nutt.) was expressed in camelina seeds. Up to 40% of short chain laurate (C12:0) and myristate (C14:0) were present in TAGs of the seed oil of the transgenics. The total oil content and germination rate of the transgenic seeds were not affected. Analysis of positions of these two fatty acyl moieties in TAGs indicated that they were present at the sn-1 and sn-3 positions, but not sn-2, on the TAGs. Suppression of the camelina KASII genes by RNAi constructs led to higher accumulation of palmitate (C16:0), from 7.5% up to 28.5%, and further reduction of longer, unsaturated fatty acids in seed TAGs. Co-transformation of camelina with both constructs resulted in enhanced accumulation of all three medium-chain, saturated fatty acids in camelina seed oils. Our results show that a California bay gene can be successfully used to modify the oil composition in camelina seed and present a new biological alternative for jet fuel production.

Accumulation of medium-chain, saturated fatty acyl moieties in seed oils of transgenic Camelina sativa

PubMed Central

Dalal, Jyoti; Vasani, Naresh; Lopez, Harry O.; Sederoff, Heike W.

2017-01-01

With its high seed oil content, the mustard family plant Camelina sativa has gained attention as a potential biofuel source. As a bioenergy crop, camelina has many advantages. It grows on marginal land with low demand for water and fertilizer, has a relatively short life cycle, and is stress tolerant. As most other crop seed oils, camelina seed triacylglycerols (TAGs) consist of mostly long, unsaturated fatty acyl moieties, which is not desirable for biofuel processing. In our efforts to produce shorter, saturated chain fatty acyl moieties in camelina seed oil for conversion to jet fuel, a 12:0-acyl-carrier thioesterase gene, UcFATB1, from California bay (Umbellularia californica Nutt.) was expressed in camelina seeds. Up to 40% of short chain laurate (C12:0) and myristate (C14:0) were present in TAGs of the seed oil of the transgenics. The total oil content and germination rate of the transgenic seeds were not affected. Analysis of positions of these two fatty acyl moieties in TAGs indicated that they were present at the sn-1 and sn-3 positions, but not sn-2, on the TAGs. Suppression of the camelina KASII genes by RNAi constructs led to higher accumulation of palmitate (C16:0), from 7.5% up to 28.5%, and further reduction of longer, unsaturated fatty acids in seed TAGs. Co-transformation of camelina with both constructs resulted in enhanced accumulation of all three medium-chain, saturated fatty acids in camelina seed oils. Our results show that a California bay gene can be successfully used to modify the oil composition in camelina seed and present a new biological alternative for jet fuel production. PMID:28212406

Two novel thioesterases are key determinants of the bimodal distribution of acyl chain length of Cuphea palustris seed oil.

PubMed

Dehesh, K; Edwards, P; Hayes, T; Cranmer, A M; Fillatti, J

1996-01-01

The seed oil of Cuphea palustris has an unusual fatty-acyl composition, whereby the principal fatty-acyl groups, myristate (64%) and caprylate (20%), differ by more than two methylenes. We have isolated two thioesterase (TE) cDNAs from C. palustris, encoding proteins designated Cp FatB1 and Cp FatB2, which, when expressed in Escherichia coli, have TE activities specific for 8:0/10:0- and 14:0/16:0-acyl carrier protein substrates, respectively. The specific activities of the recombinant affinity-purified enzymes indicate that Cp FatB2 is kinetically superior to Cp FatB1. This result is consistent with the predominance of 14:0 in the seed oil, despite apparently equal mRNA abundance of the two transcripts in the seed. In C. palustris the expression of both sequences is confined to the seed tissues. Based on these findings we propose that these two enzymes are major factors determining the bimodal chain-length composition of C. palustris oil. Analysis of the immature and mature seed oil by reverse-phase high-performance liquid chromatography confirmed that the principal triglycerides contain both 8:0 and 14:0. This result indicates that both fatty acids are synthesized at the same time and in the same cells at all developmental stages during oil deposition, suggesting that the two TEs act together in the same fatty acid synthesis system.

Long-term dietary effects on substrate selection and muscle fiber type in very-long-chain acyl-CoA dehydrogenase deficient (VLCAD(-/-)) mice.

PubMed

Tucci, Sara; Pearson, Sonja; Herebian, Diran; Spiekerkoetter, Ute

2013-04-01

Dietary fat restriction and increased carbohydrate intake are part of treatment in very-long-chain acyl-CoA dehydrogenase (VLCAD)-deficiency, the most common defect of long-chain fatty acid oxidation. The long-term impact of these interventions is unknown. We characterized here the effects of a fat-reduced, carbohydrate-enriched diet and an increased fat intake on energy metabolism in a mouse model of VLCAD-deficiency. Wild-type and VLCAD(-/-) mice were fed one year either with a normal (5.1%), a high fat (10.6%) or a low-fat, carbohydrate-enriched (2.6%) diet. Dietary effects on genes involved in lipogenesis, energy homeostasis and substrate selection were quantified by real-time-PCR. Acylcarnitines as sign of impaired energy production were determined in dried blood spots and tissues. White skeletal muscle was analyzed for muscle fiber type as well as for glycogen and triglyceride content. Both dietary modifications induced enhanced triacylglyceride accumulation in skeletal muscle and inhibition of glucose oxidation. This was accompanied by an up-regulation of genes coding for oxidative muscle fiber type I and a marked accumulation of acylcarnitines, especially prominent in the heart (164±2.8 in VLCAD(-/-) vs. 82.3±2.1 in WT μmol/mg) under a low-fat, carbohydrate-enriched diet. We demonstrate here that both dietary interventions with respect to the fat content of the diet reverse endogenous compensatory mechanisms in muscle that have evolved in VLCAD(-/-) mice resulting in pronounced energy deficiency. In particular, the low-fat carbohydrate-enriched diet was not effective in the long term. Further experiments are necessary to define the optimal energy provision for fatty acid oxidation defects. Copyright © 2013 Elsevier B.V. All rights reserved.

Carbon and Acyl Chain Flux during Stress-induced Triglyceride Accumulation by Stable Isotopic Labeling of the Polar Microalga Coccomyxa subellipsoidea C169*

PubMed Central

Allen, James W.; DiRusso, Concetta C.; Black, Paul N.

2017-01-01

Deriving biofuels and other lipoid products from algae is a promising future technology directly addressing global issues of atmospheric CO2 balance. To better understand the metabolism of triglyceride synthesis in algae, we examined their metabolic origins in the model species, Coccomyxa subellipsoidea C169, using stable isotopic labeling. Labeling patterns arising from [U-13C]glucose, 13CO2, or D2O supplementation were analyzed by GC-MS and/or LC-MS over time courses during nitrogen starvation to address the roles of catabolic carbon recycling, acyl chain redistribution, and de novo fatty acid (FA) synthesis during the expansion of the lipid bodies. The metabolic origin of stress-induced triglyceride was found to be a continuous 8:2 ratio between de novo synthesized FA and acyl chain transfer from pre-stressed membrane lipids with little input from lipid remodeling. Membrane lipids were continually synthesized with associated acyl chain editing during nitrogen stress, in contrast to an overall decrease in total membrane lipid. The incorporation rates of de novo synthesized FA into lipid classes were measured over a time course of nitrogen starvation. The synthesis of triglycerides, phospholipids, and galactolipids followed a two-stage pattern where nitrogen starvation resulted in a 2.5-fold increase followed by a gradual decline. Acyl chain flux into membrane lipids was dominant in the first stage followed by triglycerides. These data indicate that the level of metabolic control that determines acyl chain flux between membrane lipids and triglycerides during nitrogen stress relies primarily on the Kennedy pathway and de novo FA synthesis with limited, defined input from acyl editing reactions. PMID:27903654

Carbon and Acyl Chain Flux during Stress-induced Triglyceride Accumulation by Stable Isotopic Labeling of the Polar Microalga Coccomyxa subellipsoidea C169.

PubMed

Allen, James W; DiRusso, Concetta C; Black, Paul N

2017-01-06

Deriving biofuels and other lipoid products from algae is a promising future technology directly addressing global issues of atmospheric CO 2 balance. To better understand the metabolism of triglyceride synthesis in algae, we examined their metabolic origins in the model species, Coccomyxa subellipsoidea C169, using stable isotopic labeling. Labeling patterns arising from [U- 13 C]glucose, 13 CO 2 , or D 2 O supplementation were analyzed by GC-MS and/or LC-MS over time courses during nitrogen starvation to address the roles of catabolic carbon recycling, acyl chain redistribution, and de novo fatty acid (FA) synthesis during the expansion of the lipid bodies. The metabolic origin of stress-induced triglyceride was found to be a continuous 8:2 ratio between de novo synthesized FA and acyl chain transfer from pre-stressed membrane lipids with little input from lipid remodeling. Membrane lipids were continually synthesized with associated acyl chain editing during nitrogen stress, in contrast to an overall decrease in total membrane lipid. The incorporation rates of de novo synthesized FA into lipid classes were measured over a time course of nitrogen starvation. The synthesis of triglycerides, phospholipids, and galactolipids followed a two-stage pattern where nitrogen starvation resulted in a 2.5-fold increase followed by a gradual decline. Acyl chain flux into membrane lipids was dominant in the first stage followed by triglycerides. These data indicate that the level of metabolic control that determines acyl chain flux between membrane lipids and triglycerides during nitrogen stress relies primarily on the Kennedy pathway and de novo FA synthesis with limited, defined input from acyl editing reactions. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Measurement of stable isotopic enrichment and concentration of long-chain fatty acyl-carnitines in tissue by HPLC-MS.

PubMed

Sun, Dayong; Cree, Melanie G; Zhang, Xiao-Jun; Bøersheim, Elisabet; Wolfe, Robert R

2006-02-01

We have developed a new method for the simultaneous measurements of stable isotopic tracer enrichments and concentrations of individual long-chain fatty acyl-carnitines in muscle tissue using ion-pairing high-performance liquid chromatography-electrospray ionization quadrupole mass spectrometry in the selected ion monitoring (SIM) mode. Long-chain fatty acyl-carnitines were extracted from frozen muscle tissue samples by acetonitrile/methanol. Baseline separation was achieved by reverse-phase HPLC in the presence of the volatile ion-pairing reagent heptafluorobutyric acid. The SIM capability of a single quadrupole mass analyzer allows further separation of the ions of interest from the sample matrixes, providing very clean total and selected ion chromatograms that can be used to calculate the stable isotopic tracer enrichment and concentration of long-chain fatty acyl-carnitines in a single analysis. The combination of these two separation techniques greatly simplifies the sample preparation procedure and increases the detection sensitivity. Applying this protocol to biological muscle samples proves it to be a very sensitive, accurate, and precise analytical tool.

Regulation of membrane proteins by dietary lipids: effects of cholesterol and docosahexaenoic acid acyl chain-containing phospholipids on rhodopsin stability and function.

PubMed

Bennett, Michael P; Mitchell, Drake C

2008-08-01

Purified bovine rhodopsin was reconstituted into vesicles consisting of 1-stearoyl-2-oleoyl phosphatidylcholine or 1-stearoyl-2-docosahexaenoyl phosphatidylcholine with and without 30 mol % cholesterol. Rhodopsin stability was examined using differential scanning calorimetry (DSC). The thermal unfolding transition temperature (T(m)) of rhodopsin was scan rate-dependent, demonstrating the presence of a rate-limited component of denaturation. The activation energy of this kinetically controlled process (E(a)) was determined from DSC thermograms by four separate methods. Both T(m) and E(a) varied with bilayer composition. Cholesterol increased the T(m) both the presence and absence of docosahexaenoic acid acyl chains (DHA). In contrast, cholesterol lowered E(a) in the absence of DHA, but raised E(a) in the presence of 20 mol % DHA-containing phospholipid. The relative acyl chain packing order was determined from measurements of diphenylhexatriene fluorescence anisotropy decay. The T(m) for thermal unfolding was inversely related to acyl chain packing order. Rhodopsin kinetic stability (E(a)) was reduced in highly ordered or disordered membranes. Maximal kinetic stability was found within the range of acyl chain order found in native bovine rod outer segment disk membranes. The results demonstrate that membrane composition has distinct effects on the thermal versus kinetic stabilities of membrane proteins, and suggests that a balance between membrane constituents with opposite effects on acyl chain packing, such as DHA and cholesterol, may be required for maximum protein stability.

Fatty acyl-CoA reductases of birds

PubMed Central

2011-01-01

Background Birds clean and lubricate their feathers with waxes that are produced in the uropygial gland, a holocrine gland located on their back above the tail. The type and the composition of the secreted wax esters are dependent on the bird species, for instance the wax ester secretion of goose contains branched-chain fatty acids and unbranched fatty alcohols, whereas that of barn owl contains fatty acids and alcohols both of which are branched. Alcohol-forming fatty acyl-CoA reductases (FAR) catalyze the reduction of activated acyl groups to fatty alcohols that can be esterified with acyl-CoA thioesters forming wax esters. Results cDNA sequences encoding fatty acyl-CoA reductases were cloned from the uropygial glands of barn owl (Tyto alba), domestic chicken (Gallus gallus domesticus) and domestic goose (Anser anser domesticus). Heterologous expression in Saccharomyces cerevisiae showed that they encode membrane associated enzymes which catalyze a NADPH dependent reduction of acyl-CoA thioesters to fatty alcohols. By feeding studies of transgenic yeast cultures and in vitro enzyme assays with membrane fractions of transgenic yeast cells two groups of isozymes with different properties were identified, termed FAR1 and FAR2. The FAR1 group mainly synthesized 1-hexadecanol and accepted substrates in the range between 14 and 18 carbon atoms, whereas the FAR2 group preferred stearoyl-CoA and accepted substrates between 16 and 20 carbon atoms. Expression studies with tissues of domestic chicken indicated that FAR transcripts were not restricted to the uropygial gland. Conclusion The data of our study suggest that the identified and characterized avian FAR isozymes, FAR1 and FAR2, can be involved in wax ester biosynthesis and in other pathways like ether lipid synthesis. PMID:22151413

Polymorphism in 'L' shaped lipids: structure of N-, O-diacylethanolamines with mixed acyl chains.

PubMed

Tarafdar, Pradip K; Swamy, Musti J

2009-11-01

Although solid state polymorphism in lipids has been established by spectroscopic and calorimetric studies long ago, only in a few cases crystal structures of different polymorphs of the same compound have been reported, possibly due to difficulties in obtaining high quality single crystals of individual polymorphs. Recent studies show that N-, O-diacylethanolamines (DAEs) can be derived by the O-acylation of the stress-related lipids, the N-acylethanolamines under physiological conditions. In this study, two DAEs with mixed acyl chains, namely N-palmitoyl, O-octanoylethanolamine and N-palmitoyl, O-decanoylethanolamine have been synthesized and their three-dimensional structures were determined. Both the compounds were found to adopt 'L' shaped structures and exist in two polymorphic forms, alpha and beta. In the alpha form a mixed-type chain packing has been observed whereas in the beta form the chain packing is symmetric. Similar polymorphic forms are likely to exist in other 'L' shaped lipids such as 1,3-diacylglycerols and ceramides, where polymorphism has been detected earlier, but three-dimensional structures - which can give precise information about the packing at atomic resolution - have not been reported.

The activities of acyl-CoA:1-acyl-lysophospholipid acyltransferase(s) in human platelets.

PubMed Central

Bakken, A M; Farstad, M

1992-01-01

The activities of acyl-CoA:1-acyl-lysophospholipid acyltransferases (EC 2.3.1.23) have been studied in human platelet lysates by using endogenously formed [14C]acyl-CoA from [14C]fatty acid, ATP and CoA in the presence of 1-acyl-lysophosphatidyl-choline (lysoPC), -ethanolamine (lysoPE), -serine (lysoPS) or -inositol (lysoPI). Linoleic acid as fatty acid substrate had the highest affinity to acyl-CoA:1-acyl-lysophospholipid acyltransferase with lysoPC as variable substrate, followed by eicosapentaenoic acid (EPA) and arachidonic acid (AA). The activity at optimal conditions was 7.4, 7.3 and 7.2 nmol/min per 10(9) platelets with lysoPC as substrate, with linoleic acid, AA and EPA respectively. EPA and AA were incorporated into all lyso-forms. Linoleic acid was also incorporated into lysoPE at a high rate, but less into lysoPS and lysoPI. DHA was incorporated into lysoPC and lysoPE, but only slightly into lysoPI and lysoPS. Whereas incorporation of all fatty acids tested was maximal for lysoPC and lysoPI at 200 and 80 microM respectively, maximal incorporation needed over 500 microM for lysoPE and lysoPS. The optimal concentration for [14C]fatty acid substrates was in the range 15-150 microM for all lysophospholipids. Competition experiments with equimolar concentrations of either lysoPC and lysoPI or lysoPE resulted in formation of [14C]PC almost as if lysoPI or lysoPE were not added to the assay medium. PMID:1471991

Two novel thioesterases are key determinants of the bimodal distribution of acyl chain length of Cuphea palustris seed oil.

PubMed Central

Dehesh, K; Edwards, P; Hayes, T; Cranmer, A M; Fillatti, J

1996-01-01

The seed oil of Cuphea palustris has an unusual fatty-acyl composition, whereby the principal fatty-acyl groups, myristate (64%) and caprylate (20%), differ by more than two methylenes. We have isolated two thioesterase (TE) cDNAs from C. palustris, encoding proteins designated Cp FatB1 and Cp FatB2, which, when expressed in Escherichia coli, have TE activities specific for 8:0/10:0- and 14:0/16:0-acyl carrier protein substrates, respectively. The specific activities of the recombinant affinity-purified enzymes indicate that Cp FatB2 is kinetically superior to Cp FatB1. This result is consistent with the predominance of 14:0 in the seed oil, despite apparently equal mRNA abundance of the two transcripts in the seed. In C. palustris the expression of both sequences is confined to the seed tissues. Based on these findings we propose that these two enzymes are major factors determining the bimodal chain-length composition of C. palustris oil. Analysis of the immature and mature seed oil by reverse-phase high-performance liquid chromatography confirmed that the principal triglycerides contain both 8:0 and 14:0. This result indicates that both fatty acids are synthesized at the same time and in the same cells at all developmental stages during oil deposition, suggesting that the two TEs act together in the same fatty acid synthesis system. PMID:8587983

Biochemical characterization and substrate specificity of jojoba fatty acyl-CoA reductase and jojoba wax synthase.

PubMed

Miklaszewska, Magdalena; Banaś, Antoni

2016-08-01

Wax esters are used in industry for production of lubricants, pharmaceuticals and cosmetics. The only natural source of wax esters is jojoba oil. A much wider variety of industrial wax esters-containing oils can be generated through genetic engineering. Biotechnological production of tailor-made wax esters requires, however, a detailed substrate specificity of fatty acyl-CoA reductases (FAR) and wax synthases (WS), the two enzymes involved in wax esters synthesis. In this study we have successfully characterized the substrate specificity of jojoba FAR and jojoba WS. The genes encoding both enzymes were expressed heterologously in Saccharomyces cerevisiae and the activity of tested enzymes was confirmed by in vivo studies and in vitro assays using microsomal preparations from transgenic yeast. Jojoba FAR exhibited the highest in vitro activity toward 18:0-CoA followed by 20:1-CoA and 22:1-CoA. The activity toward other 11 tested acyl-CoAs was low or undetectable as with 18:2-CoA and 18:3-CoA. In assays characterizing jojoba WS combinations of 17 fatty alcohols with 14 acyl-CoAs were tested. The enzyme displayed the highest activity toward 14:0-CoA and 16:0-CoA in combination with C16-C20 alcohols as well as toward C18 acyl-CoAs in combination with C12-C16 alcohols. 20:1-CoA was efficiently utilized in combination with most of the tested alcohols. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Molecular cloning and characterization of two mouse peroxisome proliferator-activated receptor alpha (PPARalpha)-regulated peroxisomal acyl-CoA thioesterases.

PubMed

Westin, Maria A K; Alexson, Stefan E H; Hunt, Mary C

2004-05-21

Peroxisomes are organelles that function in the beta-oxidation of long- and very long-chain acyl-CoAs, bile acid-CoA intermediates, prostaglandins, leukotrienes, thromboxanes, dicarboxylic fatty acids, pristanic acid, and xenobiotic carboxylic acids. The very long- and long-chain acyl-CoAs are mainly chain-shortened and then transported to mitochondria for further metabolism. We have now identified and characterized two peroxisomal acyl-CoA thioesterases, named PTE-Ia and PTE-Ic, that hydrolyze acyl-CoAs to the free fatty acid and coenzyme A. PTE-Ia and PTE-Ic show 82% sequence identity at the amino acid level, and a putative peroxisomal type 1 targeting signal of -AKL was identified at the carboxyl-terminal end of both proteins. Localization experiments using green fluorescent fusion protein showed PTE-Ia and PTE-Ic to be localized in peroxisomes. Despite their high level of sequence identity, we show that PTE-Ia is mainly active on long-chain acyl-CoAs, whereas PTE-Ic is mainly active on medium-chain acyl-CoAs. Lack of regulation of enzyme activity by free CoASH suggests that PTE-Ia and PTE-Ic regulate intraperoxisomal levels of acyl-CoA, and they may have a function in termination of beta-oxidation of fatty acids of different chain lengths. Tissue expression studies revealed that PTE-Ia is highly expressed in kidney, whereas PTE-Ic is most highly expressed in spleen, brain, testis, and proximal and distal intestine. Both PTE-Ia and PTE-Ic were highly up-regulated in mouse liver by treatment with the peroxisome proliferator WY-14,643 and by fasting in a peroxisome proliferator-activated receptor alpha-dependent manner. These data show that PTE-Ia and PTE-Ic have different functions based on different substrate specificities and tissue expression.

Recognition of Acyl Carrier Proteins by Ketoreductases in Assembly Line Polyketide Synthases

PubMed Central

Ostrowski, Matthew P.; Cane, David E.; Khosla, Chaitan

2016-01-01

Ketoreductases (KRs) are the most widespread tailoring domains found in individual modules of assembly line polyketide synthases (PKSs), and are responsible for controlling the configurations of both the α-methyl and β-hydroxyl stereogenic centers in the growing polyketide chain. Because they recognize substrates that are covalently bound to acyl carrier proteins (ACPs) within the same PKS module, we sought to quantify the extent to which protein-protein recognition contributes to the turnover of these oxidoreductive enzymes using stand-alone domains from the 6-deoxyerythronolide B synthase (DEBS). Reduced 2-methyl-3-hydroxyacyl-ACP substrates derived from two enantiomeric acyl chains and four distinct ACP domains were synthesized and presented to four distinct KR domains. Two KRs, from DEBS modules 2 and 5, displayed little preference for oxidation of substrates tethered to their cognate ACP domains over those attached to the other ACP domains tested. In contrast, the KR from DEBS module 1 showed a ca. 10-50-fold preference for substrate attached to its native ACP domain, whereas the KR from DEBS module 6 actually displayed a ca. 10-fold preference for the ACP from DEBS module 5. Our findings suggest that recognition of the ACP by a KR domain is unlikely to affect the rate of native assembly line polyketide biosynthesis. In some cases, however, unfavorable KR-ACP interactions may suppress the rate of substrate processing when KR domains are swapped to construct hybrid PKS modules. PMID:27118242

Purification and characterization of an amidohydrolase for N4-long-chain fatty acyl derivatives of 1-beta-D-arabinofuranosylcytosine from mouse liver microsomes.

PubMed

Hori, K; Tsuruo, T; Tsukagoshi, S; Sakurai, Y

1984-03-01

N4-Long-chain fatty acyl-1-beta-D-arabinofuranosylcytosine amidohydrolase, a metabolizing enzyme for N4-acyl derivatives of 1-beta-D-arabinofuranosylcytosine with long-chain fatty acids, was purified from mouse liver microsomes. The purification was accomplished by solubilization of liver microsomes with Triton X-100, diethylaminoethyl cellulose chromatography, gel filtrations, hydroxyapatite chromatography, and concanavalin A:Sepharose chromatography. On sodium dodecyl sulfate:polyacrylamide gel electrophoresis, the purified enzyme preparation produced a single protein band with a molecular weight of 54,000. The enzyme had an optimal pH of 9.0, and the Michaelis constant for N4-palmitoyl-1-beta-D-arabinofuranosylcytosine was 67 microM. The thiols such as dithiothreitol or 2-mercaptoethanol stabilized the enzyme and stimulated its activity. p-Chloromercuribenzoate, N-ethylmaleimide, diisopropylfluorophosphate, and phenylmethylsulfonyl fluoride strongly inhibited the reaction. Bovine serum albumin markedly stimulated the enzyme activity, whereas detergents such as Triton X-100, deoxycholate, and sodium dodecyl sulfate had little effect. The enzyme did not require monovalent or divalent cations. Among the series of N4-acyl derivatives of 1-beta-D-arabinofuranosylcytosine with different chain lengths of acyl residues, the purified enzyme preferentially hydrolyzed the derivatives with long-chain fatty acids (C12 to C18), and N4-palmitoyl-1-beta-D-arabinofuranosylcytosine was the most susceptible. The purified enzyme was inactive on various N-acylamino acids, amides, oligopeptides, proteins, N-acylsphingosines (ceramides), triglyceride, lecithin, and lysolecithin. These results suggest that N4-long-chain fatty acyl-1-beta-D-arabinofuranosylcytosine amidohydrolase may be a new type of linear amidase.

Two fatty acyl reductases involved in moth pheromone biosynthesis

PubMed Central

Antony, Binu; Ding, Bao-Jian; Moto, Ken’Ichi; Aldosari, Saleh A.; Aldawood, Abdulrahman S.

2016-01-01

Fatty acyl reductases (FARs) constitute an evolutionarily conserved gene family found in all kingdoms of life. Members of the FAR gene family play diverse roles, including seed oil synthesis, insect pheromone biosynthesis, and mammalian wax biosynthesis. In insects, FAR genes dedicated to sex pheromone biosynthesis (pheromone-gland-specific fatty acyl reductase, pgFAR) form a unique clade that exhibits substantial modifications in gene structure and possesses unique specificity and selectivity for fatty acyl substrates. Highly selective and semi-selective ‘single pgFARs’ produce single and multicomponent pheromone signals in bombycid, pyralid, yponomeutid and noctuid moths. An intriguing question is how a ‘single reductase’ can direct the synthesis of several fatty alcohols of various chain lengths and isomeric forms. Here, we report two active pgFARs in the pheromone gland of Spodoptera, namely a semi-selective, C14:acyl-specific pgFAR and a highly selective, C16:acyl-specific pgFAR, and demonstrate that these pgFARs play a pivotal role in the formation of species-specific signals, a finding that is strongly supported by functional gene expression data. The study envisages a new area of research for disclosing evolutionary changes associated with C14- and C16-specific FARs in moth pheromone biosynthesis. PMID:27427355

Substrate Effects for Atomic Chain Electronics

NASA Technical Reports Server (NTRS)

Yamada, Toshishige; Saini, Subhash (Technical Monitor)

1998-01-01

A substrate for future atomic chain electronics, where adatoms are placed at designated positions and form atomically precise device components, is studied theoretically. The substrate has to serve as a two-dimensional template for adatom mounting with a reasonable confinement barrier and also provide electronic isolation, preventing unwanted coupling between independent adatom structures. For excellent structural stability, we demand chemical bonding between the adatoms and substrate atoms, but then good electronic isolation may not be guaranteed. Conditions are clarified for good isolation. Because of the chemical bonding, fundamental adatom properties are strongly influenced: a chain with group IV adatoms having two chemical bonds, or a chain with group III adatoms having one chemical bond is semiconducting. Charge transfer from or to the substrate atoms brings about unintentional doping, and the electronic properties have to be considered for the entire combination of the adatom and substrate systems even if the adatom modes are well localized at the surface.

Understanding Acyl Chain and Glycerolipid Metabolism in Plants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ohlrogge, John B.

2013-11-05

Progress is reported in these areas: acyl-editing in initial eukaryotic lipid assembly in soybean seeds; identification and characterization of two Arabidopsis thaliana lysophosphatidyl acyltransferases with preference for lysophosphatidylethanolamine; and characterization and subcellular distribution of lysolipid acyl transferase activity of pea leaves.

Gas Phase Dissociation Behavior of Acyl-Arginine Peptides.

PubMed

McGee, William M; McLuckey, Scott A

2013-11-15

The gas phase dissociation behavior of peptides containing acyl-arginine residues is investigated. These acylations are generated via a combination of ion/ion reactions between arginine-containing peptides and N -hydroxysuccinimide (NHS) esters and subsequent tandem mass spectrometry (MS/MS). Three main dissociation pathways of acylated arginine, labeled Paths 1-3, have been identified and are dependent on the acyl groups. Path 1 involves the acyl-arginine undergoing deguanidination, resulting in the loss of the acyl group and dissociation of the guanidine to generate an ornithine residue. This pathway generates selective cleavage sites based on the recently discussed "ornithine effect". Path 2 involves the coordinated losses of H 2 O and NH 3 from the acyl-arginine side chain while maintaining the acylation. We propose that Path 2 is initiated via cyclization of the δ-nitrogen of arginine and the C-terminal carbonyl carbon, resulting in rapid rearrangement from the acyl-arginine side chain and the neutral losses. Path 3 occurs when the acyl group contains α-hydrogens and is observed as a rearrangement to regenerate unmodified arginine while the acylation is lost as a ketene.

On Substrate for Atomic Chain Electronics

NASA Technical Reports Server (NTRS)

Yamada, Toshishige; Bauschlicher, Charles W., Jr.; Partridge, Harry; Saini, Subhash (Technical Monitor)

1998-01-01

A substrate for future atomic chain electronics, where adatoms are placed at designated positions and form atomically precise device components, is studied theoretically. The substrate has to serve as a two-dimensional template for adatom mounting with a reasonable confinement barrier and also provide electronic isolation, preventing unwanted coupling between independent adatom structures. However, the two requirements conflict. For excellent electronic isolation, we may seek adatom confinement via van der Waals interaction without chemical bonding to the substrate atoms, but the confinement turns out to be very weak and hence unsatisfactory. An alternative chemical bonding scheme with excellent structural strength is examined, but even fundamental adatom chain properties such as whether chains are semiconducting or metallic are strongly influenced by the nature of the chemical bonding, and electronic isolation is not always achieved. Conditions for obtaining semiconducting chains with well-localized surface-modes, leading to good isolation, are clarified and discussed.

High-throughput assay for long chain fatty acyl-CoA elongase using homogeneous scintillation proximity format.

PubMed

Shimamura, Ken; Miyamoto, Yasuhisa; Kitazawa, Hidefumi; Kobayashi, Tsutomu; Kotani, Hidehito; Tokita, Shigeru

2009-04-01

Elongase of very-long-chain fatty acid (Elovl) 6 is a rate-limiting enzyme that is responsible for the elongation of long-chain fatty acids such as palmitoic acid (C16). Elovl6 is abundantly expressed in liver and adipose tissue, and the expression levels in these tissues are up-regulated in obese animals. Furthermore, Elovl6-deficient mice display improved glucose homeostasis and insulin sensitivity, suggesting that Elovl6 might be a potential therapeutic target for metabolic disorders. From the drug discovery point of view, it is critical to establish a high-throughput screening (HTS) assay for the identification of therapeutic agents. Conventional assay methods for fatty acid elongases include an extraction step for respective radioactive products from the reaction mixtures, which is labor-intensive and not feasible for HTS. In this study, we utilized the acyl-coenzyme A (CoA) binding protein (ACBP) as a molecular probe to detect radioactive long-chain acyl-CoA, a direct product of Elovl6. Recombinant ACBP binds stearoyl-CoA but not malonyl-CoA, enabling specific detection of the radioactive product in the homogenous reaction mixture without the liquid extraction step. Finally, combination of ACBP and scintillation proximity assay beads led to specific detection of Elovl6 activity with appropriate window and reproducibility amenable to HTS (signal-to-background noise ratio of approximately 13.0-fold, Z' = 0.85). The assay system described here has the potential to enable identification of small compounds that modify fatty acid elongase activity and assessment of the therapeutic potential of acyl-CoA elongases.

The very-long-chain hydroxy fatty acyl-CoA dehydratase PASTICCINO2 is essential and limiting for plant development

PubMed Central

Bach, Liên; Michaelson, Louise V.; Haslam, Richard; Bellec, Yannick; Gissot, Lionel; Marion, Jessica; Da Costa, Marco; Boutin, Jean-Pierre; Miquel, Martine; Tellier, Frédérique; Domergue, Frederic; Markham, Jonathan E.; Beaudoin, Frederic; Napier, Johnathan A.; Faure, Jean-Denis

2008-01-01

Very-long-chain fatty acids (VLCFAs) are synthesized as acyl-CoAs by the endoplasmic reticulum-localized elongase multiprotein complex. Two Arabidopsis genes are putative homologues of the recently identified yeast 3-hydroxy-acyl-CoA dehydratase (PHS1), the third enzyme of the elongase complex. We showed that Arabidopsis PASTICCINO2 (PAS2) was able to restore phs1 cytokinesis defects and sphingolipid long chain base overaccumulation. Conversely, the expression of PHS1 was able to complement the developmental defects and the accumulation of long chain bases of the pas2–1 mutant. The pas2–1 mutant was characterized by a general reduction of VLCFA pools in seed storage triacylglycerols, cuticular waxes, and complex sphingolipids. Most strikingly, the defective elongation cycle resulted in the accumulation of 3-hydroxy-acyl-CoA intermediates, indicating premature termination of fatty acid elongation and confirming the role of PAS2 in this process. We demonstrated by in vivo bimolecular fluorescence complementation that PAS2 was specifically associated in the endoplasmic reticulum with the enoyl-CoA reductase CER10, the fourth enzyme of the elongase complex. Finally, complete loss of PAS2 function is embryo lethal, and the ectopic expression of PHS1 led to enhanced levels of VLCFAs associated with severe developmental defects. Altogether these results demonstrate that the plant 3-hydroxy-acyl-CoA dehydratase PASTICCINO2 is an essential and limiting enzyme in VLCFA synthesis but also that PAS2-derived VLCFA homeostasis is required for specific developmental processes. PMID:18799749

Role for phospholipid acyl chains and cholesterol in pulmonary infections and inflammation

PubMed Central

Shaikh, Saame Raza; Fessler, Michael B.

2016-01-01

Bacterial and viral respiratory tract infections result in millions of deaths worldwide and are currently the leading cause of death from infection. Acute inflammation is an essential element of host defense against infection, but can be damaging to the host when left unchecked. Effective host defense requires multiple lipid mediators, which collectively have proinflammatory and/or proresolving effects on the lung. During pulmonary infections, phospholipid acyl chains and cholesterol can be chemically and enzymatically oxidized, as well as truncated and modified, producing complex mixtures of bioactive lipids. We review recent evidence that phospholipids and cholesterol and their derivatives regulate pulmonary innate and adaptive immunity during infection. We first highlight data that oxidized phospholipids generated in the lung during infection stimulate pattern recognition receptors, such as TLRs and scavenger receptors, thereby amplifying the pulmonary inflammatory response. Next, we discuss evidence that oxidation of endogenous pools of cholesterol during pulmonary infections produces oxysterols that also modify the function of both innate and adaptive immune cells. Last, we conclude with data that n-3 polyunsaturated fatty acids, both in the form of phospholipid acyl chains and through enzymatic processing into endogenous proresolving lipid mediators, aid in the resolution of lung inflammation through distinct mechanisms. Unraveling the complex mechanisms of induction and function of distinct classes of bioactive lipids, both native and modified, may hold promise for developing new therapeutic strategies for improving pulmonary outcomes in response to infection. PMID:27286794

Genomic analysis of branched chain fatty acid and acyl sugar production in Solanum pennellii and Nicotiana benthamiana

USDA-ARS?s Scientific Manuscript database

Acyl sugars are extracellular epidermal lipids that are exuded from glandular trichomes and coat the aerial organs of many species in the Solanaceae. These highly viscous surfactants, which often contain branched-chain fatty acids (BCFA), play an important defensive role against pest and insects. ...

Modulation of cellulase activity by charged lipid bilayers with different acyl chain properties for efficient hydrolysis of ionic liquid-pretreated cellulose.

PubMed

Mihono, Kai; Ohtsu, Takeshi; Ohtani, Mai; Yoshimoto, Makoto; Kamimura, Akio

2016-10-01

The stability of cellulase activity in the presence of ionic liquids (ILs) is critical for the enzymatic hydrolysis of insoluble cellulose pretreated with ILs. In this work, cellulase was incorporated in the liposomes composed of negatively charged 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) and zwitterionic phosphatidylcholines (PCs) with different length and degree of unsaturation of the acyl chains. The liposomal cellulase-catalyzed reaction was performed at 45°C in the acetate buffer solution (pH 4.8) with 2.0g/L CC31 as cellulosic substrate. The crystallinity of CC31 was reduced by treating with 1-butyl-3-methylimidazolium chloride ([Bmim]Cl) at 120°C for 30min. The liposomal cellulase continuously catalyzed hydrolysis of the pretreated CC31 for 48h producing glucose in the presence of 15wt% [Bmim]Cl. The charged lipid membranes were interactive with [Bmim](+), as elucidated by the [Bmim]Cl-induced alterations in fluorescence polarization of the membrane-embedded 1,6-diphenyl-1,3,5-hexatriene (DPH) molecules. The charged membranes offered the microenvironment where inhibitory effects of [Bmim]Cl on the cellulase activity was relieved. The maximum glucose productivity GP of 10.8 mmol-glucose/(hmol-lipid) was obtained at the reaction time of 48h with the cellulase incorporated in the liposomes ([lipid]=5.0mM) composed of 50mol% POPG and 1,2-dilauroyl-sn-glycero-3-phosohocholine (DLPC) with relatively short and saturated acyl chains. Copyright © 2016 Elsevier B.V. All rights reserved.

Oxidative acylation using thioacids

NASA Technical Reports Server (NTRS)

Liu, R.; Orgel, L. E.

1997-01-01

Several important prebiotic reactions, including the coupling of amino acids into polypeptides by the formation of amide linkages, involve acylation. Theae reactions present a challenge to the understanding of prebiotic synthesis. Condensation reactions relying on dehydrating agents are either inefficient in aqueous solution or require strongly acidic conditions and high temperatures. Activated amino acids such as thioester derivatives have therefore been suggested as likely substrates for prebiotic peptide synthesis. Here we propose a closely related route to amide bond formation involving oxidative acylation by thioacids. We find that phenylalanine, leucine and phenylphosphate are acylated efficiently in aqueous solution by thioacetic acid and an oxidizing agent. From a prebiotic point of view, oxidative acylation has the advantage of proceeding efficiently in solution and under mild conditions. We anticipate that oxidative acylation should prove to be a general method for activating carboxylic acids, including amino acids.

Identification of N-Acyl Phosphatidylserine Molecules in Eukaryotic Cells

PubMed Central

Guan, Ziqiang; Li, Shengrong; Smith, Dale C.; Shaw, Walter A.; Raetz, Christian R. H.

2008-01-01

While profiling the lipidome of the mouse brain by mass spectrometry, we discovered a novel family of N-acyl phosphatidylserine (N-acyl-PS) molecules. These N-acyl-PS species were enriched by DEAE-cellulose column chromatography, and they were then characterized by accurate mass measurements, tandem mass spectrometry, liquid chromatography/mass spectrometry, and comparison to an authentic standard. Mouse brain N-acyl-PS molecules are heterogeneous and constitute about 0.1 % of the total lipid. In addition to various ester-linked fatty acyl chains on their glycerol backbones, the complexity of the N-acyl-PS series is further increased by the presence of diverse amide-linked N-acyl chains, which include saturated, mono-unsaturated and poly-unsaturated species. N-acyl-PS molecular species were also detected in the lipids of pig brain, mouse RAW264.7 macrophage tumor cells and yeast, but not E. coli. N-acyl-PSs may be biosynthetic precursors of N-acyl serine molecules, such as the recently reported signaling lipid N-arachidonoyl serine from bovine brain. We suggest that a phospholipase D might cleave N-acyl-PS to generate N-acyl serine, in analogy to the biosynthesis of the endocannabinoid N-arachidonoyl ethanolamine (anadamide) from N-arachidonoyl phosphatidylethanolamine. PMID:18031065

A comparison of the metabolic fate of Fatty acids of different chain lengths in developing oilseeds.

PubMed

Battey, J F; Ohlrogge, J B

1989-07-01

To determine if medium and long chain fatty acids can be appropriately metabolized by species that normally produce 16 and 18 carbon fatty acids, homogenates of developing Cuphea wrightii, Carthamus tinctorius, and Crambe abyssinica seeds were incubated with radiolabeled lauric, palmitic, oleic, and erucic acids. In all three species, acyl-CoA synthetase showed broad substrate specificity in synthesis of acyl-coenzyme A (CoA) from any of the fatty acids presented. In Carthamus, two- to fivefold less of the foreign FAs, lauric, and erucic acid was incorporated into acyl-CoAs than palmitic and oleic acid. Lauric and erucic acid also supported less glycerolipid synthesis in Carthamus than palmitic and oleic acid, but the rate of acyl-CoA synthesis did not control rate of glycerolipid synthesis. In all species examined, medium and long chain fatty acids were incorporated predominantly into triacylglycerols and were almost excluded from phospholipid synthesis, whereas palmitic and oleic acid were found predominantly in polar lipids. However, the rate of esterification of unusual fatty acids to triacylglycerol is slow in species that do not normally synthesize these acyl substrates.

A Comparison of the Metabolic Fate of Fatty Acids of Different Chain Lengths in Developing Oilseeds

PubMed Central

Battey, James F.; Ohlrogge, John B.

1989-01-01

To determine if medium and long chain fatty acids can be appropriately metabolized by species that normally produce 16 and 18 carbon fatty acids, homogenates of developing Cuphea wrightii, Carthamus tinctorius, and Crambe abyssinica seeds were incubated with radiolabeled lauric, palmitic, oleic, and erucic acids. In all three species, acyl-CoA synthetase showed broad substrate specificity in synthesis of acyl-coenzyme A (CoA) from any of the fatty acids presented. In Carthamus, two- to fivefold less of the foreign FAs, lauric, and erucic acid was incorporated into acyl-CoAs than palmitic and oleic acid. Lauric and erucic acid also supported less glycerolipid synthesis in Carthamus than palmitic and oleic acid, but the rate of acyl-CoA synthesis did not control rate of glycerolipid synthesis. In all species examined, medium and long chain fatty acids were incorporated predominantly into triacylglycerols and were almost excluded from phospholipid synthesis, whereas palmitic and oleic acid were found predominantly in polar lipids. However, the rate of esterification of unusual fatty acids to triacylglycerol is slow in species that do not normally synthesize these acyl substrates. PMID:16666885

Friedel-Crafts Acylation with Amides

PubMed Central

Raja, Erum K.; DeSchepper, Daniel J.; Nilsson Lill, Sten O.; Klumpp, Douglas A.

2012-01-01

Friedel-Crafts acylation has been known since the 1870s and it is an important organic synthetic reaction leading to aromatic ketone products. Friedel-Crafts acylation is usually done with carboxylic acid chlorides or anhydrides while amides are generally not useful substrates in these reactions. Despite being the least reactive carboxylic acid derivative, we have found a series of amides capable of providing aromatic ketones in good yields (55–96%, 17 examples). We propose a mechanism involving diminished C-N resonance through superelectrophilic activation and subsequent cleavage to acyl cations. PMID:22690740

Transpeptidation reactions of a specific substrate catalyzed by the streptomyces R61 DD-peptidase: characterization of a chromogenic substrate and acyl acceptor design.

PubMed

Kumar, Ish; Pratt, R F

2005-08-02

The Streptomyces R61 dd-peptidase, a functional model for penicillin-binding proteins, catalyzes the hydrolysis and aminolysis of d-alanyl-d-alanine-terminating peptides by specific amines. In vivo, this reaction completes bacterial cell wall biosynthesis. For in vitro studies of this enzyme to date, various nonspecific acyl-donor substrates have been employed. Recently, however, a peptidoglycan-mimetic peptide substrate, glycyl-l-alpha-amino-epsilon-pimelyl-d-alanyl-d-alanine, has been described that is much more specific for this enzyme. In this paper, we describe the synthesis and kinetic characterization of an analogous thiolester substrate, 3-(N-glycyl-l-cysteinyl)-propanoyl-d-alanyl-d-thiolactate, that the enzyme hydrolyzes and aminolyzes very efficiently (k(cat)/K(m) = 1.0 x 10(7) s(-)(1) M(-)(1)). Direct or indirect, by means of a thiol trap, spectrophotometric monitoring of the reactions of this substrate is readily achieved. Deacylation of the enzyme is rate-determining under substrate saturation conditions, and therefore the aminolysis reaction can be directly studied. The results show that d-amino acids and certain Gly-l-Xaa dipeptides and tripeptides may act as acyl acceptors at the active site of the enzyme. d-Phenylalanine and Gly-l-Phe were the most effective d-amino acid and dipeptide acceptors, respectively. On the basis of the dual specificity of the active site for acceptors (d-amino acids and Gly-l-Xaa peptides), "dual function" acceptors were designed and synthesized. Two of these, aminomalon-(N-ethyl)amide and aminomalon-(N-phenethyl)amide, were particularly effective. It did seem, however, that the observed rates of reaction of these very effective acceptors may be limited by some common, possibly physical, step. More extended, peptidoglycan-like, acceptors were found to be essentially unreactive. The reasons for this counterintuitive behavior are discussed.

Effects of substrate fatty acids on products of lecithin hydrolysis and acyl-CoA-independent transacylation with cholesterol by aortic enzyme preparations.

PubMed

Patelski, J; Pioruńska-Stolzmann, M

1985-01-01

The acyl composition of substrates and products of enzymatic hydrolysis and transacylation of lecithin with cholesterol in the arterial wall was investigated. Saturated acyl residues predominated in lysolecithin and unsaturated ones in acids released by hydrolysis of egg lecithin. In the reaction system with cholesterol, saturated acyls predominated in both lysolecithin and acids released whereas unsaturated ones were more abundant in newly formed acylcholesterols. Mainly unsaturated acyls were present in the hydrolysis products from soybean lecithin in the reaction systems with and without cholesterol. For acylcholesterols formed in the presence of either lecithin, the percent values are in the numerical order of C18:2 greater than C18:1 greater than C16:0 greater than or equal to C18:0. It It is concluded that acyl preferences and interactions in the enzyme-catalyzed reactions studied may contribute to the different accumulation and removal of the compounds involved from the artery.

Thermal properties of partially hydrolyzed starch-glycerophosphatidylcholine complexes with various acyl chains.

PubMed

Siswoyo, Tri Agus; Morita, Naofumi

2003-05-07

Complexes of starch and monoacyl-sn-glycerophosphatidylcholine (GPC) containing various acyl (myristoyl, palmitoyl, and stearoyl) chains were subjected to hydrolysis with glucoamylase (EC 3.2.1.3). The enzyme hydrolyzed approximately 40% of starch control and 20-28% of starch-GPC complexes. Among the GPCs examined, 1- and 2-monomyristoyl-sn-GPC showed the highest resistance to enzyme hydrolysis, and the hydrolysis rate of starch-GPCs was greater with longer chains. Enzymatic hydrolysis strongly affected the thermal properties of the starch. After enzymatic hydrolysis of starch-GPC complexes for 24 h, their thermograms had broader peaks with lower enthalpies than the corresponding starch without enzyme; however, the starch-GPC complexes showed little change. The surface of starch-GPC granules was less eroded. These results showed that the increasing amount of starch-GPC complexes could be more resistant to hydrolysis.

Structure of YciA from Haemophilus influenzae (HI0827), a Hexameric Broad Specificity Acyl-Coenzyme A Thioesterase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Willis, Mark A.; Zhuang, Zhihao; Song, Feng

2008-04-02

The crystal structure of HI0827 from Haemophilus influenzae Rd KW20, initially annotated 'hypothetical protein' in sequence databases, exhibits an acyl-coenzyme A (acyl-CoA) thioesterase 'hot dog' fold with a trimer of dimers oligomeric association, a novel assembly for this enzyme family. In studies described in the preceding paper [Zhuang, Z., Song, F., Zhao, H., Li, L., Cao, J., Eisenstein, E., Herzberg, O., and Dunaway-Mariano, D. (2008) Biochemistry 47, 2789-2796], HI0827 is shown to be an acyl-CoA thioesterase that acts on a wide range of acyl-CoA compounds. Two substrate binding sites are located across the dimer interface. The binding sites are occupiedmore » by two CoA molecules, one with full occupancy and the second only partially occupied. The CoA molecules, acquired from HI0827-expressing Escherichia coli cells, remained tightly bound to the enzyme through the protein purification steps. The difference in CoA occupancies indicates a different substrate affinity for each of the binding sites, which in turn implies that the enzyme might be subject to allosteric regulation. Mutagenesis studies have shown that the replacement of the putative catalytic carboxylate Asp44 with an alanine residue abolishes activity. The impact of this mutation is seen in the crystal structure of D44A HI0827. Whereas the overall fold and assembly of the mutant protein are the same as those of the wild-type enzyme, the CoA ligands are absent. The dimer interface is perturbed, and the channel that accommodates the thioester acyl chain is more open and wider than that observed in the wild-type enzyme. A model of intact substrate bound to wild-type HI0827 provides a structural rationale for the broad substrate range.« less

Clarification of the Mechanism of Acylation Reaction and Origin of Substrate Specificity of the Serine-Carboxyl Peptidase Sedolisin through QM/MM Free Energy Simulations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Qin; Yao, Jianzhuang; Wiodawer, Alexander

2011-01-01

Quantum mechanical/molecular mechanical (QM/MM) free energy simulations are applied for understanding the mechanism of the acylation reaction catalyzed by sedolisin, a representative serine-carboxyl peptidase, leading to the acyl-enzyme (AE) and first product from the enzyme-catalyzed reaction. One of the interesting questions to be addressed in this work is the origin of the substrate specificity of sedolisin that shows a relatively high activity on the substrates with Glu at P1 site. It is shown that the bond making and breaking events of the acylation reaction involving a peptide substrate (LLE*FL) seem to be accompanied by local conformational changes, proton transfers asmore » well as the formation of alternative hydrogen bonds. The results of the simulations indicate that the conformational change of Glu at P1 site and its formation of a low barrier hydrogen bond with Asp-170 (along with the transient proton transfer) during the acylation reaction might play a role in the relatively high specificity for the substrate with Glu at P1 site. The role of some key residues in the catalysis is confirmed through free energy simulations. Glu-80 is found to act as a general base to accept a proton from Ser-287 during the nucleophilic attack and then as a general acid to protonate the leaving group (N H of P1 -Phe) during the cleavage of the scissile peptide bond. Another acidic residue, Asp-170, acts as a general acid catalyst to protonate the carbonyl of P1-Glu during the formation of the tetrahedral intermediate and as a general base for the formation of the acyl-enzyme. The energetic results from the free energy simulations support the importance of proton transfer from Asp-170 to the carbonyl of P1-Glu in the stabilization of the tetrahedral intermediate and the formation of a low-barrier hydrogen bond between the carboxyl group of P1-Glu and Asp-170 in the lowering of the free energy barrier for the cleavage of the peptide bond. Detailed analyses of the proton

AMP-forming acetyl-CoA synthetases in Archaea show unexpected diversity in substrate utilization

PubMed Central

Ingram-Smith, Cheryl; Smith, Kerry S.

2007-01-01

Adenosine monophosphate (AMP)-forming acetyl-CoA synthetase (ACS; acetate:CoA ligase (AMP-forming), EC 6.2.1.1) is a key enzyme for conversion of acetate to acetyl-CoA, an essential intermediate at the junction of anabolic and catabolic pathways. Phylogenetic analysis of putative short and medium chain acyl-CoA synthetase sequences indicates that the ACSs form a distinct clade from other acyl-CoA synthetases. Within this clade, the archaeal ACSs are not monophyletic and fall into three groups composed of both bacterial and archaeal sequences. Kinetic analysis of two archaeal enzymes, an ACS from Methanothermobacter thermautotrophicus (designated as MT-ACS1) and an ACS from Archaeoglobus fulgidus (designated as AF-ACS2), revealed that these enzymes have very different properties. MT-ACS1 has nearly 11-fold higher affinity and 14-fold higher catalytic efficiency with acetate than with propionate, a property shared by most ACSs. However, AF-ACS2 has only 2.3-fold higher affinity and catalytic efficiency with acetate than with propionate. This enzyme has an affinity for propionate that is almost identical to that of MT-ACS1 for acetate and nearly tenfold higher than the affinity of MT-ACS1 for propionate. Furthermore, MT-ACS1 is limited to acetate and propionate as acyl substrates, whereas AF-ACS2 can also utilize longer straight and branched chain acyl substrates. Phylogenetic analysis, sequence alignment and structural modeling suggest a molecular basis for the altered substrate preference and expanded substrate range of AF-ACS2 versus MT-ACS1. PMID:17350930

Fatty acyl chain order in lecithin model membranes determined from proton magnetic resonance.

PubMed

Bloom, M; Burnell, E E; MacKay, A L; Nichol, C P; Valic, M I; Weeks, G

1978-12-26

Proton magnetic resonance (1H NMR) has been used to compare the local orientational order of acyl chains in phospholipid bilayers of multilamellar and small sonicated vesicular membranes of dipalmitoyllecithin (DPL) at 50 degrees C and egg yolk lecithin (EYL) at 31 degrees C. The orientational order of the multilamellar systems was characterized using deuterium magnetic resonance order parameters and 1H NMR second moments. 1H NMR line shapes in the vesicle samples were calculated using vesicle size distributions, determined directly using electron microscopy, and a theory of motional narrowing, which takes into account the symmetry properties of the bilayer systems. The predicted non-Lorentzian line shapes and widths were found to be in good agreement with experimental results, indicating that the local orientational order (called "packing" by many workers) in the bilayers of small vesicles and in multilamellar membranes is substantially the same. This results was found to be true not only for the largest 1H NMR line associated with the nonterminal methylene protons but also for the resolved 1H NMR lines due to the alpha-CH2 and the terminal CH3 positions on the acyl chain. Analysis of the vesicle 1H NMR spectra of EYL taken with different medium viscosities yielded a value of approximately 4 X 10(-8) cm2 s-1 for the lateral diffusion constant of the phospholipid molecules at 31 degrees C.

AHL signaling molecules with a large acyl chain enhance biofilm formation on sulfur and metal sulfides by the bioleaching bacterium Acidithiobacillus ferrooxidans.

PubMed

González, Alex; Bellenberg, Sören; Mamani, Sigde; Ruiz, Lina; Echeverría, Alex; Soulère, Laurent; Doutheau, Alain; Demergasso, Cecilia; Sand, Wolfgang; Queneau, Yves; Vera, Mario; Guiliani, Nicolas

2013-04-01

Biofilm formation plays a pivotal role in bioleaching activities of bacteria in both industrial and natural environments. Here, by visualizing attached bacterial cells on energetic substrates with different microscopy techniques, we obtained the first direct evidence that it is possible to positively modulate biofilm formation of the extremophilic bacterium Acidithiobacillus ferrooxidans on sulfur and pyrite surfaces by using Quorum Sensing molecules of the N-acylhomoserine lactone type (AHLs). Our results revealed that AHL-signaling molecules with a long acyl chain (12 or 14 carbons) increased the adhesion of A. ferrooxidans cells to these substrates. In addition, Card-Fish experiments demonstrated that C14-AHL improved the adhesion of indigenous A. ferrooxidans cells from a mixed bioleaching community to pyrite. Finally, we demonstrated that this improvement of cell adhesion is correlated with an increased production of extracellular polymeric substances. Our results open up a promising means to develop new strategies for the improvement of bioleaching efficiency and metal recovery, which could also be used to control environmental damage caused by acid mine/rock drainage.

Illuminating structure and acyl donor sites of a physiological transglutaminase substrate from Streptomyces mobaraensis.

PubMed

Juettner, Norbert E; Schmelz, Stefan; Bogen, Jan P; Happel, Dominic; Fessner, Wolf-Dieter; Pfeifer, Felicitas; Fuchsbauer, Hans-Lothar; Scrima, Andrea

2018-05-01

Transglutaminase from Streptomyces mobaraensis (MTG) has become a powerful tool to covalently and highly specifically link functional amines to glutamine donor sites of therapeutic proteins. However, details regarding the mechanism of substrate recognition and interaction of the enzyme with proteinaceous substrates still remain mostly elusive. We have determined the crystal structure of the Streptomyces papain inhibitory protein (SPI p ), a substrate of MTG, to study the influence of various substrate amino acids on positioning glutamine to the active site of MTG. SPI p exhibits a rigid, thermo-resistant double-psi-beta-barrel fold that is stabilized by two cysteine bridges. Incorporation of biotin cadaverine identified Gln-6 as the only amine acceptor site on SPI p accessible for MTG. Substitution of Lys-7 demonstrated that small and hydrophobic residues in close proximity to Gln-6 favor MTG-mediated modification and are likely to facilitate introduction of the substrate into the front vestibule of MTG. Moreover, exchange of various surface residues of SPI p for arginine and glutamate/aspartate outside the glutamine donor region influences the efficiency of modification by MTG. These results suggest the occurrence of charged contact areas between MTG and the acyl donor substrates beyond the front vestibule, and pave the way for protein engineering approaches to improve the properties of artificial MTG-substrates used in biomedical applications. © 2018 The Protein Society.

Palmitoyl-acyl carrier protein (ACP) thioesterase and the evolutionary origin of plant acyl-ACP thioesterases.

PubMed Central

Jones, A; Davies, H M; Voelker, T A

1995-01-01

Acyl-acyl carrier protein (ACP) thioesterases play an essential role in chain termination during de novo fatty acid synthesis and in the channeling of carbon flux between the two lipid biosynthesis pathways in plants. We have discovered that there are two distinct but related thioesterase gene classes in higher plants, termed FatA and FatB, whose evolutionary divergence appears to be ancient. FatA encodes the already described 18:1-ACP thioesterase. In contrast, FatB representatives encode thioesterases preferring acyl-ACPs having saturated acyl groups. We unexpectedly obtained a 16:0-ACP thioesterase cDNA from Cuphea hookeriana seed, which accumulate predominantly 8:0 and 10:0. The 16:0 thioesterase transcripts were found in non-seed tissues, and expression in transgenic Brassica napus led to the production of a 16:0-rich oil. We present evidence that this type of FatB gene is ancient and ubiquitous in plants and that specialized plant medium-chain thioesterases have evolved independently from such enzymes several times during angiosperm evolution. Also, the ubiquitous 18:1-ACP thioesterase appears to be a derivative of a 16:0 thioesterase. PMID:7734968

Palmitoyl-acyl carrier protein (ACP) thioesterase and the evolutionary origin of plant acyl-ACP thioesterases.

PubMed

Jones, A; Davies, H M; Voelker, T A

1995-03-01

Acyl-acyl carrier protein (ACP) thioesterases play an essential role in chain termination during de novo fatty acid synthesis and in the channeling of carbon flux between the two lipid biosynthesis pathways in plants. We have discovered that there are two distinct but related thioesterase gene classes in higher plants, termed FatA and FatB, whose evolutionary divergence appears to be ancient. FatA encodes the already described 18:1-ACP thioesterase. In contrast, FatB representatives encode thioesterases preferring acyl-ACPs having saturated acyl groups. We unexpectedly obtained a 16:0-ACP thioesterase cDNA from Cuphea hookeriana seed, which accumulate predominantly 8:0 and 10:0. The 16:0 thioesterase transcripts were found in non-seed tissues, and expression in transgenic Brassica napus led to the production of a 16:0-rich oil. We present evidence that this type of FatB gene is ancient and ubiquitous in plants and that specialized plant medium-chain thioesterases have evolved independently from such enzymes several times during angiosperm evolution. Also, the ubiquitous 18:1-ACP thioesterase appears to be a derivative of a 16:0 thioesterase.

The role of acyl carrier protein isoforms from Cuphea lanceolata seeds in the de-novo biosynthesis of medium-chain fatty acids.

PubMed

Schütt, B S; Brummel, M; Schuch, R; Spener, F

1998-06-01

To investigate the role of acyl carrier protein (ACP) in determining the fate of the acyl moieties linked to it in the course of de-novo fatty acid biosynthesis in higher plants, we carried out in vitro experiments to reconstitute the fatty acid synthase (FAS) reaction in extracts of spinach (Spinacia oleracea L.) leaves, rape (Brassica napus L.) seeds and Cuphea lanceolata Ait. seeds. The action of two major C. lanceolata ACP isoforms (ACP 1 and ACP 2) compared to ACP from Escherichia coli was monitored by saponification of the corresponding FAS products with subsequent analysis of the liberated fatty acids by high-performance liquid chromatography. In a second approach the preference of the medium-chain acyl-ACP-specific thioesterase (EC 3.1.2.14) of C. lanceolata seeds for the hydrolysis of acyl-ACPs prepared from the three ACP types was investigated. Both ACP isoforms from C. lanceolata seeds supported the synthesis of medium-chain fatty acids in a reconstituted FAS reaction of spinach leaf extracts. Compared to the isoform ACP 1, ACP 2 was more effective in supporting the synthesis of such fatty acids in the FAS reaction of rape seed extracts and caused a higher accumulation of FAS products in all experiments. No preference of the medium-chain thioesterase for one specific ACP isoform was observed. The results indicate that the presence of ACP 2 is essential for the synthesis of decanoic acid in C. lanceolata seeds, and its expression in the phase of accumulation of high levels of this fatty acid provides an additional and highly efficient cofactor for stimulating the FAS reaction.

Acyl carrier proteins from sunflower (Helianthus annuus L.) seeds and their influence on FatA and FatB acyl-ACP thioesterase activities.

PubMed

Aznar-Moreno, Jose A; Venegas-Calerón, Mónica; Martínez-Force, Enrique; Garcés, Rafael; Salas, Joaquín J

2016-08-01

The kinetics of acyl-ACP thioesterases from sunflower importantly changed when endogenous ACPs were used. Sunflower FatB was much more specific towards saturated acyl-ACPs when assayed with them. Acyl carrier proteins (ACPs) are small (~9 kDa), soluble, acidic proteins involved in fatty acid synthesis in plants and bacteria. ACPs bind to fatty acids through a thioester bond, generating the acyl-ACP lipoproteins that are substrates for fatty acid synthase (FAS) complexes, and that are required for fatty acid chain elongation, acting as important intermediates in de novo fatty acid synthesis in plants. Plants, usually express several ACP isoforms with distinct functionalities. We report here the cloning of three ACPs from developing sunflower seeds: HaACP1, HaACP2, and HaACP3. These proteins were plastidial ACPs expressed strongly in seeds, and as such they are probably involved in the synthesis of sunflower oil. The recombinant sunflower ACPs were expressed in bacteria but they were lethal to the prokaryote host. Thus, they were finally produced using the GST gene fusion system, which allowed the apo-enzyme to be produced and later activated to the holo form. Radiolabelled acyl-ACPs from the newly cloned holo-ACP forms were also synthesized and used to characterize the activity of recombinant sunflower FatA and FatB thioesterases, important enzymes in plant fatty acids synthesis. The activity of these enzymes changed significantly when the endogenous ACPs were used. Thus, FatA importantly increased its activity levels, whereas FatB displayed a different specificity profile, with much high activity levels towards saturated acyl-CoA derivatives. All these data pointed to an important influence of the ACP moieties on the activity of enzymes involved in lipid synthesis.

Sunflower (Helianthus annuus) long-chain acyl-coenzyme A synthetases expressed at high levels in developing seeds.

PubMed

Aznar-Moreno, Jose A; Venegas Calerón, Mónica; Martínez-Force, Enrique; Garcés, Rafael; Mullen, Robert; Gidda, Satinder K; Salas, Joaquín J

2014-03-01

Long chain fatty acid synthetases (LACSs) activate the fatty acid chains produced by plastidial de novo biosynthesis to generate acyl-CoA derivatives, important intermediates in lipid metabolism. Oilseeds, like sunflower, accumulate high levels of triacylglycerols (TAGs) in their seeds to nourish the embryo during germination. This requires that sunflower seed endosperm supports very active glycerolipid synthesis during development. Sunflower seed plastids produce large amounts of fatty acids, which must be activated through the action of LACSs, in order to be incorporated into TAGs. We cloned two different LACS genes from developing sunflower endosperm, HaLACS1 and HaLACS2, which displayed sequence homology with Arabidopsis LACS9 and LACS8 genes, respectively. These genes were expressed at high levels in developing seeds and exhibited distinct subcellular distributions. We generated constructs in which these proteins were fused to green fluorescent protein and performed transient expression experiments in tobacco cells. The HaLACS1 protein associated with the external envelope of tobacco chloroplasts, whereas HaLACS2 was strongly bound to the endoplasmic reticulum. Finally, both proteins were overexpressed in Escherichia coli and recovered as active enzymes in the bacterial membranes. Both enzymes displayed similar substrate specificities, with a very high preference for oleic acid and weaker activity toward stearic acid. On the basis of our findings, we discuss the role of these enzymes in sunflower oil synthesis. © 2013 Scandinavian Plant Physiology Society.

Acyl chain unsaturation modulates distribution of lecithin molecular species between mixed micelles and vesicles in model bile. Implications for particle structure and metastable cholesterol solubilities.

PubMed

Cohen, D E; Carey, M C

1991-08-01

We determined the distribution of lecithin molecular species between vesicles and mixed micelles in cholesterol super-saturated model biles (molar taurocholate-lecithin-cholesterol ratio 67:23:10, 3 g/dl, 0.15 M NaCl, pH approximately 6-7) that contained equimolar synthetic lecithin mixtures or egg yolk or soybean lecithins. After apparent equilibration (48 h), biles were fractionated by Superose 6 gel filtration chromatography at 20 degrees C, and lecithin molecular species in the vesicle and mixed micellar fractions were quantified as benzoyl diacylglycerides by high performance liquid chromatography. With binary lecithin mixtures, vesicles were enriched with lecithins containing the most saturated sn-1 or sn-2 chains by as much as 2.4-fold whereas mixed micelles were enriched in the more unsaturated lecithins. Vesicles isolated from model biles composed of egg yolk (primarily sn-1 16:0 and 18:0 acyl chains) or soy bean (mixed saturated and unsaturated sn-1 acyl chains) lecithins were selectively enriched (6.5-76%) in lecithins with saturated sn-1 acyl chains whereas mixed micelles were enriched with lecithins composed of either sn-1 18:1, 18:2, and 18:3 unsaturated or sn-2 20:4, 22:4, and 22:6 polyunsaturated chains. Gel filtration, lipid analysis, and quasielastic light scattering revealed that apparent micellar cholesterol solubilities and metastable vesicle cholesterol/lecithin molar ratios were as much as 60% and 100% higher, respectively, in biles composed of unsaturated lecithins. Acyl chain packing constraints imposed by distinctly different particle geometries most likely explain the asymmetric distribution of lecithin molecular species between vesicles and mixed micelles in model bile as well as the variations in apparent micellar cholesterol solubilities and vesicle cholesterol/lecithin molar ratios.(ABSTRACT TRUNCATED AT 250 WORDS)

Recurrent Ventricular Tachycardia in Medium-Chain Acyl-Coenzyme A Dehydrogenase Deficiency.

PubMed

Bala, P; Ferdinandusse, S; Olpin, S E; Chetcuti, P; Morris, A A M

2016-01-01

We report a baby with medium-chain acyl-coenzyme A dehydrogenase (MCAD) deficiency who presented on day 2 with poor feeding and lethargy. She was floppy with hypoglycaemia (1.8 mmol/l) and hyperammonaemia (182 μmol/l). Despite correction of these and a continuous intravenous infusion of glucose at 4.5-6.2 mg/kg/min, she developed generalised tonic clonic seizures on day 3. She also suffered two episodes of pulseless ventricular tachycardia, from which she was resuscitated successfully. Unfortunately, she died on day 5, following a third episode of pulseless ventricular tachycardia. Arrhythmias are generally thought to be rarer in MCAD deficiency than in disorders of long-chain fatty acid oxidation. This is, however, the sixth report of ventricular tachyarrhythmias in MCAD deficiency. Five of these involved neonates and it may be that patients with MCAD deficiency are particularly prone to ventricular arrhythmias in the newborn period. Three of the patients (including ours) had normal blood glucose concentrations at the time of the arrhythmias and had been receiving intravenous glucose for many hours. These cases suggest that arrhythmias can be induced by medium-chain acylcarnitines or other metabolites accumulating in MCAD deficiency. Ventricular tachyarrhythmias can occur in MCAD deficiency, especially in neonates.

Anatomy of the β-branching enzyme of polyketide biosynthesis and its interaction with an acyl-ACP substrate.

PubMed

Maloney, Finn P; Gerwick, Lena; Gerwick, William H; Sherman, David H; Smith, Janet L

2016-09-13

Alkyl branching at the β position of a polyketide intermediate is an important variation on canonical polyketide natural product biosynthesis. The branching enzyme, 3-hydroxy-3-methylglutaryl synthase (HMGS), catalyzes the aldol addition of an acyl donor to a β-keto-polyketide intermediate acceptor. HMGS is highly selective for two specialized acyl carrier proteins (ACPs) that deliver the donor and acceptor substrates. The HMGS from the curacin A biosynthetic pathway (CurD) was examined to establish the basis for ACP selectivity. The donor ACP (CurB) had high affinity for the enzyme (Kd = 0.5 μM) and could not be substituted by the acceptor ACP. High-resolution crystal structures of HMGS alone and in complex with its donor ACP reveal a tight interaction that depends on exquisite surface shape and charge complementarity between the proteins. Selectivity is explained by HMGS binding to an unusual surface cleft on the donor ACP, in a manner that would exclude the acceptor ACP. Within the active site, HMGS discriminates between pre- and postreaction states of the donor ACP. The free phosphopantetheine (Ppant) cofactor of ACP occupies a conserved pocket that excludes the acetyl-Ppant substrate. In comparison with HMG-CoA (CoA) synthase, the homologous enzyme from primary metabolism, HMGS has several differences at the active site entrance, including a flexible-loop insertion, which may account for the specificity of one enzyme for substrates delivered by ACP and the other by CoA.

Inhibition of Long Chain Fatty Acyl-CoA Synthetase (ACSL) and Ischemia Reperfusion Injury

PubMed Central

Prior, Allan M.; Zhang, Man; Blakeman, Nina; Datta, Palika; Pham, Hung; Young, Lindon H.; Weis, Margaret T.; Hua, Duy H.

2014-01-01

Various triacsin C analogs, containing different alkenyl chains and carboxylic acid bioisoteres including 4-aminobenzoic acid, isothiazolidine dioxide, hydroxylamine, hydroxytriazene, and oxadiazolidine dione, were synthesized and their inhibitions of long chain fatty acyl-CoA synthetase (ACSL) were examined. Two methods, a cell-based assay of ACSL activity and an in situ [14C]-palmitate incorporation into extractable lipids were used to study the inhibition. Using an in vivo leukocyte recruitment inhibition protocol, the translocation of one or more cell adhesion molecules from the cytoplasm to the plasma membrane on either the endothelium or leukocyte or both was inhibited by inhibitors 1, 9, and triacsin C. The results suggest that inhibition of ACSL may attenuate the vascular inflammatory component associated with ischemia reperfusion injury and lead to a decrease of infarct expansion. PMID:24480468

The Physiology of Protein S-acylation

PubMed Central

Chamberlain, Luke H.; Shipston, Michael J.

2015-01-01

Protein S-acylation, the only fully reversible posttranslational lipid modification of proteins, is emerging as a ubiquitous mechanism to control the properties and function of a diverse array of proteins and consequently physiological processes. S-acylation results from the enzymatic addition of long-chain lipids, most typically palmitate, onto intracellular cysteine residues of soluble and transmembrane proteins via a labile thioester linkage. Addition of lipid results in increases in protein hydrophobicity that can impact on protein structure, assembly, maturation, trafficking, and function. The recent explosion in global S-acylation (palmitoyl) proteomic profiling as a result of improved biochemical tools to assay S-acylation, in conjunction with the recent identification of enzymes that control protein S-acylation and de-acylation, has opened a new vista into the physiological function of S-acylation. This review introduces key features of S-acylation and tools to interrogate this process, and highlights the eclectic array of proteins regulated including membrane receptors, ion channels and transporters, enzymes and kinases, signaling adapters and chaperones, cell adhesion, and structural proteins. We highlight recent findings correlating disruption of S-acylation to pathophysiology and disease and discuss some of the major challenges and opportunities in this rapidly expanding field. PMID:25834228

Arabidopsis CER8 encodes LONG-CHAIN ACYL-COA SYNTHETASE 1 (LACS1) that has overlapping functions with LACS2 in plant wax and cutin synthesis.

PubMed

Lü, Shiyou; Song, Tao; Kosma, Dylan K; Parsons, Eugene P; Rowland, Owen; Jenks, Matthew A

2009-08-01

Plant cuticle is an extracellular lipid-based matrix of cutin and waxes, which covers aerial organs and protects them from many forms of environmental stress. We report here the characterization of CER8/LACS1, one of nine Arabidopsis long-chain acyl-CoA synthetases thought to activate acyl chains. Mutations in LACS1 reduced the amount of wax in all chemical classes on the stem and leaf, except in the very long-chain fatty acid (VLCFA) class wherein acids longer than 24 carbons (C(24)) were elevated more than 155%. The C(16) cutin monomers on lacs1 were reduced by 37% and 22%, whereas the C(18) monomers were increased by 28% and 20% on stem and leaf, respectively. Amounts of wax and cutin on a lacs1-1 lacs2-3 double mutant were much lower than on either parent, and lacs1-1 lacs2-3 had much higher cuticular permeability than either parent. These additive effects indicate that LACS1 and LACS2 have overlapping functions in both wax and cutin synthesis. We demonstrated that LACS1 has synthetase activity for VLCFAs C(20)-C(30), with highest activity for C(30) acids. LACS1 thus appears to function as a very long-chain acyl-CoA synthetase in wax metabolism. Since C(16) but not C(18) cutin monomers are reduced in lacs1, and C(16) acids are the next most preferred acid (behind C(30)) by LACS1 in our assays, LACS1 also appears to be important for the incorporation of C(16) monomers into cutin polyester. As such, LACS1 defines a functionally novel acyl-CoA synthetase that preferentially modifies both VLCFAs for wax synthesis and long-chain (C(16)) fatty acids for cutin synthesis.

Only One of the Five Ralstonia solanacearum Long-Chain 3-Ketoacyl-Acyl Carrier Protein Synthase Homologues Functions in Fatty Acid Synthesis

PubMed Central

Cheng, Juanli; Ma, Jincheng; Lin, Jinshui; Fan, Zhen-Chuan; Cronan, John E.

2012-01-01

Ralstonia solanacearum, a major phytopathogenic bacterium, causes a bacterial wilt disease in diverse plants. Although fatty acid analyses of total membranes of R. solanacearum showed that they contain primarily palmitic (C16:0), palmitoleic (C16:1) and cis-vaccenic (C18:1) acids, little is known regarding R. solanacearum fatty acid synthesis. The R. solanacearum GMI1000 genome is unusual in that it contains four genes (fabF1, fabF2, fabF3, and fabF4) annotated as encoding 3-ketoacyl-acyl carrier protein synthase II homologues and one gene (fabB) annotated as encoding 3-ketoacyl-acyl carrier protein synthase I. We have analyzed this puzzling apparent redundancy and found that only one of these genes, fabF1, encoded a long-chain 3-ketoacyl-acyl carrier protein synthase, whereas the other homologues did not play roles in R. solanacearum fatty acid synthesis. Mutant strains lacking fabF1 are nonviable, and thus, FabF1 is essential for R. solanacearum fatty acid biosynthesis. Moreover, R. solanacearum FabF1 has the activities of both 3-ketoacyl-acyl carrier protein synthase II and 3-ketoacyl-acyl carrier protein synthase I. PMID:22194290

Aldehyde-forming fatty acyl-CoA reductase from cyanobacteria: expression, purification and characterization of the recombinant enzyme.

PubMed

Lin, Fengming; Das, Debasis; Lin, Xiaoxia N; Marsh, E Neil G

2013-10-01

Long-chain acyl-CoA reductases (ACRs) catalyze a key step in the biosynthesis of hydrocarbon waxes. As such they are attractive as components in engineered metabolic pathways for 'drop in' biofuels. Most ACR enzymes are integral membrane proteins, but a cytosolic ACR was recently discovered in cyanobacteria. The ACR from Synechococcus elongatus was overexpressed in Escherichia coli, purified and characterized. The enzyme was specific for NADPH and catalyzed the reduction of fatty acyl-CoA esters to the corresponding aldehydes, rather than alcohols. Stearoyl-CoA was the most effective substrate, being reduced more rapidly than either longer or shorter chain acyl-CoAs. ACR required divalent metal ions, e.g. Mg(2+), for activity and was stimulated ~ 10-fold by K(+). The enzyme was inactivated by iodoacetamide and was acylated on incubation with stearoyl-CoA, suggesting that reduction occurs through an enzyme-thioester intermediate. Consistent with this, steady state kinetic analysis indicates that the enzyme operates by a 'ping-pong' mechanism with kcat = 0.36 ± 0.023 min(-1), K(m)(stearoyl-CoA) = 31.9 ± 4.2 μM and K(m)(NADPH) = 35.6 ± 4.9 μM. The slow turnover number measured for ACR poses a challenge for its use in biofuel applications where highly efficient enzymes are needed. © 2013 FEBS.

Novel inhibitory action of tunicamycin homologues suggests a role for dynamic protein fatty acylation in growth cone-mediated neurite extension

PubMed Central

1994-01-01

In neuronal growth cones, the advancing tips of elongating axons and dendrites, specific protein substrates appear to undergo cycles of posttranslational modification by covalent attachment and removal of long-chain fatty acids. We show here that ongoing fatty acylation can be inhibited selectively by long-chain homologues of the antibiotic tunicamycin, a known inhibitor of N-linked glycosylation. Tunicamycin directly inhibits transfer of palmitate to protein in a cell-free system, indicating that tunicamycin inhibition of protein palmitoylation reflects an action of the drug separate from its previously established effects on glycosylation. Tunicamycin treatment of differentiated PC12 cells or dissociated rat sensory neurons, under conditions in which protein palmitoylation is inhibited, produces a prompt cessation of neurite elongation and induces a collapse of neuronal growth cones. These growth cone responses are rapidly reversed by washout of the antibiotic, even in the absence of protein synthesis, or by addition of serum. Two additional lines of evidence suggest that the effects of tunicamycin on growth cones arise from its ability to inhibit protein long-chain acylation, rather than its previously established effects on protein glycosylation and synthesis. (a) The abilities of different tunicamycin homologues to induce growth cone collapse very systematically with the length of the fatty acyl side- chain of tunicamycin, in a manner predicted and observed for the inhibition of protein palmitoylation. Homologues with fatty acyl moieties shorter than palmitic acid (16 hydrocarbons), including potent inhibitors of glycosylation, are poor inhibitors of growth cone function. (b) The tunicamycin-induced impairment of growth cone function can be reversed by the addition of excess exogenous fatty acid, which reverses the inhibition of protein palmitoylation but has no effect on the inhibition of protein glycosylation. These results suggest an important role for

Long-term correction of very long-chain acyl-coA dehydrogenase deficiency in mice using AAV9 gene therapy.

PubMed

Keeler, Allison M; Conlon, Thomas; Walter, Glenn; Zeng, Huadong; Shaffer, Scott A; Dungtao, Fu; Erger, Kirsten; Cossette, Travis; Tang, Qiushi; Mueller, Christian; Flotte, Terence R

2012-06-01

Very long-chain acyl-coA dehydrogenase (VLCAD) is the rate-limiting step in mitochondrial fatty acid oxidation. VLCAD-deficient mice and patients clinical symptoms stem from not only an energy deficiency but also long-chain metabolite accumulations. VLCAD-deficient mice were treated systemically with 1 × 10(12) vector genomes of recombinant adeno-associated virus 9 (rAAV9)-VLCAD. Biochemical correction was observed in vector-treated mice beginning 2 weeks postinjection, as characterized by a significant drop in long-chain fatty acyl accumulates in whole blood after an overnight fast. Changes persisted through the termination point around 20 weeks postinjection. Magnetic resonance spectroscopy (MRS) and tandem mass spectrometry (MS/MS) revealed normalization of intramuscular lipids in treated animals. Correction was not observed in liver tissue extracts, but cardiac muscle extracts showed significant reduction of long-chain metabolites. Disease-specific phenotypes were characterized, including thermoregulation and maintenance of euglycemia after a fasting cold challenge. Internal body temperatures of untreated VLCAD(-/-) mice dropped below 20 °C and the mice became lethargic, requiring euthanasia. In contrast, all rAAV9-treated VLCAD(-/-) mice and the wild-type controls maintained body temperatures. rAAV9-treated VLCAD(-/-) mice maintained euglycemia, whereas untreated VLCAD(-/-) mice suffered hypoglycemia following a fasting cold challenge. These promising results suggest rAAV9 gene therapy as a potential treatment for VLCAD deficiency in humans.

Medium-chain fatty acid synthesis in lactating-rabbit mammary gland. Intracellular concentration and specificity of medium-chain acyl thioester hydrolase.

PubMed Central

Knudsen, J

1979-01-01

The concentration of medium-chain acyl thioester hydrolase and of fatty acid synthetase was determined by rocket immunoelectrophoresis in nine different particle-free supernatant fractions from lactating-rabbit mammary gland. The molar ratio of the hydrolase to fatty acid synthetase was 1.99 +/- 0.66 (mean +/- S.D.). A rate-limiting concentration of malonyl-CoA was required to ensure the predominant synthesis of medium-chain fatty acids when 2 mol of the hydrolase was added per mol of fatty acid synthetase. The interaction of the hydrolase with fatty acid synthetase was concentration-dependent, though an optimum concentration of hydrolase to synthetase could not be obtained. The lactating-rabbit mammary gland hydrolase altered the pattern of fatty acids synthesized by fatty acid synthetases prepared from cow, goat, sheep and rabbit lactating mammary glands, rabbit liver and cow adipose tissue. PMID:574008

Substrate Efflux Propensity Is the Key Determinant of Ca2+-independent Phospholipase A-β (iPLAβ)-mediated Glycerophospholipid Hydrolysis*

PubMed Central

Batchu, Krishna Chaithanya; Hokynar, Kati; Jeltsch, Michael; Mattonet, Kenny; Somerharju, Pentti

2015-01-01

The A-type phospholipases (PLAs) are key players in glycerophospholipid (GPL) homeostasis and in mammalian cells; Ca2+-independent PLA-β (iPLAβ) in particular has been implicated in this essential process. However, the regulation of this enzyme, which is necessary to avoid futile competition between synthesis and degradation, is not understood. Recently, we provided evidence that the efflux of the substrate molecules from the bilayer is the rate-limiting step in the hydrolysis of GPLs by some secretory (nonhomeostatic) PLAs. To study whether this is the case with iPLAβ as well, a mass spectrometric assay was employed to determine the rate of hydrolysis of multiple saturated and unsaturated GPL species in parallel using micelles or vesicle bilayers as the macrosubstrate. With micelles, the hydrolysis decreased with increasing acyl chain length independent of unsaturation, and modest discrimination between acyl positional isomers was observed, presumably due to the differences in the structure of the sn-1 and sn-2 acyl-binding sites of the protein. In striking contrast, no significant discrimination between positional isomers was observed with bilayers, and the rate of hydrolysis decreased with the acyl chain length logarithmically and far more than with micelles. These data provide compelling evidence that efflux of the substrate molecule from the bilayer, which also decreases monotonously with acyl chain length, is the rate-determining step in iPLAβ-mediated hydrolysis of GPLs in membranes. This finding is intriguing as it may help to understand how homeostatic PLAs are regulated and how degradation and biosynthesis are coordinated. PMID:25713085

Engineering acyl carrier protein to enhance production of shortened fatty acids.

PubMed

Liu, Xueliang; Hicks, Wade M; Silver, Pamela A; Way, Jeffrey C

2016-01-01

The acyl carrier protein (ACP) is an essential and ubiquitous component of microbial synthesis of fatty acids, the natural precursor to biofuels. Natural fatty acids usually contain long chains of 16 or more carbon atoms. Shorter carbon chains, with increased fuel volatility, are desired for internal combustion engines. Engineering the length specificity of key proteins in fatty acid metabolism, such as ACP, may enable microbial synthesis of these shorter chain fatty acids. We constructed a homology model of the Synechococcus elongatus ACP, showing a hydrophobic pocket harboring the growing acyl chain. Amino acids within the pocket were mutated to increase steric hindrance to the acyl chain. Certain mutant ACPs, when over-expressed in Escherichia coli, increased the proportion of shorter chain lipids; I75 W and I75Y showed the strongest effects. Expression of I75 W and I75Y mutant ACPs also increased production of lauric acid in E. coli that expressed the C12-specific acyl-ACP thioesterase from Cuphea palustris. We engineered the specificity of the ACP, an essential protein of fatty acid metabolism, to alter the E. coli lipid pool and enhance production of medium-chain fatty acids as biofuel precursors. These results indicate that modification of ACP itself could be combined with enzymes affecting length specificity in fatty acid synthesis to enhance production of commodity chemicals based on fatty acids.

Alteration of the fatty acid profile of Streptomyces coelicolor by replacement of the initiation enzyme 3-ketoacyl acyl carrier protein synthase III (FabH).

PubMed

Li, Yongli; Florova, Galina; Reynolds, Kevin A

2005-06-01

The first elongation step of fatty acid biosynthesis by a type II dissociated fatty acid synthases is catalyzed by 3-ketoacyl-acyl carrier protein (ACP) synthase III (KASIII, FabH). This enzyme, encoded by the fabH gene, catalyzes a decarboxylative condensation between an acyl coenzyme A (CoA) primer and malonyl-ACP. In organisms such as Escherichia coli, which generate only straight-chain fatty acids (SCFAs), FabH has a substrate preference for acetyl-CoA. In streptomycetes and other organisms which produce a mixture of both SCFAs and branched-chain fatty acids (BCFAs), FabH has been shown to utilize straight- and branched-chain acyl-CoA substrates. We report herein the generation of a Streptomyces coelicolor mutant (YL/ecFabH) in which the chromosomal copy of the fabH gene has been replaced and the essential process of fatty acid biosynthesis is initiated by plasmid-based expression of the E. coli FabH (bearing only 35% amino acid identity to the Streptomyces enzyme). The YL/ecFabH mutant produces predominantly SCFAs (86%). In contrast, BCFAs predominate (approximately 70%) in both the S. coelicolor parental strain and S. coelicolor YL/sgFabH (a deltafabH mutant carrying a plasmid expressing the Streptomyces glaucescens FabH). These results provide the first unequivocal evidence that the substrate specificity of FabH observed in vitro is a determinant of the fatty acid made in an organism. The YL/ecFabH strain grows significantly slower on both solid and liquid media. The levels of FabH activity in cell extracts of YL/ecFabH were also significantly lower than those in cell extracts of YL/sgFabH, suggesting that a decreased rate of fatty acid synthesis may account for the observed decreased growth rate. The production of low levels of BCFAs in YL/ecFabH suggests either that the E. coli FabH is more tolerant of different acyl-CoAs substrates than previously thought or that there is an additional pathway for initiation of BCFA biosynthesis in Streptomyces coelicolor.

Fatty Acyl Incorporation in the Biosynthesis of WAP-8294A, a Group of Potent Anti-MRSA Cyclic Lipodepsipeptides

PubMed Central

Chen, Haotong; Olson, Andrew S.; Su, Wei; Dussault, Patrick H.; Du, Liangcheng

2015-01-01

WAP-8294A is a family of at least 20 cyclic lipodepsipeptides exhibiting potent anti-MRSA activity. These compounds differ mainly in the hydroxylated fatty acyl chain; WAP-8294A2, the most potent member of the family that reached clinical trials, is based on (R)-3-hydroxy-7-methyloctanoic acid. It is unclear how the acyl group is incorporated because no acyl-CoA ligase (ACL) gene is present in the WAP-8294A gene cluster in Lysobacter enzymogenes OH11. Here, we identified seven putative ACL genes in the OH11 genome and showed that the yield of WAP-8294A2 was impacted by multiple ACL genes with the ACL6 gene having the most significant effect. We then investigated several (R)-3-hydroxy fatty acids and their acyl SNAC (N-acetylcysteamine) thioesters as substrates for the ACLs. Feeding (R)-3-hydroxy-7-methyloctanoate-SNAC to the ACL6 gene deletion mutant restored the production of WAP-8294A2. Finally, we heterologously expressed the seven ACL genes in E. coli and purified six of the proteins. While these enzymes exhibit a varied level of activity in vitro, ACL6 showed the highest catalytic efficiency in converting (R)-3-hydroxy-7-methyloctanoic acid to its CoA thioester when incubated with coenzyme A and ATP. These results provided both in vivo and in vitro evidence to support the fact that ACL6 is the main player for fatty acyl activation and incorporation in WAP-8294A2 biosynthesis. The results also suggest that the molecular basis for the acyl chain diversity in the WAP-8294A family is the presence of functionally overlapping ACLs. PMID:26726302

Fatty Acyl Incorporation in the Biosynthesis of WAP-8294A, a Group of Potent Anti-MRSA Cyclic Lipodepsipeptides.

PubMed

Chen, Haotong; Olson, Andrew S; Su, Wei; Dussault, Patrick H; Du, Liangcheng

WAP-8294A is a family of at least 20 cyclic lipodepsipeptides exhibiting potent anti-MRSA activity. These compounds differ mainly in the hydroxylated fatty acyl chain; WAP-8294A2, the most potent member of the family that reached clinical trials, is based on ( R )-3-hydroxy-7-methyloctanoic acid. It is unclear how the acyl group is incorporated because no acyl-CoA ligase (ACL) gene is present in the WAP-8294A gene cluster in Lysobacter enzymogenes OH11. Here, we identified seven putative ACL genes in the OH11 genome and showed that the yield of WAP-8294A2 was impacted by multiple ACL genes with the ACL6 gene having the most significant effect. We then investigated several ( R )-3-hydroxy fatty acids and their acyl SNAC ( N -acetylcysteamine) thioesters as substrates for the ACLs. Feeding ( R )-3-hydroxy-7-methyloctanoate-SNAC to the ACL6 gene deletion mutant restored the production of WAP-8294A2. Finally, we heterologously expressed the seven ACL genes in E. coli and purified six of the proteins. While these enzymes exhibit a varied level of activity in vitro , ACL6 showed the highest catalytic efficiency in converting ( R )-3-hydroxy-7-methyloctanoic acid to its CoA thioester when incubated with coenzyme A and ATP. These results provided both in vivo and in vitro evidence to support the fact that ACL6 is the main player for fatty acyl activation and incorporation in WAP-8294A2 biosynthesis. The results also suggest that the molecular basis for the acyl chain diversity in the WAP-8294A family is the presence of functionally overlapping ACLs.

Anti-proliferative effects of O-acyl-low-molecular-weight heparin derivatives on bovine pulmonary artery smooth muscle cells.

PubMed

Garg, Hari G; Mrabat, Hicham; Yu, Lunyin; Hales, Charles A; Li, Boyangzi; Moore, Casey N; Zhang, Fuming; Linhardt, Robert J

2011-08-01

Heparin (HP) inhibits the growth of several cell types in vitro including bovine pulmonary artery (BPA) smooth muscle cells (SMCs). In initial studies we discovered that an O-hexanoylated low-molecular-weight (LMW) HP derivative having acyl groups with 6-carbon chain length was more potent inhibitor of BPA-SMCs than the starting HP. We prepared several O-acylated LMWHP derivatives having 4-, 6-, 8-, 10-, 12-, and 18- carbon acyl chain lengths to determine the optimal acyl chain length for maximum anti-proliferative properties of BPA-SMCs. The starting LMWHP was prepared from unfractionated HP by sodium periodate treatment followed by sodium borohydride reduction. The tri-n-butylammonium salt of this LMWHP was O-acylated with butanoic, hexanoic, octanoic, decanoic, dodecanoic, and stearyl anhydrides separately to give respective O-acylated LMWHP derivatives. Gradient polyacrylamide gel electrophoresis (PAGE) was used to examine the average molecular weights of those O-acylated LMWHP derivatives. NMR analysis indicated the presence of one O-acyl group per disaccharide residue. Measurement of the inhibition of BPA-SMCS as a function of O-acyl chain length shows two optima, at a carbon chain length of 6 (O-hexanoylated LMWHP) and at a carbon chain length 12-18 (O-dodecanoyl and O-stearyl LMWHPs). A solution competition SPR study was performed to test the ability of different O-acylated LMWHP derivatives to inhibit fibroblast growth factor (FGF) 1 and FGF2 binding to surface-immobilized heparin. All the LMWHP derivatives bound to FGF1 and FGF2 but each exhibited slightly different binding affinity.

Versatility of acyl-acyl carrier protein synthetases

DOE PAGES

Beld, Joris; Finzel, Kara; Burkart, Michael D.

2014-10-09

The acyl carrier protein (ACP) requires posttranslational modification with a 4'-phosphopantetheine arm for activity, and this thiol-terminated modification carries cargo between enzymes in ACP-dependent metabolic pathways. In this paper, we show that acyl-ACP synthetases (AasSs) from different organisms are able to load even, odd, and unnatural fatty acids onto E. coli ACP in vitro. Vibrio harveyi AasS not only shows promiscuity for the acid substrate, but also is active upon various alternate carrier proteins. AasS activity also extends to functional activation in living organisms. We show that exogenously supplied carboxylic acids are loaded onto ACP and extended by the E.more » coli fatty acid synthase, including unnatural fatty acid analogs. These analogs are further integrated into cellular lipids. Finally, in vitro characterization of four different adenylate-forming enzymes allowed us to disambiguate CoA-ligases and AasSs, and further in vivo studies show the potential for functional application in other organisms.« less

Pulmonary lung surfactant synthetic peptide concentration-dependent modulation of DPPC and POPG acyl chain order in a DPPC:POPG:palmitic acid lipid mixture.

PubMed

Krill, S L; Gupta, S L; Smith, T

1994-05-06

Lung surfactant-associated protein interaction with lipid matrices and the effects on lipid thermotropic phase behavior are areas of active research. Many studies limit the lipids to a single or two-component system. The current investigation utilizes a three-lipid component matrix (DPPC:POPG:palmitic acid) to investigate the impact of a synthetic surfactant protein B fragment (SP-B 53-78 DiACM) on the dynamic surface activity of the lipid admixture as measured by a Wilhelmy surface balance. Also, the modulation of the individual lipid acyl chain order by the peptide within the lipid matrix is studied through the use of thermal perturbation FTIR spectroscopy. The data clearly demonstrate a concentration-dependent effect of the peptide on the surface activity with an improvement in the dynamic surface tension diagram characteristics (decreased surface tension and increased collapse plateau) especially at low, 0.36 M%, peptide concentrations. These effects are diminished upon further addition of the peptide. FTIR spectral data demonstrate that the peptide addition results in a significant increase in the acyl chain order of the DPPC and POPG components as measured by the position of the methylene stretching vibrational bands. DPPC is most sensitive to the peptide presence, while the palmitic acid is least affected. The transition temperatures of the individual lipids are also increased with the addition of the peptide. The presence of POPG in the matrix achieves the surface activity similarly seen with natural lung surfactant relative to a DPPC/palmitic acid lipid matrix alone. Its presence increases the sensitivity of the DPPC acyl chains to the presence of the peptide. These effects on the chain order are most probably related to the increased acyl chain fluidity which POPG imparts to the lipid matrix because of the presence of the cis double bond. The phosphatidylglycerol headgroup also adds a negative charge to the lipid matrix which enhances the peptide

Chlamydia trachomatis growth and development requires the activity of host Long-chain Acyl-CoA Synthetases (ACSLs).

PubMed

Recuero-Checa, Maria A; Sharma, Manu; Lau, Constance; Watkins, Paul A; Gaydos, Charlotte A; Dean, Deborah

2016-03-18

The obligate-intracellular pathogen Chlamydia trachomatis (Ct) has undergone considerable genome reduction with consequent dependence on host biosynthetic pathways, metabolites and enzymes. Long-chain acyl-CoA synthetases (ACSLs) are key host-cell enzymes that convert fatty acids (FA) into acyl-CoA for use in metabolic pathways. Here, we show that the complete host ACSL family [ACSL1 and ACSL3-6] translocates into the Ct membrane-bound vacuole, termed inclusion, and remains associated with membranes of metabolically active forms of Ct throughout development. We discovered that three different pharmacologic inhibitors of ACSL activity independently impede Ct growth in a dose-dependent fashion. Using an FA competition assay, host ACSLs were found to activate Ct branched-chain FAs, suggesting that one function of the ACSLs is to activate Ct FAs and host FAs (recruited from the cytoplasm) within the inclusion. Because the ACSL inhibitors can deplete lipid droplets (LD), we used a cell line where LD synthesis was switched off to evaluate whether LD deficiency affects Ct growth. In these cells, we found no effect on growth or on translocation of ACSLs into the inclusion. Our findings support an essential role for ACSL activation of host-cell and bacterial FAs within the inclusion to promote Ct growth and development, independent of LDs.

A retrospective review of anesthesia and perioperative care in children with medium-chain acyl-CoA dehydrogenase deficiency.

PubMed

Allen, Claire; Perkins, Russell; Schwahn, Bernd

2017-01-01

Medium-chain acyl-CoA dehydrogenase deficiency is the most common genetically determined disorder of mitochondrial fatty acid oxidation. Decompensation can result in hypoglycemia, seizures, coma, and death but may be prevented by ensuring glycogen stores do not become depleted. Perioperative care is of interest as surgery, fasting, and infection may all trigger decompensation and the safety of anesthetic agents has been questioned. Current guidelines from the British Inherited Metabolic Disease Group advise on administering fluid containing 10% glucose during the perioperative period. To review the management of anesthesia and perioperative care for children with medium-chain acyl-CoA dehydrogenase deficiency and determine the frequency and nature of any complications. A retrospective review of case notes of children with medium-chain acyl-CoA dehydrogenase deficiency undergoing anesthesia between 1997 and 2014. Fourteen patients underwent 21 episodes of anesthesia. In 20 episodes, the patient received a glucose-containing fluid during their perioperative fast, of which eight received fluid containing 10% dextrose throughout the entire perioperative period. No episodes of hypoglycemia or decompensation occurred, but perioperative hyperglycemia occurred in five episodes. A propofol bolus was administered at induction in 16 episodes and volatile agents were administered for maintenance of anesthesia in all episodes without any observed complications. In one episode, delayed offset of atracurium was reported. Perioperative metabolic decompensation and hypoglycemia appear to be uncommon in children who are well and receive glucose supplementation. Hyperglycemia may occur as a consequence of surgery and glucose supplementation. Propofol boluses and volatile anesthetic agents were used without any apparent complications. Prolonged action of atracurium was reported in one case, suggesting that nondepolarizing muscle relaxants may have delayed offset in this patient group

Hydrolysis of short-chain phosphatidylcholines by bee venom phospholipase A2.

PubMed

Raykova, D; Blagoev, B

1986-01-01

In order to find out the aggregation state of the substrate, preferred by bee venom phospholipase A2 (EC 3.1.1.4), its action on short-chain phosphatidylcholines with two identical (C6-C10) fatty acids has been tested. The rate of hydrolysis as a function of acyl chain length showed a maximum at dioctanoylphosphatidylcholine. The effects of alcohols, NaCl and Triton X-100, which affect the aggregation state of phospholipids in water, were also studied. The addition of n-alcohol led to a significant inhibition of the hydrolysis of the substrates present in micellar form and activated the hydrolysis of substrates which form liposomes. The inhibitory effect increased with increasing length of the aliphatic carbon chain of the alcohol. Triton X-100 at low Triton/phospholipid molar ratios enhanced enzyme activity. These results do not agree with the accepted idea that bee venom phospholipase A2 hydrolyzes short-chain lecithins in their molecularly dispersed form and that micelles cannot act as substrates. The data indicate that short-chain lecithins in the aggregated state are hydrolyzed and that the requirements of bee venom phospholipase A2 for the aggregation state of the substrate are not strict.

The Drug-Resistant Variant P167S Expands the Substrate Profile of CTX-M β-Lactamases for Oxyimino-Cephalosporin Antibiotics by Enlarging the Active Site upon Acylation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Patel, Meha P.; Hu, Liya; Stojanoski, Vlatko

β-Lactamases are enzymes produced by bacterial cells that provide resistance to β-lactam antibiotics. The CTX-M class of β-lactamases provides resistance against the antibiotic, cefotaxime, but not a related oxyimino-cephalosporin antibiotic, ceftazidime. β-lactamases that carry the P167S substitution, however, have been reported to provide ceftazidime resistance. The mechanism by which the P167S substitution expands the substrate profile of CTX-M enzymes is not known. In this study, CTX-M-14 was used as the model enzyme to study the structural changes caused by the P167S mutation that may accelerate ceftazidime turnover. X-ray crystallography was used to determine the structures of the CTX-M-14 P167S apo-enzymemore » along with the structures of the S70G/P167S, E166A/P167S and E166A mutant enzymes complexed with ceftazidime as well as the E166A/P167S apo-enzyme. The S70G and E166A mutations allow the capture of the enzyme-substrate complex and acylated forms of the ceftazidime molecule, respectively. The results showed a large conformational change in the Ω-loop of the CTX-M-14 ceftazidime acyl-enzyme complex of the P167S mutant but not in the enzyme-substrate complex suggesting the conformational change occurs upon acylation. The conformational change results in a larger active site cavity that prevents steric clash between the aminothiazole ring of ceftazidime and the Asn170 residue in the Ω-loop, allowing for accommodation of ceftazidime for hydrolysis. In addition, the conformational change in the Ω-loop was not observed in the E166A/P167S apoenzyme, suggesting the presence of acylated ceftazidime influences the conformational change. Finally, the E166A acyl-enzyme structure with ceftazidime did not exhibit the altered Ω-loop conformation, indicating the P167S substitution is required for the change. Taken together, the results reveal that the P167S substitution and the presence of acylated ceftazidime both drive the structure towards a

The Drug-Resistant Variant P167S Expands the Substrate Profile of CTX-M β-Lactamases for Oxyimino-Cephalosporin Antibiotics by Enlarging the Active Site upon Acylation

DOE PAGES

Patel, Meha P.; Hu, Liya; Stojanoski, Vlatko; ...

2017-06-14

β-Lactamases are enzymes produced by bacterial cells that provide resistance to β-lactam antibiotics. The CTX-M class of β-lactamases provides resistance against the antibiotic, cefotaxime, but not a related oxyimino-cephalosporin antibiotic, ceftazidime. β-lactamases that carry the P167S substitution, however, have been reported to provide ceftazidime resistance. The mechanism by which the P167S substitution expands the substrate profile of CTX-M enzymes is not known. In this study, CTX-M-14 was used as the model enzyme to study the structural changes caused by the P167S mutation that may accelerate ceftazidime turnover. X-ray crystallography was used to determine the structures of the CTX-M-14 P167S apo-enzymemore » along with the structures of the S70G/P167S, E166A/P167S and E166A mutant enzymes complexed with ceftazidime as well as the E166A/P167S apo-enzyme. The S70G and E166A mutations allow the capture of the enzyme-substrate complex and acylated forms of the ceftazidime molecule, respectively. The results showed a large conformational change in the Ω-loop of the CTX-M-14 ceftazidime acyl-enzyme complex of the P167S mutant but not in the enzyme-substrate complex suggesting the conformational change occurs upon acylation. The conformational change results in a larger active site cavity that prevents steric clash between the aminothiazole ring of ceftazidime and the Asn170 residue in the Ω-loop, allowing for accommodation of ceftazidime for hydrolysis. In addition, the conformational change in the Ω-loop was not observed in the E166A/P167S apoenzyme, suggesting the presence of acylated ceftazidime influences the conformational change. Finally, the E166A acyl-enzyme structure with ceftazidime did not exhibit the altered Ω-loop conformation, indicating the P167S substitution is required for the change. Taken together, the results reveal that the P167S substitution and the presence of acylated ceftazidime both drive the structure towards a

Mitochondrial fatty acid biosynthesis and muscle fiber plasticity in very long-chain acyl-CoA dehydrogenase-deficient mice.

PubMed

Tucci, Sara; Mingirulli, Nadja; Wehbe, Zeinab; Dumit, Verónica I; Kirschner, Janbernd; Spiekerkoetter, Ute

2018-01-01

The white skeletal muscle of very long-chain acyl-CoA-dehydrogenase-deficient (VLCAD -/- ) mice undergoes metabolic modification to compensate for defective β-oxidation in a progressive and time-dependent manner by upregulating glucose oxidation. This metabolic regulation seems to be accompanied by morphologic adaptation of muscle fibers toward the glycolytic fiber type II with the concomitant upregulation of mitochondrial fatty acid biosynthesis (mFASII) and lipoic acid biosynthesis. Dietary supplementation of VLCAD -/- mice with different medium-chain triglycerides over 1 year revealed that odd-chain species has no effect on muscle fiber switch, whereas even-chain species inhibit progressive metabolic adaptation. Our study shows that muscle may undergo adaptive mechanisms that are modulated by dietary supplementation. We describe for the first time a concomitant change of mFASII in this muscular adaptation process. © 2017 Federation of European Biochemical Societies.

A Land-Plant-Specific Glycerol-3-Phosphate Acyltransferase Family in Arabidopsis: Substrate Specificity, sn-2 Preference, and Evolution1[W][OA

PubMed Central

Yang, Weili; Simpson, Jeffrey P.; Li-Beisson, Yonghua; Beisson, Fred; Pollard, Mike; Ohlrogge, John B.

2012-01-01

Arabidopsis (Arabidopsis thaliana) has eight glycerol-3-phosphate acyltransferase (GPAT) genes that are members of a plant-specific family with three distinct clades. Several of these GPATs are required for the synthesis of cutin or suberin. Unlike GPATs with sn-1 regiospecificity involved in membrane or storage lipid synthesis, GPAT4 and -6 are unique bifunctional enzymes with both sn-2 acyltransferase and phosphatase activity resulting in 2-monoacylglycerol products. We present enzymology, pathway organization, and evolutionary analysis of this GPAT family. Within the cutin-associated clade, GPAT8 is demonstrated as a bifunctional sn-2 acyltransferase/phosphatase. GPAT4, -6, and -8 strongly prefer C16:0 and C18:1 ω-oxidized acyl-coenzyme As (CoAs) over unmodified or longer acyl chain substrates. In contrast, suberin-associated GPAT5 can accommodate a broad chain length range of ω-oxidized and unsubstituted acyl-CoAs. These substrate specificities (1) strongly support polyester biosynthetic pathways in which acyl transfer to glycerol occurs after oxidation of the acyl group, (2) implicate GPAT specificities as one major determinant of cutin and suberin composition, and (3) argue against a role of sn-2-GPATs (Enzyme Commission 2.3.1.198) in membrane/storage lipid synthesis. Evidence is presented that GPAT7 is induced by wounding, produces suberin-like monomers when overexpressed, and likely functions in suberin biosynthesis. Within the third clade, we demonstrate that GPAT1 possesses sn-2 acyltransferase but not phosphatase activity and can utilize dicarboxylic acyl-CoA substrates. Thus, sn-2 acyltransferase activity extends to all subbranches of the Arabidopsis GPAT family. Phylogenetic analyses of this family indicate that GPAT4/6/8 arose early in land-plant evolution (bryophytes), whereas the phosphatase-minus GPAT1 to -3 and GPAT5/7 clades diverged later with the appearance of tracheophytes. PMID:22864585

Acyl-Lipid Metabolism

PubMed Central

Li-Beisson, Yonghua; Shorrosh, Basil; Beisson, Fred; Andersson, Mats X.; Arondel, Vincent; Bates, Philip D.; Baud, Sébastien; Bird, David; DeBono, Allan; Durrett, Timothy P.; Franke, Rochus B.; Graham, Ian A.; Katayama, Kenta; Kelly, Amélie A.; Larson, Tony; Markham, Jonathan E.; Miquel, Martine; Molina, Isabel; Nishida, Ikuo; Rowland, Owen; Samuels, Lacey; Schmid, Katherine M.; Wada, Hajime; Welti, Ruth; Xu, Changcheng; Zallot, Rémi; Ohlrogge, John

2013-01-01

Acyl lipids in Arabidopsis and all other plants have a myriad of diverse functions. These include providing the core diffusion barrier of the membranes that separates cells and subcellular organelles. This function alone involves more than 10 membrane lipid classes, including the phospholipids, galactolipids, and sphingolipids, and within each class the variations in acyl chain composition expand the number of structures to several hundred possible molecular species. Acyl lipids in the form of triacylglycerol account for 35% of the weight of Arabidopsis seeds and represent their major form of carbon and energy storage. A layer of cutin and cuticular waxes that restricts the loss of water and provides protection from invasions by pathogens and other stresses covers the entire aerial surface of Arabidopsis. Similar functions are provided by suberin and its associated waxes that are localized in roots, seed coats, and abscission zones and are produced in response to wounding. This chapter focuses on the metabolic pathways that are associated with the biosynthesis and degradation of the acyl lipids mentioned above. These pathways, enzymes, and genes are also presented in detail in an associated website (ARALIP: http://aralip.plantbiology.msu.edu/). Protocols and methods used for analysis of Arabidopsis lipids are provided. Finally, a detailed summary of the composition of Arabidopsis lipids is provided in three figures and 15 tables. PMID:23505340

Acyl-Lipid Metabolism

PubMed Central

Li-Beisson, Yonghua; Shorrosh, Basil; Beisson, Fred; Andersson, Mats X.; Arondel, Vincent; Bates, Philip D.; Baud, Sébastien; Bird, David; DeBono, Allan; Durrett, Timothy P.; Franke, Rochus B.; Graham, Ian A.; Katayama, Kenta; Kelly, Amélie A.; Larson, Tony; Markham, Jonathan E.; Miquel, Martine; Molina, Isabel; Nishida, Ikuo; Rowland, Owen; Samuels, Lacey; Schmid, Katherine M.; Wada, Hajime; Welti, Ruth; Xu, Changcheng; Zallot, Rémi; Ohlrogge, John

2010-01-01

Acyl lipids in Arabidopsis and all other plants have a myriad of diverse functions. These include providing the core diffusion barrier of the membranes that separates cells and subcellular organelles. This function alone involves more than 10 membrane lipid classes, including the phospholipids, galactolipids, and sphingolipids, and within each class the variations in acyl chain composition expand the number of structures to several hundred possible molecular species. Acyl lipids in the form of triacylglycerol account for 35% of the weight of Arabidopsis seeds and represent their major form of carbon and energy storage. A layer of cutin and cuticular waxes that restricts the loss of water and provides protection from invasions by pathogens and other stresses covers the entire aerial surface of Arabidopsis. Similar functions are provided by suberin and its associated waxes that are localized in roots, seed coats, and abscission zones and are produced in response to wounding. This chapter focuses on the metabolic pathways that are associated with the biosynthesis and degradation of the acyl lipids mentioned above. These pathways, enzymes, and genes are also presented in detail in an associated website (ARALIP: http://aralip.plantbiology.msu.edu/). Protocols and methods used for analysis of Arabidopsis lipids are provided. Finally, a detailed summary of the composition of Arabidopsis lipids is provided in three figures and 15 tables. PMID:22303259

Medium-Chain Acyl-CoA Deficiency: Outlines from Newborn Screening, In Silico Predictions, and Molecular Studies

PubMed Central

Catarzi, Serena; Caciotti, Anna; Thusberg, Janita; Tonin, Rodolfo; Malvagia, Sabrina; la Marca, Giancarlo; Pasquini, Elisabetta; Cavicchi, Catia; Ferri, Lorenzo; Donati, Maria A.; Baronio, Federico; Guerrini, Renzo; Mooney, Sean D.; Morrone, Amelia

2013-01-01

Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is a disorder of fatty acid oxidation characterized by hypoglycemic crisis under fasting or during stress conditions, leading to lethargy, seizures, brain damage, or even death. Biochemical acylcarnitines data obtained through newborn screening by liquid chromatography-tandem mass spectrometry (LC-MS/MS) were confirmed by molecular analysis of the medium-chain acyl-CoA dehydrogenase (ACADM) gene. Out of 324.000 newborns screened, we identified 14 MCADD patients, in whom, by molecular analysis, we found a new nonsense c.823G>T (p.Gly275∗) and two new missense mutations: c.253G>C (p.Gly85Arg) and c.356T>A (p.Val119Asp). Bioinformatics predictions based on both phylogenetic conservation and functional/structural software were used to characterize the new identified variants. Our findings confirm the rising incidence of MCADD whose existence is increasingly recognized due to the efficacy of an expanded newborn screening panel by LC-MS/MS making possible early specific therapies that can prevent possible crises in at-risk infants. We noticed that the “common” p.Lys329Glu mutation only accounted for 32% of the defective alleles, while, in clinically diagnosed patients, this mutation accounted for 90% of defective alleles. Unclassified variants (UVs or VUSs) are especially critical when considering screening programs. The functional and pathogenic characterization of genetic variants presented here is required to predict their medical consequences in newborns. PMID:24294134

Enzymatic Acylation of Anthocyanins Isolated from Alpine Bearberry ( Arctostaphylos alpina) and Lipophilic Properties, Thermostability, and Antioxidant Capacity of the Derivatives.

PubMed

Yang, Wei; Kortesniemi, Maaria; Yang, Baoru; Zheng, Jie

2018-03-21

Cyanidin-3- O-galactoside (cy-gal) isolated from alpine bearberry ( Arctostaphylos alpine L.) was enzymatically acylated with saturated fatty acids of different chain lengths with Candida antarctica lipase immobilized on acrylic resin (Novozyme 435). The acylation reaction was optimized by considering the reaction medium, acyl donor, substrate molar ratio, reaction temperature, and reaction time. The highest conversion yield of 73% was obtained by reacting cy-gal with lauric acid (molar ratio of 1:10) in tert-butanol at 60 °C for 72 h. A novel compound was synthesized, which was identified as cyanidin-3- O-(6″-dodecanoyl)galactoside by mass spectrometry and nuclear magnetic resonance. Introducing lauric acid into cy-gal significantly improved both the lipophilicity and thermostability and substantially preserved the ultraviolet-visible absorbance and antioxidant properties. The research provides important insight in expanding the application of natural anthocyanins in the cosmetic and food industries.

Defluoridation potential of jute fibers grafted with fatty acyl chain

NASA Astrophysics Data System (ADS)

Manna, Suvendu; Saha, Prosenjit; Roy, Debasis; Sen, Ramkrishna; Adhikari, Basudam

2015-11-01

Waterborne fluoride is usually removed from water by coagulation, adsorption, ion exchange, electro dialysis or reverse osmosis. These processes are often effective over narrow pH ranges, release ions considered hazardous to human health or produce large volumes of toxic sludge that are difficult to handle and dispose. Although plant matters have been shown to remove waterborne fluoride, they suffer from poor removal efficiency. Following from the insight that interaction between microbial carbohydrate biopolymers and anionic surfaces is often facilitated by lipids, an attempt has been made to enhance fluoride adsorption efficiency of jute by grafting the lignocellulosic fiber with fatty acyl chains found in vegetable oils. Fluoride removal efficiency of grafted jute was found to be comparable or higher than those of alternative defluoridation processes. Infrared and X-ray photoelectron spectroscopic evidence indicated that hydrogen bonding, protonation and C-F bonding were responsible for fluoride accumulation on grafted jute. Adsorption based on grafted jute fibers appears to be an economical, sustainable and eco-friendly alternative technique for removing waterborne fluoride.

Chlamydia trachomatis growth and development requires the activity of host Long-chain Acyl-CoA Synthetases (ACSLs)

PubMed Central

Recuero-Checa, Maria A.; Sharma, Manu; Lau, Constance; Watkins, Paul A.; Gaydos, Charlotte A.; Dean, Deborah

2016-01-01

The obligate-intracellular pathogen Chlamydia trachomatis (Ct) has undergone considerable genome reduction with consequent dependence on host biosynthetic pathways, metabolites and enzymes. Long-chain acyl-CoA synthetases (ACSLs) are key host-cell enzymes that convert fatty acids (FA) into acyl-CoA for use in metabolic pathways. Here, we show that the complete host ACSL family [ACSL1 and ACSL3–6] translocates into the Ct membrane-bound vacuole, termed inclusion, and remains associated with membranes of metabolically active forms of Ct throughout development. We discovered that three different pharmacologic inhibitors of ACSL activity independently impede Ct growth in a dose-dependent fashion. Using an FA competition assay, host ACSLs were found to activate Ct branched-chain FAs, suggesting that one function of the ACSLs is to activate Ct FAs and host FAs (recruited from the cytoplasm) within the inclusion. Because the ACSL inhibitors can deplete lipid droplets (LD), we used a cell line where LD synthesis was switched off to evaluate whether LD deficiency affects Ct growth. In these cells, we found no effect on growth or on translocation of ACSLs into the inclusion. Our findings support an essential role for ACSL activation of host-cell and bacterial FAs within the inclusion to promote Ct growth and development, independent of LDs. PMID:26988341

A role for long-chain acyl-CoA synthetase-4 (ACSL4) in diet-induced phospholipid remodeling and obesity-associated adipocyte dysfunction

USDA-ARS?s Scientific Manuscript database

OBJECTIVE: Regulation of fatty acid (FA) metabolism is central to adipocyte dysfunction during diet-induced obesity (DIO). Long-chain acyl-CoA synthetase-4 (ACSL4) has been hypothesized to modulate the metabolic fates of polyunsaturated FA (PUFA), including arachidonic acid (AA), but the in vivo act...

Dynamic properties of individual water molecules in a hydrophobic pore lined with acyl chains: a molecular dynamics study.

PubMed

Qi, Z; Sokabe, M

1998-03-30

Recently, a certain class of synthetic molecules has been shown to form ion channels, the pore of which is lined with hydrophobic acyl chains [M. Sokabe, in: F. Oosawa, H. Hayashi, T. Yoshioka (Eds.), Transmembrane Signaling and Sensation, JSSP/VNU Science Press BV, Tokyo, 1984, p. 119; F. Hayashi, M. Sokabe, M. Takagi, K. Hayashi, U. Kishimoto, Biochim. Biophys. Acta, 510 (1978) 305; M.J. Pregel, L. Jullien, J. Canceill, L. Lacombe, J.M. Lehn, J. Chem. Soc. Perkin Trans., 2 (1995) 417; Y. Tanaka, Y. Kobuke, M. Sokabe, Angew. Chem. Int. Ed. Engl., 34 (1995) 693; M. Sokabe, Z. Qi, K. Donowaki, H. Ishida, K. Okubo, Biophys. J., 70 (1996) A201; H. Ishida, K. Donowaki, Y. Inoue, Z. Qi, M. Sokabe, Chem. Lett. (1997) p. 953]. As an initial step towards understanding the physical mechanisms of ion permeation across such a hydrophobic pore, systematic molecular dynamics simulations were performed to investigate dynamic and energetic properties of water molecules inside the pore using a dimer of alanine-N'-acylated cyclic peptide as a channel model. Dynamic energy profiles for water molecules indicated that the energy barrier at the middle region of the pore is approximately 2-3 kcal/mol higher than that in the cap water region which was defined as a vicinity region of the channel entrance. Energetics analyses demonstrated that the mutual interactions among intrapore water molecules are the major factor to give favorable interaction (negative energy contribution) for themselves. The pore, despite being lined with acyl chains, has a favorable van der Waals interaction with intrapore water molecules. These results may help to explain why water-filled channels can be formed by the hydrophobic helices in natural channels.

Acyl chain conformational ordering of individual components in liquid-crystalline bilayers of mixtures of phosphatidylcholines and phosphatidic acids. A comparative FTIR and 2H NMR study

NASA Astrophysics Data System (ADS)

Ziegler, Wolfgang; Blume, Alfred

1995-09-01

The conformational ordering of the acyl chains of all possible binary 1:1 mixtures containing the phospholipids DMPC, DMPA, DPPC, and DPPA was investigated using FTIR and 2H NMR spectroscopy. One of the components was always labelled with perdeuterated chains to be able to observe the individual behaviour of the two components. From the temperature dependence of the frequencies of the symmetric and antisymmetric CH 2- and CD 2-stretching vibrations the transition temperatures were determined. The integral intensities of the conformation sensitive CH 2-wagging bands at ca. 1368 cm -1(gtg' and gtg sequences), 1356 cm -1 (double gauche), and 1342 cm -1 (end gauche) can be converted to numbers of gauche conformers per acyl chain using calibration factors published by Senak et al. J. Phys. Chem. 95 (1991) 2565. The 2H NMR quadrupolar splittings of the CD 2-segments of the perdeuterated lipid components are affected not only by trans-gauche isomerizations but also by long axis rotations and restricted wobbling motions of the acyl chains. The values of the average gauche probability overlinep3 from FTIR spectroscopy and the average order parameters overlineSCD, the order parameter of the terminal methyl groups SCDCD 3 and the average order parameter for the plateau region overlineSCDPlat of components in the mixtures are compared to those of the pure lipids evaluated in a previous publication Tuchtenhagen et al. Eur. Biophys. J. 23 (1994) 323. The conformational behaviour of lipids in mixtures is mainly influenced by head groups interactions, PAs always being more ordered than the corresponding PCs. Depending on absolute chain length and on chain length differences between the two components different conformational behaviour is observed for the two components in the mixtures, indicating non-ideal mixing and formation of micro-domains in the liquid-crystalline phase. Increases in order at the chain ends with a concomitant decrease in probabilities for end gauche

Kinetics of bacterial phospholipase C activity at micellar interfaces: effect of substrate aggregate microstructure and a model for the kinetic parameters.

PubMed

Singh, Jasmeet; Ranganathan, Radha; Hajdu, Joseph

2008-12-25

Activity at micellar interfaces of bacterial phospholipase C from Bacillus cereus on phospholipids solubilized in micelles was investigated with the goal of elucidating the role of the interface microstructure and developing further an existing kinetic model. Enzyme kinetics and physicochemical characterization of model substrate aggregates were combined, thus enabling the interpretation of kinetics in the context of the interface. Substrates were diacylphosphatidylcholine of different acyl chain lengths in the form of mixed micelles with dodecyldimethylammoniopropanesulfonate. An early kinetic model, reformulated to reflect the interfacial nature of the kinetics, was applied to the kinetic data. A better method of data treatment is proposed, use of which makes the presence of microstructure effects quite transparent. Models for enzyme-micelle binding and enzyme-lipid binding are developed, and expressions incorporating the microstructural properties are derived for the enzyme-micelle dissociation constant K(s) and the interface Michaelis-Menten constant, K(M). Use of these expressions in the interface kinetic model brings excellent agreement between the kinetic data and the model. Numerical values for the thermodynamic and kinetic parameters are determined. Enzyme-lipid binding is found to be an activated process with an acyl chain length dependent free energy of activation that decreases with micelle lipid molar fraction with a coefficient of about -15RT and correlates with the tightness of molecular packing in the substrate aggregate. Thus, the physical insight obtained includes a model for the kinetic parameters that shows that these parameters depend on the substrate concentration and acyl chain length of the lipid. Enzyme-micelle binding is indicated to be hydrophobic and solvent mediated with a dissociation constant of 1.2 mM.

Kinetics of acyl transfer reactions in organic media catalysed by Candida antarctica lipase B.

PubMed

Martinelle, M; Hult, K

1995-09-06

The acyl transfer reactions catalysed by Candida antartica lipase B in organic media followed a bi-bi ping-pong mechanism, with competitive substrate inhibition by the alcohols used as acyl acceptors. The effect of organic solvents on Vm and Km was investigated. The Vm values in acetonitrile was 40-50% of those in heptane. High Km values in acetonitrile compared to those in heptane could partly be explained by an increased solvation of the substrates in acetonitrile. Substrate solvation caused a 10-fold change in substrate specificity, defined as (Vm/Km)ethyl octanoate/(Vm/Km)octanoic acid, going from heptane to acetonitrile. Deacylation was the rate determining step for the acyl transfer in heptane with vinyl- and ethyl octanoate as acyl donors and (R)-2-octanol as acyl acceptor. With 1-octanol, a rate determining deacylation step in heptane was indicated using the same acyl donors. Using 1-octanol as acceptor in heptane, S-ethyl thiooctanoate had a 25- to 30-fold lower Vm/Km value and vinyl octanoate a 4-fold higher Vm/Km value than that for ethyl octanoate. The difference showed to be a Km effect for vinyl octanoate and mainly a Km effect for S-ethyl thiooctanoate. The Vm values of the esterification of octanoic acid with different alcohols was 10-30-times lower than those for the corresponding transesterification of ethyl octanoate. The low activity could be explained by a low pH around the enzyme caused by the acid or a withdrawing of active enzyme by nonproductive binding by the acid.

Chlamydia trachomatis Scavenges Host Fatty Acids for Phospholipid Synthesis via an Acyl-Acyl Carrier Protein Synthetase*

PubMed Central

Yao, Jiangwei; Dodson, V. Joshua; Frank, Matthew W.; Rock, Charles O.

2015-01-01

The obligate intracellular parasite Chlamydia trachomatis has a reduced genome but relies on de novo fatty acid and phospholipid biosynthesis to produce its membrane phospholipids. Lipidomic analyses showed that 8% of the phospholipid molecular species synthesized by C. trachomatis contained oleic acid, an abundant host fatty acid that cannot be made by the bacterium. Mass tracing experiments showed that isotopically labeled palmitic, myristic, and lauric acids added to the medium were incorporated into C. trachomatis-derived phospholipid molecular species. HeLa cells did not elongate lauric acid, but infected HeLa cell cultures elongated laurate to myristate and palmitate. The elongated fatty acids were incorporated exclusively into C. trachomatis-produced phospholipid molecular species. C. trachomatis has adjacent genes encoding the separate domains of the bifunctional acyl-acyl carrier protein (ACP) synthetase/2-acylglycerolphosphoethanolamine acyltransferase gene (aas) of Escherichia coli. The CT775 gene encodes an acyltransferase (LpaT) that selectively transfers fatty acids from acyl-ACP to the 1-position of 2-acyl-glycerophospholipids. The CT776 gene encodes an acyl-ACP synthetase (AasC) with a substrate preference for palmitic compared with oleic acid in vitro. Exogenous fatty acids were elongated and incorporated into phospholipids by Escherichia coli-expressing AasC, illustrating its function as an acyl-ACP synthetase in vivo. These data point to an AasC-dependent pathway in C. trachomatis that selectively scavenges host saturated fatty acids to be used for the de novo synthesis of its membrane constituents. PMID:26195634

Unmasking tandem site interaction in human acetylcholinesterase. Substrate activation with a cationic acetanilide substrate.

PubMed

Johnson, Joseph L; Cusack, Bernadette; Davies, Matthew P; Fauq, Abdul; Rosenberry, Terrone L

2003-05-13

Acetylcholinesterase (AChE) contains a narrow and deep active site gorge with two sites of ligand binding, an acylation site (or A-site) at the base of the gorge, and a peripheral site (or P-site) near the gorge entrance. The P-site contributes to catalytic efficiency by transiently binding substrates on their way to the acylation site, where a short-lived acyl enzyme intermediate is produced. A conformational interaction between the A- and P-sites has recently been found to modulate ligand affinities. We now demonstrate that this interaction is of functional importance by showing that the acetylation rate constant of a substrate bound to the A-site is increased by a factor a when a second molecule of substrate binds to the P-site. This demonstration became feasible through the introduction of a new acetanilide substrate analogue of acetylcholine, 3-(acetamido)-N,N,N-trimethylanilinium (ATMA), for which a = 4. This substrate has a low acetylation rate constant and equilibrates with the catalytic site, allowing a tractable algebraic solution to the rate equation for substrate hydrolysis. ATMA affinities for the A- and P-sites deduced from the kinetic analysis were confirmed by fluorescence titration with thioflavin T as a reporter ligand. Values of a >1 give rise to a hydrolysis profile called substrate activation, and the AChE site-specific mutant W86F, and to a lesser extent wild-type human AChE itself, showed substrate activation with acetylthiocholine as the substrate. Substrate activation was incorporated into a previous catalytic scheme for AChE in which a bound P-site ligand can also block product dissociation from the A-site, and two additional features of the AChE catalytic pathway were revealed. First, the ability of a bound P-site ligand to increase the substrate acetylation rate constant varied with the structure of the ligand: thioflavin T accelerated ATMA acetylation by a factor a(2) of 1.3, while propidium failed to accelerate. Second, catalytic rate

Medium-chain triglycerides impair lipid metabolism and induce hepatic steatosis in very long-chain acyl-CoA dehydrogenase (VLCAD)-deficient mice.

PubMed

Tucci, Sara; Primassin, Sonja; Ter Veld, Frank; Spiekerkoetter, Ute

2010-09-01

A medium-chain-triglyceride (MCT)-based diet is mainstay of treatment in very-long-chain acyl-CoA dehydrogenase deficiency (VLCADD), a long-chain fatty acid beta-oxidation defect. Beneficial effects have been reported with an MCT-bolus prior to exercise. Little is known about the impact of a long-term MCT diet on hepatic lipid metabolism. Here we investigate the effects of MCT-supplementation on liver and blood lipids in the murine model of VLCADD. Wild-type (WT) and VLCAD-knock-out (KO) mice were fed (1) a long-chain triglyceride (LCT)-diet over 5weeks, (2) an MCT diet over 5 weeks and (3) an LCT diet plus MCT-bolus. Blood and liver lipid content were determined. Expression of genes regulating lipogenesis was analyzed by RT-PCR. Under the LCT diet, VLCAD-KO mice accumulated significantly higher blood cholesterol concentrations compared to WT mice. The MCT-diet induced severe hepatic steatosis, significantly higher serum free fatty acids and impaired hepatic lipid mobilization in VLCAD-KO mice. Expression at mRNA level of hepatic lipogenic genes was up-regulated. The long-term MCT diet stimulates lipogenesis and impairs hepatic lipid metabolism in VLCAD-KO mice. These results suggest a critical reconsideration of a long-term MCT-modified diet in human VLCADD. In contrast, MCT in situations of increased energy demand appears to be a safer treatment alternative.

The activity of Rhizomuchor miehei lipase as a biocatalyst in enzymatic acylation of cyclic alcohol

NASA Astrophysics Data System (ADS)

Iftitah, Elvina Dhiaul; Srihardyastuti, Arie; Ariefin, Mokhamat

2017-03-01

We report the activity of Rhizomuchor miehei lipase (RML) as a biocatalyst, in particular the investigations concerning the effort of substrate-structure reactivity on the enzymatic acylation. The acylation was studied using acetic anhydride as an acyl donor and performed in n-hexane as a solvent. The selectivity of the enzymatic acylation was revealed by Gas Chromatography-Mass Spectra. We observed that, RML has shown different behavior when catalyzing the acylation of isopulegol and mixture of isopulegol and citronellal (ratio 1:1). The chemoselectivity for the O-acylation was improved when the acyl acceptor included mixture of isopulegol and citronellal

An insight on acyl migration in solvent-free ethanolysis of model triglycerides using Novozym 435.

PubMed

Sánchez, Daniel Alberto; Tonetto, Gabriela Marta; Ferreira, María Luján

2016-02-20

In this work, the ethanolysis of triglycerides catalyzed by immobilized lipase was studied, focusing on the secondary reaction of acyl migration. The catalytic tests were performed in a solvent-free reaction medium using Novozym 435 as biocatalyst. The selected experimental variables were biocatalyst loading (5-20mg), reaction time (30-90min), and chain length of the fatty acids in triglycerides with and without unsaturation (short (triacetin), medium (tricaprylin) and long (tripalmitin/triolein)). The formation of 2-monoglyceride by ethanolysis of triglycerides was favored by long reaction times and large biocatalyst loading with saturated short- to medium-chain triglycerides. In the case of long-chain triglycerides, the formation of this monoglyceride was widely limited by acyl migration. In turn, acyl migration increased the yield of ethyl esters and minimized the content of monoglycerides and diglycerides. Thus, the enzymatic synthesis of biodiesel was favored by long-chain triglycerides (which favor the acyl migration), long reaction times and large biocatalyst loading. The conversion of acylglycerides made from long-chain fatty acids with unsaturation was relatively low due to limitations in their access to the active site of the lipase. Copyright © 2016 Elsevier B.V. All rights reserved.

Mechanism and regulation of mycobactin fatty acyl-AMP ligase FadD33.

PubMed

Vergnolle, Olivia; Xu, Hua; Blanchard, John S

2013-09-27

Mycobacterial siderophores are critical components for bacterial virulence in the host. Pathogenic mycobacteria synthesize iron chelating siderophores named mycobactin and carboxymycobactin to extract intracellular macrophage iron. The two siderophores differ in structure only by a lipophilic aliphatic chain attached on the ε-amino group of the lysine mycobactin core, which is transferred by MbtK. Prior to acyl chain transfer, the lipophilic chain requires activation by a specific fatty acyl-AMP ligase FadD33 (also known as MbtM) and is then loaded onto phosphopantetheinylated acyl carrier protein (holo-MbtL) to form covalently acylated MbtL. We demonstrate that FadD33 prefers long chain saturated lipids and initial velocity studies showed that FadD33 proceeds via a Bi Uni Uni Bi ping-pong mechanism. Inhibition experiments suggest that, during the first half-reaction (adenylation), fatty acid binds first to the free enzyme, followed by ATP and the release of pyrophosphate to form the adenylate intermediate. During the second half-reaction (ligation), holo-MbtL binds to the enzyme followed by the release of products AMP and acylated MbtL. In addition, we characterized a post-translational regulation mechanism of FadD33 by the mycobacterial protein lysine acetyltransferase in a cAMP-dependent manner. FadD33 acetylation leads to enzyme inhibition, which can be reversed by the NAD(+)-dependent deacetylase, MSMEG_5175 (DAc1). To the best of our knowledge, this is the first time that bacterial siderophore synthesis has been shown to be regulated via post-translational protein acetylation.

Growth Studies of Probiotic Bacteria on Short Chain Glucomannan, a Potential Prebiotic Substrate

DTIC Science & Technology

2012-12-05

PROBIOTIC BACTERIA ON SHORT CHAIN GLUCOMANNAN, A POTENTIAL PREBIOTIC SUBSTRATE by Wayne S. Muller Steve Arcidiacono Adam Liebowitz Ken Racicot... PROBIOTIC BACTERIA ON SHORT CHAIN GLUCOMANNAN, A POTENTIAL PREBIOTIC SUBSTRATE 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER PE...commercial prebiotic substrates. All three substrates had similar degree of polymerization (DP) of 2-9. Five probiotic bacteria were evaluated for

Chimeric Fatty Acyl-Acyl Carrier Protein Thioesterases Provide Mechanistic Insight into Enzyme Specificity and Expression.

PubMed

Ziesack, Marika; Rollins, Nathan; Shah, Aashna; Dusel, Brendon; Webster, Gordon; Silver, Pamela A; Way, Jeffrey C

2018-05-15

Medium-chain fatty acids are commodity chemicals. Increasing and modifying the activity of thioesterases (TEs) on medium-chain fatty acyl-acyl carrier protein (acyl-ACP) esters may enable a high-yield microbial production of these molecules. The plant Cuphea palustris harbors two distinct TEs: C. palustris FatB1 ( Cp FatB1) (C 8 specificity, lower activity) and Cp FatB2 (C 14 specificity, higher activity) with 78% sequence identity. We combined structural features from these two enzymes to create several chimeric TEs, some of which showed nonnatural fatty acid production as measured by an enzymatic assay and gas chromatography-mass spectrometry (GC-MS). Notably, chimera 4 exhibited an increased C 8 fatty acid production in correlation with improved microbial expression. This chimera led us to identify Cp FatB2-specific amino acids between positions 219 and 272 that lead to higher protein levels. Chimera 7 produced a broad range of fatty acids and appeared to combine a fatty acid binding pocket with long-chain specificity and an ACP interaction site that may activate fatty acid extrusion. Using homology modeling and in silico docking with ACP, we identified a "positive patch" within amino acids 162 to 218, which may direct the ACP interaction and regulate access to short-chain fatty acids. On the basis of this modeling, we transplanted putative ACP interaction sequences from Cp FatB1 into Cp FatB2 and created a chimeric thioesterase that produced medium-chain as well as long-chain fatty acids. Thus, the engineering of chimeric enzymes and characterizing their microbial activity and chain-length specificity suggested mechanistic insights into TE functions and also generated thioesterases with potentially useful properties. These observations may inform a rational engineering of TEs to allow alkyl chain length control. IMPORTANCE Medium-chain fatty acids are important commodity chemicals. These molecules are used as plastic precursors and in shampoos and other

Kinetics of reactions of the Actinomadura R39 DD-peptidase with specific substrates.

PubMed

Adediran, S A; Kumar, Ish; Nagarajan, Rajesh; Sauvage, Eric; Pratt, R F

2011-01-25

The Actinomadura R39 DD-peptidase catalyzes the hydrolysis and aminolysis of a number of small peptides and depsipeptides. Details of its substrate specificity and the nature of its in vivo substrate are not, however, well understood. This paper describes the interactions of the R39 enzyme with two peptidoglycan-mimetic substrates 3-(D-cysteinyl)propanoyl-D-alanyl-D-alanine and 3-(D-cysteinyl)propanoyl-D-alanyl-D-thiolactate. A detailed study of the reactions of the former substrate, catalyzed by the enzyme, showed DD-carboxypeptidase, DD-transpeptidase, and DD-endopeptidase activities. These results confirm the specificity of the enzyme for a free D-amino acid at the N-terminus of good substrates and indicated a preference for extended D-amino acid leaving groups. The latter was supported by determination of the structural specificity of amine nucleophiles for the acyl-enzyme generated by reaction of the enzyme with the thiolactate substrate. It was concluded that a specific substrate for this enzyme, and possibly the in vivo substrate, may consist of a partly cross-linked peptidoglycan polymer where a free side chain N-terminal un-cross-linked amino acid serves as the specific acyl group in an endopeptidase reaction. The enzyme is most likely a DD-endopeptidase in vivo. pH-rate profiles for reactions of the enzyme with peptides, the thiolactate named above, and β-lactams indicated the presence of complex proton dissociation pathways with sticky substrates and/or protons. The local structure of the active site may differ significantly for reactions of peptides and β-lactams. Solvent kinetic deuterium isotope effects indicate the presence of classical general acid/base catalysis in both acylation and deacylation; there is no evidence of the low fractionation factor active site hydrogen found previously in class A and C β-lactamases.

Molecular structure, supramolecular organization and thermotropic phase behavior of N-acylglycine alkyl esters with matched acyl and alkyl chains.

PubMed

Reddy, S Thirupathi; Swamy, Musti J

2017-11-01

N-Acylglycines (NAGs), the endogenous single-tailed lipids present in rat brain and other mammalian tissues, play significant roles in cell physiology and exhibit interesting pharmacological properties. In the present study, a homologous series of N-acylglycine alkyl esters (NAGEs) with matched chains were synthesized and characterized. Results of differential scanning calorimetric studies revealed that all NAGEs exhibit a single sharp phase transition and that the transition enthalpy and entropy show a linear dependence on the N-acyl and ester alkyl chain length. The structure of N-myristoylglycine myristyl ester (NMGME), solved by single-crystal X-ray diffraction, showed that the molecule adopts a linear geometry and revealed that the structure of N-myristoyl glycyl moiety in NMGME is identical to that in N-myristoylglycine. The molecules are packed in layers with the polar functional groups of the ester and amide functionalities located at the center of the layer. The crystal packing is stabilized by NH⋯O hydrogen bonds between the amide CO and NH groups of adjacent molecules as well as by CH⋯O hydrogen bonds between the amide carbonyl and the methylene CH adjacent to the ester carbonyl of neighboring molecules as well as between ester carbonyl and methylene group of the glycine moiety of adjacent molecules. Powder X-ray diffraction studies showed a linear dependence of the d-spacings on the acyl chain length, suggesting that all NAGEs adopt a structure similar to the packing exhibited in the crystal lattice of NMGME. Copyright © 2017 Elsevier B.V. All rights reserved.

Regulation of Long-Chain N-Acyl-Homoserine Lactones in Agrobacterium vitis

PubMed Central

Hao, Guixia; Burr, Thomas J.

2006-01-01

Homologs of quorum-sensing luxR and luxI regulatory genes, avsR and avsI, were identified in Agrobacterium vitis strain F2/5. Compared to other LuxI proteins from related species, the deduced AvsI shows the greatest identity to SinI (71%) from Sinorhizobium meliloti Rm1021. AvsR possesses characteristic autoinducer binding and helix-turn-helix DNA binding domains and shares a high level of identity with SinR (38%) from Rm1021. Site-directed mutagenesis of avsR and avsI was performed, and both genes are essential for hypersensitive-like response (HR) and necrosis. Two hypothetical proteins (ORF1 and ORF2) that are positioned downstream of avsR-avsI are also essential for the phenotypes. Profiles of N-acyl-homoserine lactones (AHLs) isolated from the wild type and mutants revealed that disruption of avsI, ORF1, or ORF2 abolished the production of long-chain AHLs. Disruption of avsR reduces long-chain AHLs. Expression of a cloned avsI gene in A. tumefaciens strain NT1 resulted in synthesis of long-chain AHLs. The necrosis and HR phenotypes of the avsI and avsR mutants were fully complemented with cloned avsI. The addition of synthetic AHLs (C16:1 and 3-O-C16:1) complemented grape necrosis in the avsR, avsI, ORF1, and ORF2 mutants. It was determined by reverse transcriptase PCR that the expression level of avsI is regulated by avsR but not by aviR or avhR, two other luxR homologs which were previously shown to be associated with induction of a tobacco hypersensitive response and grape necrosis. We further verified that avsR regulates avsI by measuring the expression of an avsI::lacZ fusion construct. PMID:16513747

Lanthanum tricyanide-catalyzed acyl silane-ketone benzoin additions.

PubMed

Tarr, James C; Johnson, Jeffrey S

2009-09-03

Lanthanum tricyanide efficiently catalyzes a benzoin-type coupling between acyl silanes and ketones. Yields range from moderate to excellent over a broad substrate scope encompassing aryl, alkyl, electron-rich, and sterically hindered ketones.

Photoaffinity Labeling of Developing Jojoba Seed Microsomal Membranes with a Photoreactive Analog of Acyl-Coenzyme A (Acyl-CoA) (Identification of a Putative Acyl-CoA:Fatty Alcohol Acyltransferase.

PubMed

Shockey, J. M.; Rajasekharan, R.; Kemp, J. D.

1995-01-01

Jojoba (Simmondsia chinensis, Link) is the only plant known that synthesizes liquid wax. The final step in liquid wax biosynthesis is catalyzed by an integral membrane enzyme, fatty acyl-coenzyme A (CoA):fatty alcohol acyltransferase, which transfers an acyl chain from acyl-CoA to a fatty alcohol to form the wax ester. To purify the acyltransferase, we have labeled the enzyme with a radioiodinated, photoreactive analog of acyl-CoA, 12-[N-(4-azidosalicyl)amino] dodecanoyl-CoA (ASD-CoA). This molecule acts as an inhibitor of acyltransferase activity in the dark and as an irreversible inhibitor upon exposure to ultraviolet light. Oleoyl-CoA protects enzymatic activity in a concentration-dependent manner. Photolysis of microsomal membranes with labeled ASD-CoA resulted in strong labeling of two polypeptides of 57 and 52 kD. Increasing concentrations of oleoyl-CoA reduced the labeling of the 57-kD polypeptide dramatically, whereas the labeling of the 52-kD polypeptide was much less responsive to oleoyl-CoA. Also, unlike the other polypeptide, the labeling of the 57-kD polypeptide was enhanced considerably when photolyzed in the presence of dodecanol. These results suggest that a 57-kD polypeptide from jojoba microsomes may be the acyl-CoA:fatty alcohol acyltransferase.

Electrochemical and PM-IRRAS Studies of the Effect of Cholesterol on the Structure of a DMPC Bilayer Supported at an Au (111) Electrode Surface, Part 1: Properties of the Acyl Chains

PubMed Central

Bin, Xiaomin; Horswell, Sarah L.; Lipkowski, Jacek

2005-01-01

Charge density measurements and polarization modulation infrared reflection absorption spectroscopy were employed to investigate the spreading of small unilamellar vesicles of a dimyristoylphosphatidylcholine (DMPC)/cholesterol (7:3 molar ratio) mixture onto an Au (111) electrode surface. The electrochemical experiments demonstrated that vesicles fuse and spread onto the Au (111) electrode surface, forming a bilayer, at rational potentials −0.4 V < (E − Epzc) < 0.4 V or field strength <6×107 V m−1. Polarization modulation infrared reflection absorption spectroscopy experiments provided information concerning the conformation and orientation of the acyl chains of DMPC molecules. Deuterated DMPC was used to subtract the contribution of C-H stretching bands of cholesterol and of the polar head region of DMPC from spectra in the C-H stretching region. The absorption spectra of the C-H stretch bands in the acyl chains were determined in this way. The properties of the DMPC/cholesterol bilayer have been compared with the properties of a pure DMPC bilayer. The presence of 30% cholesterol gives a thicker and more fluid bilayer characterized by a lower capacity and lower tilt angle of the acyl chains. PMID:15849259

Long-chain acyl-CoA synthetase 6 regulates lipid synthesis and mitochondrial oxidative capacity in human and rat skeletal muscle.

PubMed

Teodoro, Bruno G; Sampaio, Igor H; Bomfim, Lucas H M; Queiroz, André L; Silveira, Leonardo R; Souza, Anderson O; Fernandes, Anna M A P; Eberlin, Marcos N; Huang, Tai-Yu; Zheng, Donghai; Neufer, P Darrell; Cortright, Ronald N; Alberici, Luciane C

2017-02-01

Long-chain acyl-CoA synthetase 6 (ACSL6) mRNA is present in human and rat skeletal muscle, and is modulated by nutritional status: exercise and fasting decrease ACSL6 mRNA, whereas acute lipid ingestion increase its expression. ACSL6 genic inhibition in rat primary myotubes decreased lipid accumulation, as well as activated the higher mitochondrial oxidative capacity programme and fatty acid oxidation through the AMPK/PGC1-α pathway. ACSL6 overexpression in human primary myotubes increased phospholipid species and decreased oxidative metabolism. Long-chain acyl-CoA synthetases (ACSL 1 to 6) are key enzymes regulating the partitioning of acyl-CoA species toward different metabolic fates such as lipid synthesis or β-oxidation. Despite our understanding of ecotopic lipid accumulation in skeletal muscle being associated with metabolic diseases such as obesity and type II diabetes, the role of specific ACSL isoforms in lipid synthesis remains unclear. In the present study, we describe for the first time the presence of ACSL6 mRNA in human skeletal muscle and the role that ACSL6 plays in lipid synthesis in both rodent and human skeletal muscle. ACSL6 mRNA was observed to be up-regulated by acute high-fat meal ingestion in both rodents and humans. In rats, we also demonstrated that fasting and chronic aerobic training negatively modulated the ACSL6 mRNA and other genes of lipid synthesis. Similar results were obtained following ACSL6 knockdown in rat myotubes, which was associated with a decreased accumulation of TAGs and lipid droplets. Under the same knockdown condition, we further demonstrate an increase in fatty acid content, p-AMPK, mitochondrial content, mitochondrial respiratory rates and palmitate oxidation. These results were associated with increased PGC-1α, UCP2 and UCP3 mRNA and decreased reactive oxygen species production. In human myotubes, ACSL6 overexpression reduced palmitate oxidation and PGC-1α mRNA. In conclusion, ACSL6 drives acyl-CoA toward lipid

Identification of N-acyl-fumonisin B1 as new cytotoxic metabolites of fumonisin mycotoxins.

PubMed

Harrer, Henning; Laviad, Elad L; Humpf, Hans Ulrich; Futerman, Anthony H

2013-03-01

Fumonisins are mycotoxins produced by Fusarium species. The predominant derivative, fumonisin B1 (FB1), occurs in food and feed and is of health concern due to its hepatotoxic and carcinogenic effects. However, the role of FB1 metabolites on the mechanism of the toxicity, the inhibition of the ceramide synthesis, is unknown. The aim of this study was to identify new fumonisin metabolites and to evaluate their cytotoxic potential. MS, molecular biology, and in vitro enzyme assays were used to investigate fumonisin metabolism in mammalian cells overexpressing human ceramide synthase (CerS) genes. N-acyl-FB1 derivatives were detected as new metabolites in cultured cells at levels of up to 10 pmol/mg of protein. The N-acylation of FB1 and hydrolyzed FB1 was analyzed in several cell lines, including cells overexpressing CerS. The acyl-chain length of the N-acyl fumonisins depends on the CerS isoform acylating them. The N-acyl fumonisins are more cytotoxic than the parent fumonisin B1. The identification of N-acyl fumonisins with various acyl chain lengths together with the observed cytotoxicity of these compounds is a new aspect of fumonisin-related toxicity. Therefore, these new metabolites might play an important role in the mode of action of fumonisins. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Lanthanum Tricyanide-Catalyzed Acyl Silane-Ketone Benzoin Additions

PubMed Central

Tarr, James C.; Johnson, Jeffrey S.

2009-01-01

Lanthanum tricyanide efficiently catalyzes a benzoin-type coupling between acyl silanes and ketones. Yields range from moderate to excellent over a broad substrate scope encompassing aryl, alkyl, electron-rich, and sterically hindered ketones. PMID:19655731

Microbial synthesis of medium-chain chemicals from renewables.

PubMed

Sarria, Stephen; Kruyer, Nicholas S; Peralta-Yahya, Pamela

2017-12-01

Linear, medium-chain (C8-C12) hydrocarbons are important components of fuels as well as commodity and specialty chemicals. As industrial microbes do not contain pathways to produce medium-chain chemicals, approaches such as overexpression of endogenous enzymes or deletion of competing pathways are not available to the metabolic engineer; instead, fatty acid synthesis and reversed β-oxidation are manipulated to synthesize medium-chain chemical precursors. Even so, chain lengths remain difficult to control, which means that purification must be used to obtain the desired products, titers of which are typically low and rarely exceed milligrams per liter. By engineering the substrate specificity and activity of the pathway enzymes that generate the fatty acyl intermediates and chain-tailoring enzymes, researchers can boost the type and yield of medium-chain chemicals. Development of technologies to both manipulate chain-tailoring enzymes and to assay for products promises to enable the generation of g/L yields of medium-chain chemicals.

Synthesis, characterisation and physicochemical properties of hydrophobically modified inulin using long-chain fatty acyl chlorides.

PubMed

Han, Lingyu; Ratcliffe, I; Williams, P A

2017-12-15

A series of inulin derivatives were synthesized in aqueous solution using acyl chlorides with varying alkyl chain length (C10-C16). They were characterised using a number of techniques including MALDI TOF-MS, 1 H NMR and FTIR and their degree of substitution determined. The solution properties of the hydrophobically modified inulins were investigated using dye solubilisation and surface tension and it was confirmed that the molecules aggregated in solution above a critical concentration (critical aggregation concentration, CAC). The value of the CAC was found to be reasonably consistent between the different techniques and was shown to decrease with increasing hydrophobe chain length. It was found that the C10, C12 and C14 derivatives formed stable oil-in-water emulsions and the emulsion droplet size decreased with increasing alkyl chain length. The C16 derivative was not able to produce stable oil-in-water emulsions; however, it was able to form stable water-in-oil emulsions. The fact that the derivatives are able to form micellar-like aggregates and stabilise emulsions makes them suitable candidates for the encapsulation and delivery of active compounds with potential application in food, cosmetic, personal care and pharmaceutical formulations. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cellular and Molecular Responses of Dunaliella tertiolecta by Expression of a Plant Medium Chain Length Fatty Acid Specific Acyl-ACP Thioesterase

PubMed Central

Lin, Huixin; Shen, Hui; Lee, Yuan K.

2018-01-01

Metabolic engineering of microalgae to accumulate high levels of medium chain length fatty acids (MCFAs) has met with limited success. Traditional approaches employ single introduction of MCFA specific acyl-ACP thioesterases (TEs), but our current research in transgenic Dunaliella tertiolecta line has highlighted that, there is no single rate-limiting approach that can effectively increase MCFA levels. Here, we explore the accumulation of MCFAs in D. tertiolecta after transgenic expression of myristic acid biased TE (C14TE). We observe that the MCFA levels were negatively correlated to the fatty acid (FA) synthesis genes, ketoacyl-ACP synthase II (KASII), stearoyl-CoA-9-desaturase (Δ9D), and oleoyl-CoA-12-desaturase (Δ12D). To further examine the molecular mechanism of MCFA accumulation in microalgae, we investigate the transcriptomic dynamics of the MCFA producing strain of D. tertiolecta. At the transcript level, enhanced MCFA accumulation primarily involved up-regulation of photosynthetic genes and down-regulation of genes from central carbon metabolic processes, resulting in an overall decrease in carbon precursors for FA synthesis. We additionally observe that MCFA specific peroxisomal β-oxidation gene (ACX3) was greatly enhanced to prevent excessive build-up of unusual MCFA levels. Besides, long chain acyl-CoA synthetase gene (LACS) was down-regulated, likely in attempt to control fatty acyl supply flux to FA synthesis cycle. This article provides a spatial regulation model of unusual FA accumulation in microalgae and a platform for additional metabolic engineering targeting pathways from FA synthesis, FA transport, and peroxisomal β-oxidation to achieve microalgae oils with higher levels of MCFAs. PMID:29670594

Triheptanoin: long-term effects in the very long-chain acyl-CoA dehydrogenase-deficient mouse[S

PubMed Central

Tucci, Sara; Floegel, Ulrich; Beermann, Frauke; Behringer, Sidney; Spiekerkoetter, Ute

2017-01-01

A rather new approach in the treatment of long-chain fatty acid oxidation disorders is represented by triheptanoin, a triglyceride with three medium-odd-chain heptanoic acids (C7), due to its anaplerotic potential. We here investigate the effects of a 1-year triheptanoin-based diet on the clinical phenotype of very long-chain-acyl-CoA-dehydrogenase-deficient (VLCAD−/−) mice. The cardiac function was assessed in VLCAD−/− mice by in vivo MRI. Metabolic adaptations were identified by the expression of genes regulating energy metabolism and anaplerotic processes using real-time PCR, and the results were correlated with the measurement of the glycolytic enzymes pyruvate dehydrogenase and pyruvate kinase. Finally, the intrahepatic lipid accumulation and oxidative stress in response to the long-term triheptanoin diet were assessed. Triheptanoin was not able to prevent the development of systolic dysfunction in VLCAD−/− mice despite an upregulation of cardiac glucose oxidation. Strikingly, the anaplerotic effects of triheptanoin were restricted to the liver. Despite this, the hepatic lipic content was increased upon triheptanoin supplementation. Our data demonstrate that the concept of anaplerosis does not apply to all tissues equally. PMID:27884962

Ufd2p synthesizes branched ubiquitin chains to promote the degradation of substrates modified with atypical chains

PubMed Central

Liu, Chao; Liu, Weixiao; Ye, Yihong; Li, Wei

2017-01-01

Ubiquitination of a subset of proteins by ubiquitin chain elongation factors (E4), represented by Ufd2p in Saccharomyces cerevisiae, is a pivotal regulator for many biological processes. However, the mechanism of Ufd2p-mediated ubiquitination is largely unclear. Here, we show that Ufd2p catalyses K48-linked multi-monoubiquitination on K29-linked ubiquitin chains assembled by the ubiquitin ligase (Ufd4p), resulting in branched ubiquitin chains. This reaction depends on the interaction of K29-linked ubiquitin chains with two N-terminal loops of Ufd2p. Only following the addition of K48-linked ubiquitin to substrates modified with K29-linked ubiquitin chains, can the substrates be escorted to the proteasome for degradation. We demonstrate that this ubiquitin chain linkage switching reaction is essential for ERAD, oleic acid and acid pH resistance in yeast. Thus, our results suggest that Ufd2p functions by switching ubiquitin chain linkages to allow the degradation of proteins modified with a ubiquitin linkage, which is normally not targeted to the proteasome. PMID:28165462

Characterization of long-chain acyl-CoA synthetases which stimulate secretion of fatty acids in green algae Chlamydomonas reinhardtii.

PubMed

Jia, Bin; Song, Yanzi; Wu, Min; Lin, Baicheng; Xiao, Kang; Hu, Zhangli; Huang, Ying

2016-01-01

Microalgae biofuel has become the most promising renewable energy over the past few years. But limitations still exist because of its high cost. Although, efforts have been made in enhancement of lipid productivity, the major cost problem in harvesting and oil extraction is still intractable. Thus, the idea of fatty acids (FAs) secretion which can massively facilitate algae harvesting and oil extraction was investigated here. The cDNAs of two long-chain acyl-CoA synthetases (LACSs) genes were cloned from Chlamydomonas reinhardtii and named as cracs1 and cracs2. They showed different substrate adaptation in the yeast complementation experiments. Cracs2 could utilize FAs C12:0, C14:0, C16:0, C18:0, C16:1 and C18:1, while crac1 could only utilize substrate C14:0, C16:1 and C18:1. Knockdown of cracs1 and cracs2 in C. reinhardtii resulted in accumulation of intracellular lipids. The total intracellular lipids contents of transgenic algae q-15 (knockdown of cracs1) and p-13 (knockdown of cracs2) were 45 and 55 %, respectively higher than that of cc849. Furthermore, FAs secretion was discovered in both transgenic algae. Secreted FAs can reach 8.19 and 9.66 mg/10(9) cells in q-15 and p-13, respectively. These results demonstrated the possibility of FAs secretion by microalgae and may give a new strategy of low-cost oil extraction. According to our findings, we proposed that FAs secretion may also be achieved in other species besides Chlamydomonas reinhardtii by knocking-down cracs genes, which may promote the future industrial application of microalgae biofuels.

Compartmentalized acyl-CoA metabolism in skeletal muscle regulates systemic glucose homeostasis.

PubMed

Li, Lei O; Grevengoed, Trisha J; Paul, David S; Ilkayeva, Olga; Koves, Timothy R; Pascual, Florencia; Newgard, Christopher B; Muoio, Deborah M; Coleman, Rosalind A

2015-01-01

The impaired capacity of skeletal muscle to switch between the oxidation of fatty acid (FA) and glucose is linked to disordered metabolic homeostasis. To understand how muscle FA oxidation affects systemic glucose, we studied mice with a skeletal muscle-specific deficiency of long-chain acyl-CoA synthetase (ACSL)1. ACSL1 deficiency caused a 91% loss of ACSL-specific activity and a 60-85% decrease in muscle FA oxidation. Acsl1(M-/-) mice were more insulin sensitive, and, during an overnight fast, their respiratory exchange ratio was higher, indicating greater glucose use. During endurance exercise, Acsl1(M-/-) mice ran only 48% as far as controls. At the time that Acsl1(M-/-) mice were exhausted but control mice continued to run, liver and muscle glycogen and triacylglycerol stores were similar in both genotypes; however, plasma glucose concentrations in Acsl1(M-/-) mice were ∼40 mg/dL, whereas glucose concentrations in controls were ∼90 mg/dL. Excess use of glucose and the likely use of amino acids for fuel within muscle depleted glucose reserves and diminished substrate availability for hepatic gluconeogenesis. Surprisingly, the content of muscle acyl-CoA at exhaustion was markedly elevated, indicating that acyl-CoAs synthesized by other ACSL isoforms were not available for β-oxidation. This compartmentalization of acyl-CoAs resulted in both an excessive glucose requirement and severely compromised systemic glucose homeostasis. © 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

Activation of Exogenous Fatty Acids to Acyl-Acyl Carrier Protein Cannot Bypass FabI Inhibition in Neisseria*

PubMed Central

Yao, Jiangwei; Bruhn, David F.; Frank, Matthew W.; Lee, Richard E.; Rock, Charles O.

2016-01-01

Neisseria is a Gram-negative pathogen with phospholipids composed of straight chain saturated and monounsaturated fatty acids, the ability to incorporate exogenous fatty acids, and lipopolysaccharides that are not essential. The FabI inhibitor, AFN-1252, was deployed as a chemical biology tool to determine whether Neisseria can bypass the inhibition of fatty acid synthesis by incorporating exogenous fatty acids. Neisseria encodes a functional FabI that was potently inhibited by AFN-1252. AFN-1252 caused a dose-dependent inhibition of fatty acid synthesis in growing Neisseria, a delayed inhibition of growth phenotype, and minimal inhibition of DNA, RNA, and protein synthesis, showing that its mode of action is through inhibiting fatty acid synthesis. Isotopic fatty acid labeling experiments showed that Neisseria encodes the ability to incorporate exogenous fatty acids into its phospholipids by an acyl-acyl carrier protein-dependent pathway. However, AFN-1252 remained an effective antibacterial when Neisseria were supplemented with exogenous fatty acids. These results demonstrate that extracellular fatty acids are activated by an acyl-acyl carrier protein synthetase (AasN) and validate type II fatty acid synthesis (FabI) as a therapeutic target against Neisseria. PMID:26567338

Evolution of acyl-ACP-thioesterases and β-ketoacyl-ACP-synthases revealed by protein-protein interactions.

PubMed

Beld, Joris; Blatti, Jillian L; Behnke, Craig; Mendez, Michael; Burkart, Michael D

2014-08-01

The fatty acid synthase (FAS) is a conserved primary metabolic enzyme complex capable of tolerating cross-species engineering of domains for the development of modified and overproduced fatty acids. In eukaryotes, acyl-acyl carrier protein thioesterases (TEs) off-load mature cargo from the acyl carrier protein (ACP), and plants have developed TEs for short/medium-chain fatty acids. We showed that engineering plant TEs into the green microalga Chlamydomonas reinhardtii does not result in the predicted shift in fatty acid profile. Since fatty acid biosynthesis relies on substrate recognition and protein-protein interactions between the ACP and its partner enzymes, we hypothesized that plant TEs and algal ACP do not functionally interact. Phylogenetic analysis revealed major evolutionary differences between FAS enzymes, including TEs and ketoacyl synthases (KSs), in which the former is present only in some species, whereas the latter is present in all, and has a common ancestor. In line with these results, TEs appeared to be selective towards their ACP partners whereas KSs showed promiscuous behavior across bacterial, plant and algal species. Based on phylogenetic analyses, in silico docking, in vitro mechanistic crosslinking and in vivo algal engineering, we propose that phylogeny can predict effective interactions between ACPs and partner enzymes.

Evolution of acyl-ACP-thioesterases and β-ketoacyl-ACP-synthases revealed by protein-protein interactions

PubMed Central

Beld, Joris; Blatti, Jillian L.; Behnke, Craig; Mendez, Michael; Burkart, Michael D.

2014-01-01

The fatty acid synthase (FAS) is a conserved primary metabolic enzyme complex capable of tolerating cross-species engineering of domains for the development of modified and overproduced fatty acids. In eukaryotes, acyl-acyl carrier protein thioesterases (TEs) off-load mature cargo from the acyl carrier protein (ACP), and plants have developed TEs for short/medium-chain fatty acids. We showed that engineering plant TEs into the green microalga Chlamydomonas reinhardtii does not result in the predicted shift in fatty acid profile. Since fatty acid biosynthesis relies on substrate recognition and protein-protein interactions between the ACP and its partner enzymes, we hypothesized that plant TEs and algal ACP do not functionally interact. Phylogenetic analysis revealed major evolutionary differences between FAS enzymes, including TEs and ketoacyl synthases (KSs), in which the former is present only in some species, whereas the latter is present in all, and has a common ancestor. In line with these results, TEs appeared to be selective towards their ACP partners whereas KSs showed promiscuous behavior across bacterial, plant and algal species. Based on phylogenetic analyses, in silico docking, in vitro mechanistic crosslinking and in vivo algal engineering, we propose that phylogeny can predict effective interactions between ACPs and partner enzymes. PMID:25110394

Suzuki-miyaura cross-coupling in acylation reactions, scope and recent developments.

PubMed

Blangetti, Marco; Rosso, Heléna; Prandi, Cristina; Deagostino, Annamaria; Venturello, Paolo

2013-01-17

Since the first report and due to its handiness and wide scope, the Suzuki-Miyaura (SM) cross coupling reaction has become a routine methodology in many laboratories worldwide. With respect to other common transition metal catalyzed cross couplings, the SM reaction has been so far less exploited as a tool to introduce an acyl function into a specific substrate. In this review, the various approaches found in the literature will be considered, starting from the direct SM acylative coupling to the recent developments of cross coupling between boronates and acyl chlorides or anhydrides. Special attention will be dedicated to the use of masked acyl boronates, alkoxy styryl and alkoxy dienyl boronates as coupling partners. A final section will be then focused on the acyl SM reaction as key synthetic step in the framework of natural products synthesis.

Impact of fatty acyl composition and quantity of triglycerides on bioaccessibility of dietary carotenoids.

PubMed

Huo, Tianyao; Ferruzzi, Mario G; Schwartz, Steven J; Failla, Mark L

2007-10-31

A carotenoid-rich salad meal with varying amounts and types of triglycerides (TG) was digested using simulated gastric and small intestinal conditions. Xanthophylls (lutein and zeaxanthin) and carotenes (alpha-carotene, beta-carotene, and lycopene) in chyme and micelle fraction were quantified to determine digestive stability and efficiency of micellarization (bioaccessibility). Micellarization of lutein (+zeaxanthin) exceeded that of alpha- and beta-carotenes, which was greater than that of lycopene for all test conditions. Micellarization of carotenes, but not lutein (+zeaxanthin), was enhanced (P < 0.05) by addition of TG (2.5% v/w) to the meal and was dependent on fatty acyl chain length in structured TG (c18:1 > c8:0 > c4:0). The degree of unsaturation of c18 fatty acyl chains in TG added to the salad purée did not significantly alter the efficiency of micellarization of carotenoids. Relatively low amounts of triolein and canola oil (0.5-1%) were required for maximum micellarization of carotenes, but more oil (approximately 2.5%) was required when TG with medium chain saturated fatty acyl groups (e.g., trioctanoin and coconut oil) was added to the salad. Uptake of lutein and beta-carotene by Caco-2 cells also was examined by exposing cells to micelles generated during the simulated digestion of salad purée with either triolein or trioctanoin. Cell accumulation of beta-carotene was independent of fatty acyl composition of micelles, whereas lutein uptake was slightly, but significantly, increased from samples with digested triolein compared to trioctanoin. The results show that the in vitro transfer of alpha-carotene, beta-carotene, and lycopene from chyme to mixed micelles during digestion requires minimal (0.5-1%) lipid content in the meal and is affected by the length of fatty acyl chains but not the degree of unsaturation in TG. In contrast, fatty acyl chain length has limited if any impact on carotenoid uptake by small intestinal epithelial cells. These

Physical characterisation of high amylose maize starch and acylated high amylose maize starches.

PubMed

Lim, Ya-Mei; Hoobin, Pamela; Ying, DanYang; Burgar, Iko; Gooley, Paul R; Augustin, Mary Ann

2015-03-06

The particle size, water sorption properties and molecular mobility of high amylose maize starch (HAMS) and high amylose maize starch acylated with acetate (HAMSA), propionate (HAMSP) and butyrate (HAMSB) were investigated. Acylation increased the mean particle size (D(4,3)) and lowered the specific gravity (G) of the starch granules with an inverse relationship between the length of the fatty acid chain and particle size. Acylation of HAMS with fatty acids lowered the monolayer moisture content with the trend being HAMSBchain. Measurement of molecular mobility of the starch granules by NMR spectroscopy with Carr-Purcell-Meiboom-Gill (CMPG) experiments showed that T2 long was reduced in acylated starches and that drying and storage of the starch granules further reduced T2 long. Analysis of the Free Induction Decay (FID) focussing on the short components of T2 (correlated to the solid matrix), indicated that drying and subsequent storage resulted in alterations of starch at 0.33a(w) and that these changes were reduced with acylation. In vitro enzymatic digestibility of heated starch dispersions by bacterial α-amylase was increased by acylation (HAMSchain. Digestibility was enhanced with an increase in particle size, or decrease in G, and inversely proportional to the total T2 signal. It is suggested that both external surface area and an internal network of pores and channels collectively influence the digestibility of starch. Copyright © 2014 Elsevier Ltd. All rights reserved.

Photoaffinity Labeling of Developing Jojoba Seed Microsomal Membranes with a Photoreactive Analog of Acyl-Coenzyme A (Acyl-CoA) (Identification of a Putative Acyl-CoA:Fatty Alcohol Acyltransferase.

PubMed Central

Shockey, J. M.; Rajasekharan, R.; Kemp, J. D.

1995-01-01

Jojoba (Simmondsia chinensis, Link) is the only plant known that synthesizes liquid wax. The final step in liquid wax biosynthesis is catalyzed by an integral membrane enzyme, fatty acyl-coenzyme A (CoA):fatty alcohol acyltransferase, which transfers an acyl chain from acyl-CoA to a fatty alcohol to form the wax ester. To purify the acyltransferase, we have labeled the enzyme with a radioiodinated, photoreactive analog of acyl-CoA, 12-[N-(4-azidosalicyl)amino] dodecanoyl-CoA (ASD-CoA). This molecule acts as an inhibitor of acyltransferase activity in the dark and as an irreversible inhibitor upon exposure to ultraviolet light. Oleoyl-CoA protects enzymatic activity in a concentration-dependent manner. Photolysis of microsomal membranes with labeled ASD-CoA resulted in strong labeling of two polypeptides of 57 and 52 kD. Increasing concentrations of oleoyl-CoA reduced the labeling of the 57-kD polypeptide dramatically, whereas the labeling of the 52-kD polypeptide was much less responsive to oleoyl-CoA. Also, unlike the other polypeptide, the labeling of the 57-kD polypeptide was enhanced considerably when photolyzed in the presence of dodecanol. These results suggest that a 57-kD polypeptide from jojoba microsomes may be the acyl-CoA:fatty alcohol acyltransferase. PMID:12228351

Properties of Acetate Kinase Isozymes and a Branched-Chain Fatty Acid Kinase from a Spirochete

PubMed Central

Harwood, Caroline S.; Canale-Parola, Ercole

1982-01-01

Spirochete MA-2, which is anaerobic, ferments glucose, forming acetate as a major product. The spirochete also ferments (but does not utilize as growth substrates) small amounts of l-leucine, l-isoleucine, and l-valine, forming the branched-chain fatty acids isovalerate, 2-methylbutyrate, and isobutyrate, respectively, as end products. Energy generated through the fermentation of these amino acids is utilized to prolong cell survival under conditions of growth substrate starvation. A branched-chain fatty acid kinase and two acetate kinase isozymes were resolved from spirochete MA-2 cell extracts. Kinase activity was followed by measuring the formation of acyl phosphate from fatty acid and ATP. The branched-chain fatty acid kinase was active with isobutyrate, 2-methylbutyrate, isovalerate, butyrate, valerate, or propionate as a substrate but not with acetate as a substrate. The acetate kinase isozymes were active with acetate and propionate as substrates but not with longer-chain fatty acids as substrates. The acetate kinase isozymes and the branched-chain fatty acid kinase differed in nucleoside triphosphate and cation specificities. Each acetate kinase isozyme had an apparent molecular weight of approximately 125,000, whereas the branched-chain fatty acid kinase had a molecular weight of approximately 76,000. These results show that spirochete MA-2 synthesizes a branched-chain fatty acid kinase specific for leucine, isoleucine, and valine fermentation. It is likely that a phosphate branched-chain amino acids is also synthesized by spirochete MA-2. Thus, in spirochete MA-2, physiological mechanisms have evolved which serve specifically to generate maintenance energy from branched-chain amino acids. PMID:6288660

Characterization of the transacylase activity of rat liver 60-kDa lysophospholipase-transacylase. Acyl transfer from the sn-2 to the sn-1 position.

PubMed

Sugimoto, H; Yamashita, S

1999-05-18

Rat liver 60-kDa lysophospholipase-transacylase catalyzes not only the hydrolysis of 1-acyl-sn-glycero-3-phosphocholine, but also the transfer of its acyl chain to a second molecule of 1-acyl-sn-glycero-3-phosphocholine to form phosphatidylcholine (H. Sugimoto, S. Yamashita, J. Biol. Chem. 269 (1994) 6252-6258). Here we report the detailed characterization of the transacylase activity of the enzyme. The enzyme mediated three types of acyl transfer between donor and acceptor lipids, transferring acyl residues from: (1) the sn-1 to -1(3); (2) sn-1 to -2; and (3) sn-2 to -1 positions. In the sn-1 to -1(3) transfer, the sn-1 acyl residue of 1-acyl-sn-glycero-3-phosphocholine was transferred to the sn-1(3) positions of glycerol and 2-acyl-sn-glycerol, producing 1(3)-acyl-sn-glycerol and 1,2-diacyl-sn-glycerol, respectively. In the sn-1 to -2 transfer, the sn-1 acyl residue of 1-acyl-sn-glycero-3-phosphocholine was transferred to not only the sn-2 positions of 1-acyl-sn-glycero-3-phosphocholine, but also 1-acyl-sn-glycero-3-phosphoethanolamine, producing phosphatidylcholine and phosphatidylethanolamine, respectively. 1-Acyl-sn-glycero-3-phospho-myo-inositol and 1-acyl-sn-glycero-3-phosphoserine were much less effectively transacylated by the enzyme. In the sn-2 to -1 transfer, the sn-2 acyl residue of 2-acyl-sn-glycero-3-phosphocholine was transferred to the sn-1 position of 2-acyl-sn-glycero-3-phosphocholine and 2-acyl-sn-glycero-3-phosphoethanolamine, producing phosphatidylcholine and phosphatidylethanolamine, respectively. Consistently, the enzyme hydrolyzed the sn-2 acyl residue from 2-acyl-sn-glycero-3-phosphocholine. By the sn-2 to -1 transfer activity, arachidonic acid was transferred from the sn-2 position of donor lipids to the sn-1 position of acceptor lipids, thus producing 1-arachidonoyl phosphatidylcholine. When 2-arachidonoyl-sn-glycero-3-phosphocholine was used as the sole substrate, diarachidonoyl phosphatidylcholine was synthesized at a rate of 0

Characterization of acyl-coenzyme A:diacylglycerol acyltransferase (DGAT) enzyme of human small intestine.

PubMed

Hiramine, Yasushi; Tanabe, Toshizumi

2011-06-01

Acyl-coenzyme A:diacylglycerol acyltransferase (DGAT) enzyme plays a significant role in dietary triacylglycerol (TAG) absorption in the small intestine. However, the characteristics of human intestinal DGAT enzyme have not been examined in detail. The aim of our study was to characterize the human intestinal DGAT enzyme by examining acyl-CoA specificity, temperature dependency, and selectivity for 1,2-diacylglycerol (DAG) or 1,3-DAG. We detected DGAT activity of human intestinal microsome and found that the acyl-CoA specificity and temperature dependency of intestinal DGAT coincided with those of recombinant human DGAT1. To elucidate the selectivity of human intestinal DGAT to 1,2-DAG or 1,3-DAG, we conducted acyl-coenzyme A:monoacylglycerol acyltransferase assays using 1- or 2-monoacylglycerol (MAG) as substrates. When 2-MAG was used as acyl acceptor, both 1,2-DAG and TAG were generated; however, when 1-MAG was used, 1,3-DAG was predominantly observed and little TAG was detected. These findings suggest that human small intestinal DGAT, which is mainly encoded by DGAT1, utilizes 1,2-DAG as the substrate to form TAG. This study will contribute to understand the lipid absorption profile in the small intestine.

Parental Experiences of Raising a Child With Medium Chain Acyl-CoA Dehydrogenase Deficiency.

PubMed

Piercy, Hilary; Machaczek, Katarzyna; Ali, Parveen; Yap, Sufin

2017-01-01

Newborn screening enabling early diagnosis of medium chain acyl-CoA dehydrogenase deficiency (MCADD) has dramatically improved health outcomes in children with MCADD. Achieving those outcomes depends on effective management by parents. Understanding parental management strategies and associated anxieties and concerns is needed to inform provision of appropriate care and support. Semistructured interviews were conducted with a purposive sample of parents of children aged 2 to 12 years. Thematic analysis identified two main themes. Managing dietary intake examined how parents managed day-to-day dietary intake to ensure adequate intake and protection of safe fasting intervals. Managing and preventing illness events explored parental experiences of managing illness events and their approach to preventing these events. Management strategies were characterized by caution and vigilance and influenced by a lack of confidence in others to manage the condition. The study identifies the need for increased awareness of the condition, particularly in relation to emergency treatment.

Deficiency of a Retinal Dystrophy Protein, Acyl-CoA Binding Domain-containing 5 (ACBD5), Impairs Peroxisomal β-Oxidation of Very-long-chain Fatty Acids*

PubMed Central

Yagita, Yuichi; Shinohara, Kyoko; Abe, Yuichi; Nakagawa, Keiko; Al-Owain, Mohammed; Alkuraya, Fowzan S.; Fujiki, Yukio

2017-01-01

Acyl-CoA binding domain-containing 5 (ACBD5) is a peroxisomal protein that carries an acyl-CoA binding domain (ACBD) at its N-terminal region. The recent identification of a mutation in the ACBD5 gene in patients with a syndromic form of retinal dystrophy highlights the physiological importance of ACBD5 in humans. However, the underlying pathogenic mechanisms and the precise function of ACBD5 remain unclear. We herein report that ACBD5 is a peroxisomal tail-anchored membrane protein exposing its ACBD to the cytosol. Using patient-derived fibroblasts and ACBD5 knock-out HeLa cells generated via genome editing, we demonstrate that ACBD5 deficiency causes a moderate but significant defect in peroxisomal β-oxidation of very-long-chain fatty acids (VLCFAs) and elevates the level of cellular phospholipids containing VLCFAs without affecting peroxisome biogenesis, including the import of membrane and matrix proteins. Both the N-terminal ACBD and peroxisomal localization of ACBD5 are prerequisite for efficient VLCFA β-oxidation in peroxisomes. Furthermore, ACBD5 preferentially binds very-long-chain fatty acyl-CoAs (VLC-CoAs). Together, these results suggest a direct role of ACBD5 in peroxisomal VLCFA β-oxidation. Based on our findings, we propose that ACBD5 captures VLC-CoAs on the cytosolic side of the peroxisomal membrane so that the transport of VLC-CoAs into peroxisomes and subsequent β-oxidation thereof can proceed efficiently. Our study reclassifies ACBD5-related phenotype as a novel peroxisomal disorder. PMID:27899449

Laser desorption/ionization mass spectrometry of lipids using etched silver substrates.

PubMed

Schnapp, Andreas; Niehoff, Ann-Christin; Koch, Annika; Dreisewerd, Klaus

2016-07-15

Silver-assisted laser desorption/ionization mass spectrometry can be used for the analysis of small molecules. For example, adduct formation with silver cations enables the molecular analysis of long-chain hydrocarbons, which are difficult to ionize via conventional matrix-assisted laser desorption ionization (MALDI). Here we used highly porous silver foils, produced by etching with nitric acid, as sample substrates for LDI mass spectrometry. As model system for the analysis of complex lipid mixtures, cuticular extracts of fruit flies (Drosophila melanogaster) and worker bees (Apis mellifera) were investigated. The mass spectra obtained by spotting extract onto the etched silver substrates demonstrate the sensitive detection of numerous lipid classes such as long-chain saturated and unsaturated hydrocarbons, fatty acyl alcohols, wax esters, and triacylglycerols. MS imaging of cuticular surfaces with a lateral resolution of a few tens of micrometers became possible after blotting, i.e., after transferring lipids by physical contact with the substrate. The examples of pheromone-producing male hindwings of the squinting bush brown butterfly (Bicyclus anynana) and a fingermark are shown. Because the substrates are also easy to produce, they provide a viable alternative to colloidal silver nanoparticles and other so far described silver substrates. Copyright © 2016 Elsevier Inc. All rights reserved.

Plasma fatty acyl-carnitines during 8 weeks of overfeeding: relation to diet energy expenditure and body composition: the PROOF study.

PubMed

Bray, George A; Redman, Leanne M; de Jonge, Lilian; Rood, Jennifer; Sutton, Elizabeth F; Smith, Steven R

2018-06-01

Overfeeding is a strategy for evaluating the effects of excess energy intake. In this secondary analysis we tested the possibility that different levels of dietary protein might differentially modify the response of fatty acyl-carnitines to overfeeding. Twenty-three healthy adult men and women were overfed by 40% for 8 weeks while in-patients with diets containing 5% (LPD), 15% (NPD) or 25% (HPD) protein. Plasma fatty acyl-carnitines were measured by gas chromatography/mass spectrometry (GC/MS) at baseline and after 8 weeks of overfeeding. Measurements included: body composition by DXA, energy expenditure by ventilated hood and doubly-labeled water, fat cell size from subcutaneous fat biopsies, and fat distribution by CT scan. Analysis was done on 5 groups of fatty acyl-carnitines identified by principal components analysis and 6 individual short-chain fatty acyl carnitines. Higher protein intake was associated with significantly lower 8 week levels of medium chain fatty acids and C2, C4-OH and C 6:1, but higher values of C3 and C5:1 acyl-carnitines derived from essential amino acids. In contrast energy and fat intake were only weakly related to changes in fatty acyl-carnitines. A decease or smaller rise in 8 week medium chain acyl-carnitines was associated with an increase in sleeping energy expenditure (P = 0.0004), and fat free mass (P < 0.0001) and a decrease in free fatty acid concentrations (FFA) (P = 0.0067). In contrast changes in short-chain fatty acyl-carnitines were related to changes in resting energy expenditure (P = 0.0026), and fat free mass (P = 0.0007), and C4-OH was positively related to FFA (P = 0006). Protein intake was the major factor influencing changes in fatty acyl carnitines during overfeeding with higher values of most acyl-fatty acids on the low protein diet. The association of dietary protein and fat intake may explain the changes in energy expenditure and metabolic variables resulting in the observed

ECERIFERUM2-LIKE proteins have unique biochemical and physiological functions in very-long-chain fatty acid elongation.

PubMed

Haslam, Tegan M; Haslam, Richard; Thoraval, Didier; Pascal, Stéphanie; Delude, Camille; Domergue, Frédéric; Fernández, Aurora Mañas; Beaudoin, Frédéric; Napier, Johnathan A; Kunst, Ljerka; Joubès, Jérôme

2015-03-01

The extension of very-long-chain fatty acids (VLCFAs) for the synthesis of specialized apoplastic lipids requires unique biochemical machinery. Condensing enzymes catalyze the first reaction in fatty acid elongation and determine the chain length of fatty acids accepted and produced by the fatty acid elongation complex. Although necessary for the elongation of all VLCFAs, known condensing enzymes cannot efficiently synthesize VLCFAs longer than 28 carbons, despite the prevalence of C28 to C34 acyl lipids in cuticular wax and the pollen coat. The eceriferum2 (cer2) mutant of Arabidopsis (Arabidopsis thaliana) was previously shown to have a specific deficiency in cuticular waxes longer than 28 carbons, and heterologous expression of CER2 in yeast (Saccharomyces cerevisiae) demonstrated that it can modify the acyl chain length produced by a condensing enzyme from 28 to 30 carbon atoms. Here, we report the physiological functions and biochemical specificities of the CER2 homologs CER2-LIKE1 and CER2-LIKE2 by mutant analysis and heterologous expression in yeast. We demonstrate that all three CER2-LIKEs function with the same small subset of condensing enzymes, and that they have different effects on the substrate specificity of the same condensing enzyme. Finally, we show that the changes in acyl chain length caused by each CER2-LIKE protein are of substantial importance for cuticle formation and pollen coat function. © 2015 American Society of Plant Biologists. All Rights Reserved.

Synthesis of medium-chain length capsinoids from coconut oil catalyzed by Candida rugosa lipases.

PubMed

Trbojević Ivić, Jovana; Milosavić, Nenad; Dimitrijević, Aleksandra; Gavrović Jankulović, Marija; Bezbradica, Dejan; Kolarski, Dušan; Veličković, Dušan

2017-03-01

A commercial preparation of Candida rugosa lipases (CRL) was tested for the production of capsinoids by esterification of vanillyl alcohol (VA) with free fatty acids (FA) and coconut oil (CO) as acyl donors. Screening of FA chain length indicated that C8-C12 FA (the most common FA found in CO triglycerides) are the best acyl-donors, yielding 80-85% of their specific capsinoids. Hence, when CO, which is rich in these FA, was used as the substrate, a mixture of capsinoids (vanillyl caprylate, vanillyl decanoate and vanillyl laurate) was obtained. The findings presented here suggest that our experimental method can be applied for the enrichment of CO with capsinoids, thus giving it additional health promoting properties. Copyright © 2016 Elsevier Ltd. All rights reserved.

Commelinid Monocotyledon Lignins Are Acylated by p-Coumarate.

PubMed

Karlen, Steven D; Free, Heather C A; Padmakshan, Dharshana; Smith, Bronwen G; Ralph, John; Harris, Philip J

2018-06-01

Commelinid monocotyledons are a monophyletic clade differentiated from other monocotyledons by the presence of cell wall-bound ferulate and p -coumarate. The Poaceae, or grass family, is a member of this group, and most of the p -coumarate in the cell walls of this family acylates lignin. Here, we isolated and examined lignified cell wall preparations from 10 species of commelinid monocotyledons from nine families other than Poaceae, including species from all four commelinid monocotyledon orders (Poales, Zingiberales, Commelinales, and Arecales). We showed that, as in the Poaceae, lignin-linked p -coumarate occurs exclusively on the hydroxyl group on the γ-carbon of lignin unit side chains, mostly on syringyl units. Although the mechanism of acylation has not been studied directly in these species, it is likely to be similar to that in the Poaceae and involve BAHD acyl-coenzyme A:monolignol transferases. © 2018 American Society of Plant Biologists. All rights reserved.

Acyl Coenzyme A Thioesterase 7 Regulates Neuronal Fatty Acid Metabolism To Prevent Neurotoxicity

PubMed Central

Ellis, Jessica M.; Wong, G. William

2013-01-01

Numerous neurological diseases are associated with dysregulated lipid metabolism; however, the basic metabolic control of fatty acid metabolism in neurons remains enigmatic. Here we have shown that neurons have abundant expression and activity of the long-chain cytoplasmic acyl coenzyme A (acyl-CoA) thioesterase 7 (ACOT7) to regulate lipid retention and metabolism. Unbiased and targeted metabolomic analysis of fasted mice with a conditional knockout of ACOT7 in the nervous system, Acot7N−/−, revealed increased fatty acid flux into multiple long-chain acyl-CoA-dependent pathways. The alterations in brain fatty acid metabolism were concomitant with a loss of lean mass, hypermetabolism, hepatic steatosis, dyslipidemia, and behavioral hyperexcitability in Acot7N−/− mice. These failures in adaptive energy metabolism are common in neurodegenerative diseases. In agreement, Acot7N−/− mice exhibit neurological dysfunction and neurodegeneration. These data show that ACOT7 counterregulates fatty acid metabolism in neurons and protects against neurotoxicity. PMID:23459938

Acyl coenzyme A thioesterase 7 regulates neuronal fatty acid metabolism to prevent neurotoxicity.

PubMed

Ellis, Jessica M; Wong, G William; Wolfgang, Michael J

2013-05-01

Numerous neurological diseases are associated with dysregulated lipid metabolism; however, the basic metabolic control of fatty acid metabolism in neurons remains enigmatic. Here we have shown that neurons have abundant expression and activity of the long-chain cytoplasmic acyl coenzyme A (acyl-CoA) thioesterase 7 (ACOT7) to regulate lipid retention and metabolism. Unbiased and targeted metabolomic analysis of fasted mice with a conditional knockout of ACOT7 in the nervous system, Acot7(N-/-), revealed increased fatty acid flux into multiple long-chain acyl-CoA-dependent pathways. The alterations in brain fatty acid metabolism were concomitant with a loss of lean mass, hypermetabolism, hepatic steatosis, dyslipidemia, and behavioral hyperexcitability in Acot7(N-/-) mice. These failures in adaptive energy metabolism are common in neurodegenerative diseases. In agreement, Acot7(N-/-) mice exhibit neurological dysfunction and neurodegeneration. These data show that ACOT7 counterregulates fatty acid metabolism in neurons and protects against neurotoxicity.

Differences in substrate specificity of V. cholerae FabH enzymes suggest new approaches for the development of novel antibiotics and biofuels.

PubMed

Hou, Jing; Zheng, Heping; Tzou, Wen-Shyong; Cooper, David R; Chruszcz, Maksymilian; Chordia, Mahendra D; Kwon, Keehwan; Grabowski, Marek; Minor, Wladek

2018-06-19

Vibrio cholerae, the causative pathogen of the life-threatening infection cholera, encodes two copies of β-ketoacyl-ACP synthase III (vcFabH1 and vcFabH2). vcFabH1 and vcFabH2 are pathogenic proteins associated with fatty acid synthesis, lipid metabolism, and potential applications in biofuel production. Our biochemical assays characterize vcFabH1 as exhibiting specificity for acetyl-CoA and CoA thioesters with short acyl chains, similar to that observed for FabH homologs found in most Gram-negative bacteria. vcFabH2 prefers medium chain-length acyl-CoA thioesters, particularly octanoyl-CoA, which is a pattern of specificity rarely seen in bacteria. Structural characterization of one vcFabH and six vcFabH2 structures determined in either apo-form or in complex with acetyl-CoA/octanoyl-CoA indicate that the substrate binding pockets of vcFabH1 and vcFabH2 are of different sizes, accounting for variations in substrate chain-length specificity. An unusual and unique feature of vcFabH2 is its C-terminal fragment that interacts with both the substrate-entrance loop and the dimer interface of the enzyme. Our discovery of the pattern of substrate specificity of both vcFabH1 and vcFabH2 can potentially aid the development of novel antibacterial agents against V. cholerae. Additionally, the distinctive substrate preference of FabH2 in V. cholerae and related facultative anaerobes conceivably make it an attractive component of genetically engineered bacteria used for commercial biofuel production. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Mechanisms of chain adsorption on porous substrates and critical conditions of polymer chromatography.

PubMed

Cimino, Richard T; Rasmussen, Christopher J; Brun, Yefim; Neimark, Alexander V

2016-11-01

Polymer adsorption is a ubiquitous phenomenon with numerous technological and healthcare applications. The mechanisms of polymer adsorption on surfaces and in pores are complex owing to a competition between various entropic and enthalpic factors. Due to adsorption of monomers to the surface, the chain gains in enthalpy yet loses in entropy because of confining effects. This competition leads to the existence of critical conditions of adsorption when enthalpy gain and entropy loss are in balance. The critical conditions are controlled by the confining geometry and effective adsorption energy, which depends on the solvent composition and temperature. This phenomenon has important implications in polymer chromatography, since the retention at the critical point of adsorption (CPA) is chain length independent. However, the mechanisms of polymer adsorption in pores are poorly understood and there is an ongoing discussion in the theoretical literature about the very existence of CPA for polymer adsorption on porous substrates. In this work, we examine the mechanisms of chain adsorption on a model porous substrate using Monte Carlo (MC) simulations. We distinguish three adsorption mechanisms depending on the chain location: on external surface, completely confined in pores, and also partially confined in pores in so-called "flower" conformations. The free energies of different conformations of adsorbed chains are calculated by the incremental gauge cell MC method that allows one to determine the partition coefficient as a function of the adsorption potential, pore size, and chain length. We confirm the existence of the CPA for chain length independent separation on porous substrates, which is explained by the dominant contributions of the chain adsorption at the external surface, in particular in flower conformations. Moreover, we show that the critical conditions for porous and nonporous substrates are identical and depend only on the surface chemistry. The theoretical

Prolonged Fasting Identifies Heat Shock Protein 10 as a Sirtuin 3 Substrate

PubMed Central

Lu, Zhongping; Chen, Yong; Aponte, Angel M.; Battaglia, Valentina; Gucek, Marjan; Sack, Michael N.

2015-01-01

Although Sirtuin 3 (SIRT3), a mitochondrially enriched deacetylase and activator of fat oxidation, is down-regulated in response to high fat feeding, the rate of fatty acid oxidation and mitochondrial protein acetylation are invariably enhanced in this dietary milieu. These paradoxical data implicate that additional acetylation modification-dependent levels of regulation may be operational under nutrient excess conditions. Because the heat shock protein (Hsp) Hsp10-Hsp60 chaperone complex mediates folding of the fatty acid oxidation enzyme medium-chain acyl-CoA dehydrogenase, we tested whether acetylation-dependent mitochondrial protein folding contributes to this regulatory discrepancy. We demonstrate that Hsp10 is a functional SIRT3 substrate and that, in response to prolonged fasting, SIRT3 levels modulate mitochondrial protein folding. Acetyl mutagenesis of Hsp10 lysine 56 alters Hsp10-Hsp60 binding, conformation, and protein folding. Consistent with Hsp10-Hsp60 regulation of fatty acid oxidation enzyme integrity, medium-chain acyl-CoA dehydrogenase activity and fat oxidation are elevated by Hsp10 acetylation. These data identify acetyl modification of Hsp10 as a nutrient-sensing regulatory node controlling mitochondrial protein folding and metabolic function. PMID:25505263

New parasite inhibitors encompassing novel conformationally-locked 5'-acyl sulfamoyl adenosines.

PubMed

Dixit, Shailesh S; Upadhayaya, Ram Shankar; Chattopadhyaya, Jyoti

2012-08-14

We describe the design, synthesis and biological evaluation of conformationally-locked 5'-acyl sulfamoyl adenosine derivatives as new parasitic inhibitors against Trypanosoma and Leishmania. The conformationally-locked (3'-endo, North-type) nucleosides have been synthesized by covalently attaching a 4'-CH(2)-O-2' bridge () across C2'-C4' of adenosine in order to reduce the conformational flexibility of the pentose ring. This is designed to decrease the entropic penalty for complex formation with the target protein, which may improve free-energy of stabilization of the complex leading to improved potency. Conformationally-locked 5'-acyl sulfamoyl adenosine derivatives (16-22) were tested against parasitic protozoans for the first time in this work, and showed potent inhibition of Trypanosoma cruzi, Trypanosoma brucei, Trypanosoma rhodesiense and Leishmania infantum with IC(50) = 0.25-0.51 μM. In particular, the potent 5'-pentanyl acyl sulfamoyl adenosine derivative 17 (IC(50) = 0.25 μM) against intracellular L. infantum amastigotes and Trypanosoma subspecies is interesting in view of its almost insignificant cytotoxicity in murine macrophage host cells (CC(50) >4 μM) and in diploid human fibroblasts MRC-5 cell lines (CC(50) 4 μM). This work also suggests that variable alkyl chain length of the acyl group on the acylsulfamoyl side chain at 5' can modulate the toxicity of 5'-O-sulfamoylnucleoside analogues. This conformationally-locked sulfamoyl adenosine scaffold presents some interesting possibilities for further drug design and lead optimization.

ECERIFERUM2-LIKE Proteins Have Unique Biochemical and Physiological Functions in Very-Long-Chain Fatty Acid Elongation1[OPEN

PubMed Central

Haslam, Tegan M.; Haslam, Richard; Thoraval, Didier; Pascal, Stéphanie; Delude, Camille; Domergue, Frédéric; Fernández, Aurora Mañas; Beaudoin, Frédéric; Napier, Johnathan A.; Kunst, Ljerka; Joubès, Jérôme

2015-01-01

The extension of very-long-chain fatty acids (VLCFAs) for the synthesis of specialized apoplastic lipids requires unique biochemical machinery. Condensing enzymes catalyze the first reaction in fatty acid elongation and determine the chain length of fatty acids accepted and produced by the fatty acid elongation complex. Although necessary for the elongation of all VLCFAs, known condensing enzymes cannot efficiently synthesize VLCFAs longer than 28 carbons, despite the prevalence of C28 to C34 acyl lipids in cuticular wax and the pollen coat. The eceriferum2 (cer2) mutant of Arabidopsis (Arabidopsis thaliana) was previously shown to have a specific deficiency in cuticular waxes longer than 28 carbons, and heterologous expression of CER2 in yeast (Saccharomyces cerevisiae) demonstrated that it can modify the acyl chain length produced by a condensing enzyme from 28 to 30 carbon atoms. Here, we report the physiological functions and biochemical specificities of the CER2 homologs CER2-LIKE1 and CER2-LIKE2 by mutant analysis and heterologous expression in yeast. We demonstrate that all three CER2-LIKEs function with the same small subset of condensing enzymes, and that they have different effects on the substrate specificity of the same condensing enzyme. Finally, we show that the changes in acyl chain length caused by each CER2-LIKE protein are of substantial importance for cuticle formation and pollen coat function. PMID:25596184

SUMO chain formation relies on the amino-terminal region of SUMO-conjugating enzyme and has dedicated substrates in plants

PubMed Central

Tomanov, Konstantin; Nehlin, Lilian; Ziba, Ionida

2018-01-01

The small ubiquitin-related modifier (SUMO) conjugation apparatus usually attaches single SUMO moieties to its substrates, but SUMO chains have also been identified. To better define the biochemical requirements and characteristics of SUMO chain formation, mutations in surface-exposed Lys residues of Arabidopsis SUMO-conjugating enzyme (SCE) were tested for in vitro activity. Lys-to-Arg changes in the amino-terminal region of SCE allowed SUMO acceptance from SUMO-activating enzyme and supported substrate mono-sumoylation, but these mutations had significant effects on SUMO chain assembly. We found no indication that SUMO modification of SCE promotes chain formation. A substrate was identified that is modified by SUMO chain addition, showing that SCE can distinguish substrates for either mono-sumoylation or SUMO chain attachment. It is also shown that SCE with active site Cys mutated to Ser can accept SUMO to form an oxyester, but cannot transfer this SUMO moiety onto substrates, explaining a previously known dominant negative effect of this mutation. PMID:29133528

Increased Long Chain acyl-Coa Synthetase Activity and Fatty Acid Import Is Linked to Membrane Synthesis for Development of Picornavirus Replication Organelles

PubMed Central

Scott, Alison J.; Ford, Lauren A.; Pei, Zhengtong; Watkins, Paul A.; Ernst, Robert K.; Belov, George A.

2013-01-01

All positive strand (+RNA) viruses of eukaryotes replicate their genomes in association with membranes. The mechanisms of membrane remodeling in infected cells represent attractive targets for designing future therapeutics, but our understanding of this process is very limited. Elements of autophagy and/or the secretory pathway were proposed to be hijacked for building of picornavirus replication organelles. However, even closely related viruses differ significantly in their requirements for components of these pathways. We demonstrate here that infection with diverse picornaviruses rapidly activates import of long chain fatty acids. While in non-infected cells the imported fatty acids are channeled to lipid droplets, in infected cells the synthesis of neutral lipids is shut down and the fatty acids are utilized in highly up-regulated phosphatidylcholine synthesis. Thus the replication organelles are likely built from de novo synthesized membrane material, rather than from the remodeled pre-existing membranes. We show that activation of fatty acid import is linked to the up-regulation of cellular long chain acyl-CoA synthetase activity and identify the long chain acyl-CoA syntheatse3 (Acsl3) as a novel host factor required for polio replication. Poliovirus protein 2A is required to trigger the activation of import of fatty acids independent of its protease activity. Shift in fatty acid import preferences by infected cells results in synthesis of phosphatidylcholines different from those in uninfected cells, arguing that the viral replication organelles possess unique properties compared to the pre-existing membranes. Our data show how poliovirus can change the overall cellular membrane homeostasis by targeting one critical process. They explain earlier observations of increased phospholipid synthesis in infected cells and suggest a simple model of the structural development of the membranous scaffold of replication complexes of picorna-like viruses, that may be

Kinetic characterization of Escherichia coli outer membrane phospholipase A using mixed detergent-lipid micelles.

PubMed

Horrevoets, A J; Hackeng, T M; Verheij, H M; Dijkman, R; de Haas, G H

1989-02-07

The substrate specificity of Escherichia coli outer membrane phospholipase A was analyzed in mixed micelles of lipid with deoxycholate or Triton X-100. Diglycerides, monoglycerides, and Tweens 40 and 85 in Triton X-100 are hydrolyzed at rates comparable to those of phospholipids and lysophospholipids. p-Nitrophenyl esters of fatty acids with different chain lengths and triglycerides are not hydrolyzed. The minimal substrate characteristics consist of a long acyl chain esterified to a more or less hydrophilic headgroup as is the case for the substrate monopalmitoylglycol. Binding occurs via the hydrocarbon chain of the substrate; diacyl compounds are bound three to five times better than monoacyl compounds. When acting on lecithins, phospholipase A1 activity is six times higher than phospholipase A2 activity or 1-acyl lysophospholipase activity. Activity on the 2-acyl lyso compound is about two times less than that on the 1-acyl lysophospholipid. The enzyme therefore has a clear preference for the primary ester bond of phospholipids. In contrast to phospholipase A1 activity, phospholipase A2 activity is stereospecific. Only the L isomer of a lecithin analogue in which the primary acyl chain was replaced by an alkyl ether group is hydrolyzed. The D isomer of this analogue is a competitive inhibitor, bound with the same affinity as the L isomer. On these ether analogues the enzyme shows the same preference for the primary acyl chain as with the natural diester phospholipids. Despite its broad specificity, the enzyme will initially act as a phospholipase A1 in the E. coli envelope where it is embedded in phospholipids.

Molecular dynamics investigations of BioH protein substrate specificity for biotin synthesis.

PubMed

Xue, Qiao; Cui, Ying-Lu; Zheng, Qing-Chuan; Zhang, Hong-Xing

2016-05-01

BioH, an enzyme of biotin synthesis, plays an important role in fatty acid synthesis which assembles the pimelate moiety. Pimeloyl-acyl carrier protein (ACP) methyl ester, which is long known to be a biotin precursor, is the physiological substrate of BioH. Azelayl methyl ester, which has a longer chain than pimeloyl methyl ester, conjugated to ACP is also indeed accepted by BioH with very low rate of hydrolysis. To date, the substrate specificity for BioH and the molecular origin for the experimentally observed rate changes of hydrolysis by the chain elongation have remained elusive. To this end, we have investigated chain elongation effects on the structures by using the fully atomistic molecular dynamics simulations combined with binding free energy calculations. The results indicate that the substrate specificity is determined by BioH together with ACP. The added two methylenes would increase the structural flexibility by protein motions at the interface of ACP and BioH, instead of making steric clashes with the side chains of the BioH hydrophobic cavity. On the other hand, the slower hydrolysis of azelayl substrate is suggested to be associated with the loose of contacts between BioH and ACP, and with the lost electrostatic interactions of two ionic/hydrogen bonding networks at the interface of the two proteins. The present study provides important insights into the structure-function relationships of the complex of BioH with pimeloyl-ACP methyl ester, which could contribute to further understanding about the mechanism of the biotin synthetic pathway, including the catalytic role of BioH.

Very-Long-Chain Acyl-CoA Dehydrogenase Deficiency--diagnostic difficulties and own experience in multidisciplinary management.

PubMed

Stępień, Karolina M; Roberts, Mark; Hendriksz, Christian J

2015-01-01

A 19-year old female patient presented with a two-year history of muscle pain and weakness before she was admitted to an acute medical ward with rhabdomyolysis (creatine kinase of 83,344 IU/L) and normal renal function tests. Following admission she was under the care of the rheumatology and neurology teams, which investigated her thoroughly. As part of the belt-and-braces approach, both teams contacted the specialist Adult Inherited Metabolic Disorders team for advice, instigating definitive diagnostic investigations. An accurate diagnosis was required, as an inherited metabolic disorders can present in adult patients as a milder form of the disease. Very-long-chain Acyl-CoA dehydrogenase (VLCAD) deficiency should always be considered as a differential diagnosis of myopathy-related symptoms. Hence, the liaison between neurologists, rheumatologists and metabolic physicians is essential in early diagnosis and the management of patients with conditions causing myopathy.

Cloning and characterization of a novel amidase from Paracoccus sp. M-1, showing aryl acylamidase and acyl transferase activities.

PubMed

Shen, Weiliang; Chen, Honghong; Jia, Kaizhi; Ni, Jun; Yan, Xin; Li, Shunpeng

2012-05-01

A novel amidase gene, designated pamh, was cloned from Paracoccus sp. M-1. Site-directed mutagenesis and bioinformatic analysis showed that the PamH protein belonged to the amidase signature enzyme family. PamH was expressed in Escherichia coli, purified, and characterized. The molecular mass of PamH was determined to be 52 kDa with an isoelectric point of 5.13. PamH displayed its highest enzymatic activity at 45°C and at pH 8.0 and was stable within a pH range of 5.0-10.0. The PamH enzyme exhibited amidase activity, aryl acylamidase activity, and acyl transferase activity, allowing it to function across a very broad substrate spectrum. PamH was highly active on aromatic and short-chain aliphatic amides (benzamide and propionamide), moderately active on amino acid amides, and possessed weak urease activity. Of the anilides examined, only propanil was a good substrate for PamH. For propanil, the k (cat) and K (m) were 2.8 s(-1) and 158 μM, respectively, and the catalytic efficiency value (k (cat)/K (m)) was 0.018 μM(-1) s(-1). In addition, PamH was able to catalyze the acyl transfer reaction to hydroxylamine for both amide and anilide substrates, including acetamide, propanil, and 4-nitroacetanilide; the highest reaction rate was shown with isobutyramide. These characteristics make PamH an excellent candidate for environmental remediation and an important enzyme for the biosynthesis of novel amides.

Role of protein conformational mobility in enzyme catalysis: acylation of alpha-chymotrypsin by specific peptide substrates.

PubMed

Hengge, Alvan C; Stein, Ross L

2004-01-27

To probe the mechanistic origins of convex Eyring plots that have been observed for alpha-chymotrypsin (alpha-CT)-catalyzed hydrolysis of specific p-nitroanilide substrates [Case, A., and Stein, R. L. (2003) Biochemistry 42, 3335-3348], we determined the temperature-dependence of (15)N-kinetic isotope effects for the alpha-CT-catalyzed hydrolysis of N-succinyl-Phe p-nitroanilide (Suc-Phe-pNA). To provide an interpretational context for these enzymatic isotope effects, we also determined 15N-KIE for alkaline hydrolysis of p-nitroacetanilide. In 0.002 and 2 N hydroxide (30 degrees C), 15N-KIE values are 1.035 and 0.995 (+/-0.001), respectively, and are consistent with the reported [HO-]-dependent change in rate-limiting step from leaving group departure from an anionic tetrahedral intermediate in dilute base, to hydroxide attack in concentrated base. For the alpha-CT-catalyzed hydrolysis of Suc-Phe-pNA, 15N-KIE is on kc/Km and thus reflects structural features of transition states for all reaction steps up to and including acylation of the active site serine. The isotope effect at 35 degrees C is 1.014 (+/-0.001) and suggests that in the transition state for this reaction, departure of leaving group from the tetrahedral intermediate is well advanced. Significantly, 15N-KIE does not vary over the temperature range 5-45 degrees C. This result eliminates one of the competing hypotheses for the convex Eyring plot observed for this reaction, that is, a temperature-dependent change in rate-limiting step within the chemical manifold of acylation, but supports a mechanism in which an isomerization of enzyme conformation is coupled to active site chemistry. We finally suggest that the near absolute temperature-independence of 15N-KIE may point to a unique transition state for this process.

Acyl Chain-Dependent Effect of Lysophosphatidylcholine on Endothelium-Dependent Vasorelaxation

PubMed Central

Rao, Shailaja P.; Riederer, Monika; Lechleitner, Margarete; Hermansson, Martin; Desoye, Gernot; Hallström, Seth; Graier, Wolfgang F.; Frank, Saša

2013-01-01

Previously we identified palmitoyl-, oleoyl-, linoleoyl-, and arachidonoyl-lysophosphatidylcholine (LPC 16:0, 18:1, 18:2 and 20:4) as the most prominent LPC species generated by endothelial lipase (EL). In the present study, we examined the impact of those LPC on acetylcholine (ACh)- induced vascular relaxation. All tested LPC attenuated ACh-induced relaxation, measured ex vivo, using mouse aortic rings and wire myography. The rank order of potency was as follows: 18:2>20:4>16:0>18:1. The attenuating effect of LPC 16:0 on relaxation was augmented by indomethacin-mediated cyclooxygenase (COX)-inhibition and CAY10441, a prostacyclin (PGI2)- receptor (IP) antagonist. Relaxation attenuated by LPC 20:4 and 18:2 was improved by indomethacin and SQ29548, a thromboxane A2 (TXA2)- receptor antagonist. The effect of LPC 20:4 could also be improved by TXA2- and PGI2-synthase inhibitors. As determined by EIA assays, the tested LPC promoted secretion of PGI2, TXA2, PGF2α, and PGE2, however, with markedly different potencies. LPC 16:0 was the most potent inducer of superoxide anion production by mouse aortic rings, followed by LPC 18:2, 20:4 and 18:1, respectively. The strong antioxidant tempol recovered relaxation impairment caused by LPC 18:2, 18:1 and 20:4, but not by LPC 16:0. The tested LPC attenuate ACh-induced relaxation through induction of proconstricting prostanoids and superoxide anions. The potency of attenuating relaxation and the relative contribution of underlying mechanisms are strongly related to LPC acyl-chain length and degree of saturation. PMID:23741477

Commelinid Monocotyledon Lignins Are Acylated by p-Coumarate1[OPEN

PubMed Central

Free, Heather C.A.; Smith, Bronwen G.

2018-01-01

Commelinid monocotyledons are a monophyletic clade differentiated from other monocotyledons by the presence of cell wall-bound ferulate and p-coumarate. The Poaceae, or grass family, is a member of this group, and most of the p-coumarate in the cell walls of this family acylates lignin. Here, we isolated and examined lignified cell wall preparations from 10 species of commelinid monocotyledons from nine families other than Poaceae, including species from all four commelinid monocotyledon orders (Poales, Zingiberales, Commelinales, and Arecales). We showed that, as in the Poaceae, lignin-linked p-coumarate occurs exclusively on the hydroxyl group on the γ-carbon of lignin unit side chains, mostly on syringyl units. Although the mechanism of acylation has not been studied directly in these species, it is likely to be similar to that in the Poaceae and involve BAHD acyl-coenzyme A:monolignol transferases. PMID:29724771

N-Acylated and D enantiomer derivatives of a nonamer core peptide of lactoferricin B showing improved antimicrobial activity.

PubMed

Wakabayashi, H; Matsumoto, H; Hashimoto, K; Teraguchi, S; Takase, M; Hayasawa, H

1999-05-01

N-acylated or D enantiomer peptide derivatives based on the sequence RRWQWRMKK in lactoferricin B demonstrated antimicrobial activities greater than those of lactoferricin B against bacteria and fungi. The most potent peptide, conjugated with an 11-carbon-chain acyl group, showed two to eight times lower MIC than lactoferricin B.

SIRT3 and SIRT5 Regulate the Enzyme Activity and Cardiolipin Binding of Very Long-Chain Acyl-CoA Dehydrogenase

PubMed Central

Zhang, Yuxun; Bharathi, Sivakama S.; Rardin, Matthew J.; Uppala, Radha; Verdin, Eric; Gibson, Bradford W.; Goetzman, Eric S.

2015-01-01

SIRT3 and SIRT5 have been shown to regulate mitochondrial fatty acid oxidation but the molecular mechanisms behind the regulation are lacking. Here, we demonstrate that SIRT3 and SIRT5 both target human very long-chain acyl-CoA dehydrogenase (VLCAD), a key fatty acid oxidation enzyme. SIRT3 deacetylates and SIRT5 desuccinylates K299 which serves to stabilize the essential FAD cofactor in the active site. Further, we show that VLCAD binds strongly to cardiolipin and isolated mitochondrial membranes via a domain near the C-terminus containing lysines K482, K492, and K507. Acetylation or succinylation of these residues eliminates binding of VLCAD to cardiolipin. SIRT3 deacetylates K507 while SIRT5 desuccinylates K482, K492, and K507. Sirtuin deacylation of recombinant VLCAD rescues membrane binding. Endogenous VLCAD from SIRT3 and SIRT5 knockout mouse liver shows reduced binding to cardiolipin. Thus, SIRT3 and SIRT5 promote fatty acid oxidation by converging upon VLCAD to promote its activity and membrane localization. Regulation of cardiolipin binding by reversible lysine acylation is a novel mechanism that is predicted to extrapolate to other metabolic proteins that localize to the inner mitochondrial membrane. PMID:25811481

Overexpression of Human Fatty Acid Transport Protein 2/Very Long Chain Acyl-CoA Synthetase 1 (FATP2/Acsvl1) Reveals Distinct Patterns of Trafficking of Exogenous Fatty Acids

PubMed Central

Melton, Elaina M.; Cerny, Ronald L.; DiRusso, Concetta C.; Black, Paul N.

2014-01-01

In mammals, the fatty acid transport proteins (FATP1 through FATP6) are members of a highly conserved family of proteins, which function in fatty acid transport proceeding through vectorial acylation and in the activation of very long chain fatty acids, branched chain fatty acids and secondary bile acids. FATP1, 2 and 4, for example directly function in fatty acid transport and very long chain fatty acids activation while FATP5 does not function in fatty acid transport but activates secondary bile acids. In the present work, we have used stable isotopically labeled fatty acids differing in carbon length and saturation in cells expressing FATP2 to gain further insights into how this protein functions in fatty acid transport and intracellular fatty acid trafficking. Our previous studies showed the expression of FATP2 modestly increased C16:0-CoA and C20:4-CoA and significantly increased C18:3-CoA and C22:6-CoA after 4hr. The increases in C16:0-CoA and C18:3-CoA suggest FATP2 must necessarily partner with a long chain acyl CoA synthetase (Acsl) to generate C16:0-CoA and C18:3-CoA through vectorial acylation. The very long chain acyl CoA synthetase activity of FATP2 is consistent in the generation of C20:4-CoA and C22:6-CoA coincident with transport from their respective exogenous fatty acids. The trafficking of exogenous fatty acids into phosphatidic acid (PA) and into the major classes of phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidyserine (PS)) resulted in distinctive profiles, which changed with the expression of FATP2. The trafficking of exogenous C16:0 and C22:6 into PA was significant where there was 6.9- and 5.3-fold increased incorporation, respectively, over the control; C18:3 and C20:4 also trended to increase in the PA pool while there were no changes for C18:1 and C18:2. The trafficking of C18:3 into PC and PI trended higher and approached significance. In the case of C20:4, expression of

Overexpression of human fatty acid transport protein 2/very long chain acyl-CoA synthetase 1 (FATP2/Acsvl1) reveals distinct patterns of trafficking of exogenous fatty acids.

PubMed

Melton, Elaina M; Cerny, Ronald L; DiRusso, Concetta C; Black, Paul N

2013-11-01

In mammals, the fatty acid transport proteins (FATP1 through FATP6) are members of a highly conserved family of proteins, which function in fatty acid transport proceeding through vectorial acylation and in the activation of very long chain fatty acids, branched chain fatty acids and secondary bile acids. FATP1, 2 and 4, for example directly function in fatty acid transport and very long chain fatty acids activation while FATP5 does not function in fatty acid transport but activates secondary bile acids. In the present work, we have used stable isotopically labeled fatty acids differing in carbon length and saturation in cells expressing FATP2 to gain further insights into how this protein functions in fatty acid transport and intracellular fatty acid trafficking. Our previous studies showed the expression of FATP2 modestly increased C16:0-CoA and C20:4-CoA and significantly increased C18:3-CoA and C22:6-CoA after 4h. The increases in C16:0-CoA and C18:3-CoA suggest FATP2 must necessarily partner with a long chain acyl CoA synthetase (Acsl) to generate C16:0-CoA and C18:3-CoA through vectorial acylation. The very long chain acyl CoA synthetase activity of FATP2 is consistent in the generation of C20:4-CoA and C22:6-CoA coincident with transport from their respective exogenous fatty acids. The trafficking of exogenous fatty acids into phosphatidic acid (PA) and into the major classes of phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidyserine (PS)) resulted in distinctive profiles, which changed with the expression of FATP2. The trafficking of exogenous C16:0 and C22:6 into PA was significant where there was 6.9- and 5.3-fold increased incorporation, respectively, over the control; C18:3 and C20:4 also trended to increase in the PA pool while there were no changes for C18:1 and C18:2. The trafficking of C18:3 into PC and PI trended higher and approached significance. In the case of C20:4, expression of

Prolonged QT interval and lipid alterations beyond β-oxidation in very long-chain acyl-CoA dehydrogenase null mouse hearts

PubMed Central

Gélinas, Roselle; Thompson-Legault, Julie; Bouchard, Bertrand; Daneault, Caroline; Mansour, Asmaa; Gillis, Marc-Antoine; Charron, Guy; Gavino, Victor; Labarthe, François

2011-01-01

Patients with very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency frequently present cardiomyopathy and heartbeat disorders. However, the underlying factors, which may be of cardiac or extra cardiac origins, remain to be elucidated. In this study, we tested for metabolic and functional alterations in the heart from 3- and 7-mo-old VLCAD null mice and their littermate counterparts, using validated experimental paradigms, namely, 1) ex vivo perfusion in working mode, with concomitant evaluation of myocardial contractility and metabolic fluxes using 13C-labeled substrates under various conditions; as well as 2) in vivo targeted lipidomics, gene expression analysis as well as electrocardiogram monitoring by telemetry in mice fed various diets. Unexpectedly, when perfused ex vivo, working VLCAD null mouse hearts maintained values similar to those of the controls for functional parameters and for the contribution of exogenous palmitate to β-oxidation (energy production), even at high palmitate concentration (1 mM) and increased energy demand (with 1 μM epinephrine) or after fasting. However, in vivo, these hearts displayed a prolonged rate-corrected QT (QTc) interval under all conditions examined, as well as the following lipid alterations: 1) age- and condition-dependent accumulation of triglycerides, and 2) 20% lower docosahexaenoic acid (an omega-3 polyunsaturated fatty acid) in membrane phospholipids. The latter was independent of liver but affected by feeding a diet enriched in saturated fat (exacerbated) or fish oil (attenuated). Our finding of a longer QTc interval in VLCAD null mice appears to be most relevant given that such condition increases the risk of sudden cardiac death. PMID:21685264

N-Acylated and d Enantiomer Derivatives of a Nonamer Core Peptide of Lactoferricin B Showing Improved Antimicrobial Activity

PubMed Central

Wakabayashi, Hiroyuki; Matsumoto, Hiroshi; Hashimoto, Koichi; Teraguchi, Susumu; Takase, Mitsunori; Hayasawa, Hirotoshi

1999-01-01

N-acylated or d enantiomer peptide derivatives based on the sequence RRWQWRMKK in lactoferricin B demonstrated antimicrobial activities greater than those of lactoferricin B against bacteria and fungi. The most potent peptide, conjugated with an 11-carbon-chain acyl group, showed two to eight times lower MIC than lactoferricin B. PMID:10223949

Lipolysis of natural long chain and synthetic medium chain galactolipids by pancreatic lipase-related protein 2.

PubMed

Amara, Sawsan; Barouh, Nathalie; Lecomte, Jérôme; Lafont, Dominique; Robert, Sylvie; Villeneuve, Pierre; De Caro, Alain; Carrière, Frédéric

2010-04-01

Monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are the most abundant lipids in nature, mainly as important components of plant leaves and chloroplast membranes. Pancreatic lipase-related protein 2 (PLRP2) was previously found to express galactolipase activity, and it is assumed to be the main enzyme involved in the digestion of these common vegetable lipids in the gastrointestinal tract. Most of the previous in vitro studies were however performed with medium chain synthetic galactolipids as substrates. It was shown here that recombinant guinea pig (Cavia porcellus) as well as human PLRP2 hydrolyzed at high rates natural DGDG and MGDG extracted from spinach leaves. Their specific activities were estimated by combining the pH-stat technique, thin layer chromatography coupled to scanning densitometry and gas chromatography. The optimum assay conditions for hydrolysis of these natural long chain galactolipids were investigated and the optimum bile salt to substrate ratio was found to be different from that established with synthetic medium chains MGDG and DGDG. Nevertheless the length of acyl chains and the nature of the galactosyl polar head of the galactolipid did not have major effects on the specific activities of PLRP2, which were found to be very high on both medium chain [1786+/-100 to 5420+/-85U/mg] and long chain [1756+/-208 to 4167+/-167U/mg] galactolipids. Fatty acid composition analysis of natural MGDG, DGDG and their lipolysis products revealed that PLRP2 only hydrolyzed one ester bond at the sn-1 position of galactolipids. PLRP2 might be used to produce lipid and free fatty acid fractions enriched in either 16:3 n-3 or 18:3 n-3 fatty acids, both found at high levels in galactolipids. 2010 Elsevier B.V. All rights reserved.

Allometric scaling of fatty acyl chains in fowl liver, lung and kidney, but not in brain phospholipids.

PubMed

Szabó, András; Mézes, Miklós; Romvári, Róbert; Fébel, Hedvig

2010-03-01

The phospholipid (PL) fatty acyl chain (FA) composition (mol%) was determined in the kidney, liver, lung and brain of 8 avian species ranging in body mass from 150g (Japanese quail, Coturnix coturnix japonica) to 19kg (turkey, Meleagris gallopavo). In all organs except the brain, docosahexaenoic acid (C22:6 n3, DHA) was found to show a negative allometric scaling (allometric exponent: B=-0.18; -0.20 and -0.24, for kidney, liver and lung, respectively). With minor inter-organ differences, smaller birds had more n3 FAs and longer FA chains in the renal, hepatic and pulmonary PLs. Comparing our results with literature data on avian skeletal muscle, liver mitochondria and kidney microsomes and divergent mammalian tissues, the present findings in the kidney, liver and lung PLs seem to be a part of a general relationship termed "membranes as metabolic pacemakers". Marked negative allometric scaling was found furthermore for the tissue malondialdehyde concentrations in all organs except the brain (B=-0.17; -0.13 and -0.05, respectively). In the liver and kidney a strong correlation was found between the tissue MDA and DHA levels, expressing the role of DHA in shaping the allometric properties of membrane lipids. 2009 Elsevier Inc. All rights reserved.

Tandem UIMs confer Lys48 ubiquitin chain substrate preference to deubiquitinase USP25

PubMed Central

Kawaguchi, Kohei; Uo, Kazune; Tanaka, Toshiaki; Komada, Masayuki

2017-01-01

Ubiquitin-specific protease (USP) 25, belonging to the USP family of deubiquitinases, harbors two tandem ubiquitin-interacting motifs (UIMs), a ~20-amino-acid α-helical stretch that binds to ubiquitin. However, the role of the UIMs in USP25 remains unclear. Here we show that the tandem UIM region binds to Lys48-, but not Lys63-, linked ubiquitin chains, where the two UIMs played a critical and cooperative role. Purified USP25 exhibited higher ubiquitin isopeptidase activity to Lys48-, than to Lys63-, linked ubiquitin chains. Mutations that disrupted the ubiquitin-binding ability of the tandem UIMs resulted in a reduced ubiquitin isopeptidase activity of USP25, suggesting a role for the UIMs in exerting the full catalytic activity of USP25. Moreover, when mutations that convert the binding preference from Lys48- to Lys63-linked ubiquitin chains were introduced into the tandem UIM region, the USP25 mutants acquired elevated and reduced isopeptidase activity toward Lys63- and Lys48-linked ubiquitin chains, respectively. These results suggested that the binding preference of the tandem UIMs toward Lys48-linked ubiquitin chains contributes not only to the full catalytic activity but also to the ubiquitin chain substrate preference of USP25, possibly by selectively holding the Lys48-linked ubiquitin chain substrates in the proximity of the catalytic core. PMID:28327663

Acyl CoA synthetase 5 (ACSL5) ablation in mice increases energy expenditure and insulin sensitivity and delays fat absorption

USDA-ARS?s Scientific Manuscript database

Objective: The family of acyl-CoA synthetase enzymes (ACSL) activates fatty acids within cells to generate long chain fatty acyl CoA (FACoA). The differing metabolic fates of FACoAs such as incorporation into neutral lipids, phospholipids, and oxidation pathways are differentially regulated by the ...

Sexual dimorphism of lipid metabolism in very long-chain acyl-CoA dehydrogenase deficient (VLCAD-/-) mice in response to medium-chain triglycerides (MCT).

PubMed

Tucci, Sara; Flögel, Ulrich; Spiekerkoetter, Ute

2015-07-01

Medium-chain triglycerides (MCT) are widely applied in the treatment of long-chain fatty acid oxidation disorders. Previously it was shown that long-term MCT supplementation strongly affects lipid metabolism in mice. We here investigate sex-specific effects in mice with very-long-chain-acyl-CoA dehydrogenase (VLCAD) deficiency in response to a long-term MCT modified diet. We quantified blood lipids, acylcarnitines, glucose, insulin and free fatty acids, as well as tissue triglycerides in the liver and skeletal muscle under a control and an MCT diet over 1 year. In addition, visceral and hepatic fat content and muscular intramyocellular lipids (IMCL) were assessed by in vivo(1)H magnetic resonance spectroscopy (MRS) techniques. The long-term application of an MCT diet induced a marked alteration of glucose homeostasis. However, only VLCAD-/- female mice developed a severe metabolic syndrome characterized by marked insulin resistance, dyslipidemia, severe hepatic and visceral steatosis, whereas VLCAD-/- males seemed to be protected and only presented with milder insulin resistance. Moreover, the highly saturated MCT diet is associated with a decreased hepatic stearoyl-CoA desaturase 1 (SCD1) activity in females aggravating the harmful effects of a saturated MCT diet. Long-term MCT supplementation deeply affects lipid metabolism in a sexual dimorphic manner resulting in a severe metabolic syndrome only in female mice. These findings are striking since the first signs of insulin resistance already occur in female VLCAD-/- mice during their reproductive period. How these metabolic adaptations are finally regulated needs to be determined. More important, the relevance of these findings for humans under these dietary modifications needs to be investigated. Copyright © 2015 Elsevier B.V. All rights reserved.

De novo fatty acid biosynthesis and elongation in very long-chain acyl-CoA dehydrogenase-deficient mice supplemented with odd or even medium-chain fatty acids.

PubMed

Tucci, Sara; Behringer, Sidney; Spiekerkoetter, Ute

2015-11-01

An even medium-chain triglyceride (MCT)-based diet is the mainstay of treatment in very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency (VLCADD). Previous studies with magnetic resonance spectroscopy have shown an impact of MCT on the average fatty acid chain length in abdominal fat. We therefore assume that medium-chain fatty acids (MCFAs) are elongated and accumulate in tissue as long-chain fatty acids. In this study, we explored the hepatic effects of long-term supplementation with MCT or triheptanoin, an odd-chain C7-based triglyceride, in wild-type and VLCAD-deficient (VLCAD(-/-) ) mice after 1 year of supplementation as compared with a control diet. The de novo biosynthesis and elongation of fatty acids, and peroxisomal β-oxidation, were quantified by RT-PCR. This was followed by a comprehensive analysis of hepatic and cardiac fatty acid profiles by GC-MS. Long-term application of even and odd MCFAs strongly induced de novo biosynthesis and elongation of fatty acids in both wild-type and VLCAD(-/-) mice, leading to an alteration of the hepatic fatty acid profiles. We detected de novo-synthesized and elongated fatty acids, such as heptadecenoic acid (C17:1n9), eicosanoic acid (C20:1n9), erucic acid (C22:1n9), and mead acid (C20:3n9), that were otherwise completely absent in mice under control conditions. In parallel, the content of monounsaturated fatty acids was massively increased. Furthermore, we observed strong upregulation of peroxisomal β-oxidation in VLCAD(-/-) mice, especially when they were fed an MCT diet. Our data raise the question of whether long-term MCFA supplementation represents the most efficient treatment in the long term. Studies on the hepatic toxicity of triheptanoin are still ongoing. © 2015 FEBS.

Acyl carrier protein structural classification and normal mode analysis

PubMed Central

Cantu, David C; Forrester, Michael J; Charov, Katherine; Reilly, Peter J

2012-01-01

All acyl carrier protein primary and tertiary structures were gathered into the ThYme database. They are classified into 16 families by amino acid sequence similarity, with members of the different families having sequences with statistically highly significant differences. These classifications are supported by tertiary structure superposition analysis. Tertiary structures from a number of families are very similar, suggesting that these families may come from a single distant ancestor. Normal vibrational mode analysis was conducted on experimentally determined freestanding structures, showing greater fluctuations at chain termini and loops than in most helices. Their modes overlap more so within families than between different families. The tertiary structures of three acyl carrier protein families that lacked any known structures were predicted as well. PMID:22374859

Active-Site Protonation States in an Acyl-Enzyme Intermediate of a Class A β-Lactamase with a Monobactam Substrate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cooper, Jonathan B.; Weiss, Kevin L.; Coates, Leighton

The monobactam antibiotic aztreonam is used to treat cystic fibrosis patients with chronic pulmonary infections colonized by Pseudomonas aeruginosa strains expressing CTX-M extended-spectrum β-lactamases. Several active site residues in class A β-lactamases have been proposed to play key roles in monobactam hydrolysis. The protonation states of these residues have been determined previously for the apo form of a CTX-M β-lactamase. However, they have not yet been determined for a monobactam acyl-enzyme intermediate. Here we used neutron and high-resolution X-ray crystallography to probe the mechanism by which CTX-M extended-spectrum β-lactamases hydrolyze monobactam antibiotics. In these first reported structures of a classmore » A β-lactamase in acyl enzyme complex with aztreonam we directly observed most of the hydrogen atoms (as deuterium) within the active site in the captured acyl-enzyme state between Toho-1 β-lactamase and aztreonam. Although Lys 234 is fully protonated in the acyl-intermediate, we find that Lys 73 is neutral. These findings are consistent with Lys 73 being able to serve as a general base during the acylation part of the catalytic mechanism, in agreement with previous mechanistic proposals.« less

Active-Site Protonation States in an Acyl-Enzyme Intermediate of a Class A β-Lactamase with a Monobactam Substrate

DOE PAGES

Cooper, Jonathan B.; Weiss, Kevin L.; Coates, Leighton; ...

2016-10-24

The monobactam antibiotic aztreonam is used to treat cystic fibrosis patients with chronic pulmonary infections colonized by Pseudomonas aeruginosa strains expressing CTX-M extended-spectrum β-lactamases. Several active site residues in class A β-lactamases have been proposed to play key roles in monobactam hydrolysis. The protonation states of these residues have been determined previously for the apo form of a CTX-M β-lactamase. However, they have not yet been determined for a monobactam acyl-enzyme intermediate. Here we used neutron and high-resolution X-ray crystallography to probe the mechanism by which CTX-M extended-spectrum β-lactamases hydrolyze monobactam antibiotics. In these first reported structures of a classmore » A β-lactamase in acyl enzyme complex with aztreonam we directly observed most of the hydrogen atoms (as deuterium) within the active site in the captured acyl-enzyme state between Toho-1 β-lactamase and aztreonam. Although Lys 234 is fully protonated in the acyl-intermediate, we find that Lys 73 is neutral. These findings are consistent with Lys 73 being able to serve as a general base during the acylation part of the catalytic mechanism, in agreement with previous mechanistic proposals.« less

The Acyl Desaturase CER17 Is Involved in Producing Wax Unsaturated Primary Alcohols and Cutin Monomers.

PubMed

Yang, Xianpeng; Zhao, Huayan; Kosma, Dylan K; Tomasi, Pernell; Dyer, John M; Li, Rongjun; Liu, Xiulin; Wang, Zhouya; Parsons, Eugene P; Jenks, Matthew A; Lü, Shiyou

2017-02-01

We report n-6 monounsaturated primary alcohols (C 26:1 , C 28:1 , and C 30:1 homologs) in the cuticular waxes of Arabidopsis (Arabidopsis thaliana) inflorescence stem, a class of wax not previously reported in Arabidopsis. The Arabidopsis cer17 mutant was completely deficient in these monounsaturated alcohols, and CER17 was found to encode a predicted ACYL-COENZYME A DESATURASE LIKE4 (ADS4). Studies of the Arabidopsis cer4 mutant and yeast variously expressing CER4 (a predicted fatty acyl-CoA reductase) with CER17/ADS4, demonstrated CER4's principal role in synthesis of these monounsaturated alcohols. Besides unsaturated alcohol deficiency, cer17 mutants exhibited a thickened and irregular cuticle ultrastructure and increased amounts of cutin monomers. Although unsaturated alcohols were absent throughout the cer17 stem, the mutation's effects on cutin monomers and cuticle ultrastructure were much more severe in distal than basal stems, consistent with observations that the CER17/ADS4 transcript was much more abundant in distal than basal stems. Furthermore, distal but not basal stems of a double mutant deficient for both CER17/ADS4 and LONG-CHAIN ACYL-COA SYNTHETASE1 produced even more cutin monomers and a thicker and more disorganized cuticle ultrastructure and higher cuticle permeability than observed for wild type or either mutant parent, indicating a dramatic genetic interaction on conversion of very long chain acyl-CoA precursors. These results provide evidence that CER17/ADS4 performs n-6 desaturation of very long chain acyl-CoAs in both distal and basal stems and has a major function associated with governing cutin monomer amounts primarily in the distal segments of the inflorescence stem. © 2017 American Society of Plant Biologists. All Rights Reserved.

Interactions of acylated methylglucoside derivatives with CO2: simulation and calculations.

PubMed

Chang, H H; Cao, R X; Yang, C C; Wei, W L; Pang, X Y; Qiao, Y

2016-01-01

Carbohydrates have drawn considerable interest from researchers recently due to their affinity for CO2. However, most of the research in this field has focused on peracetylated derivatives. Compared with acetylated carbohydrates, which have already been studied in depth, methyl D-glucopyranoside derivatives are more stable and could have additional applications. Thus, in the present work, ab initio calculations were performed to elucidate the characteristics of the interactions of methylglucoside derivatives with CO2, and to investigate how the binding energy (ΔE) is affected by isomerization or the introduction of various acyl groups. Four methyl D-glucopyranosides (each with two anomers) bearing acetyl, propionyl, butyryl, and isobutyryl moieties, respectively, were designed as substrates, and the 1:1 complexes of a CO2 molecule with each of these sugar substrates were modeled. The results indicate that ΔE is mainly influenced by interaction distance and the number of negatively charged donors or interacting pairs in the complex; the structure of the acyl group present in the substrate is a secondary influence. Except in the case of methyl 2-O-acetyl-D-glucopyranose, the ΔE values of the α- and β-anomers of each methylglucoside were found to be almost the same. Therefore, we would expect the CO2 affinities of the four derivatives studied here to be as strong as or even stronger than that of peracetylated D-glucopyranose. Graphical Abstract The binding energy between methyl D-glucopyranoside derivatives with various substituted acyl groups and CO2 are evaluated by ab initio calculations. The strong interaction between these methyl dglucopyranoside derivatives and CO2 showed the potential of their application for CO2 capture.

Downregulation of carnitine acyl-carnitine translocase by miRNAs 132 and 212 amplifies glucose-stimulated insulin secretion.

PubMed

Soni, Mufaddal S; Rabaglia, Mary E; Bhatnagar, Sushant; Shang, Jin; Ilkayeva, Olga; Mynatt, Randall; Zhou, Yun-Ping; Schadt, Eric E; Thornberry, Nancy A; Muoio, Deborah M; Keller, Mark P; Attie, Alan D

2014-11-01

We previously demonstrated that micro-RNAs (miRNAs) 132 and 212 are differentially upregulated in response to obesity in two mouse strains that differ in their susceptibility to obesity-induced diabetes. Here we show the overexpression of miRNAs 132 and 212 enhances insulin secretion (IS) in response to glucose and other secretagogues including nonfuel stimuli. We determined that carnitine acyl-carnitine translocase (CACT; Slc25a20) is a direct target of these miRNAs. CACT is responsible for transporting long-chain acyl-carnitines into the mitochondria for β-oxidation. Small interfering RNA-mediated knockdown of CACT in β-cells led to the accumulation of fatty acyl-carnitines and enhanced IS. The addition of long-chain fatty acyl-carnitines promoted IS from rat insulinoma β-cells (INS-1) as well as primary mouse islets. The effect on INS-1 cells was augmented in response to suppression of CACT. A nonhydrolyzable ether analog of palmitoyl-carnitine stimulated IS, showing that β-oxidation of palmitoyl-carnitine is not required for its stimulation of IS. These studies establish a link between miRNA-dependent regulation of CACT and fatty acyl-carnitine-mediated regulation of IS. © 2014 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

The R117A variant of the Escherichia coli transacylase FabD synthesizes novel acyl-(acyl carrier proteins).

PubMed

Marcella, Aaron M; Barb, Adam W

2017-12-01

The commercial impact of fermentation systems producing novel and biorenewable chemicals will flourish with the expansion of enzymes engineered to synthesize new molecules. Though a small degree of natural variability exists in fatty acid biosynthesis, the molecular space accessible through enzyme engineering is fundamentally limitless. Prokaryotic fatty acid biosynthesis enzymes build carbon chains on a functionalized acyl carrier protein (ACP) that provides solubility, stability, and a scaffold for interactions with the synthetic enzymes. Here, we identify the malonyl-coenzyme A (CoA)/holo-ACP transacylase (FabD) from Escherichia coli as a platform enzyme for engineering to diversify microbial fatty acid biosynthesis. The FabD R117A variant produced novel ACP-based primer and extender units for fatty acid biosynthesis. Unlike the wild-type enzyme that is highly specific for malonyl-CoA to produce malonyl-ACP, the R117A variant synthesized acetyl-ACP, succinyl-ACP, isobutyryl-ACP, 2-butenoyl-ACP, and β-hydroxybutyryl-ACP among others from holo-ACP and the corresponding acyl-CoAs with specific activities from 3.7 to 120 nmol min -1  mg -1 . FabD R117A maintained K M values for holo-ACP (~ 40 μM) and displayed small changes in K M for acetoacetyl-CoA (110 ± 30 μM) and acetyl-CoA (200 ± 70 μM) when compared to malonyl-CoA (80 ± 30 μM). FabD R117A represents a novel catalyst that synthesizes a broad range of acyl-acyl-ACPs.

Effects of riboflavin deficiency and clofibrate treatment on the five acyl-CoA dehydrogenases in rat liver mitochondria.

PubMed

Veitch, K; Draye, J P; Van Hoof, F; Sherratt, H S

1988-09-01

Rats were maintained on a riboflavin-deficient diet or on a diet containing clofibrate (0.5%, w/w). The activities of the mitochondrial FAD-dependent straight-chain acyl-CoA dehydrogenases (butyryl-CoA, octanoyl-CoA and palmitoyl-CoA) and the branched-chain acyl-CoA dehydrogenases (isovaleryl-CoA and isobutyryl-CoA) involved in the degradation of branched-chain acyl-CoA esters derived from branched-chain amino acids were assayed in liver mitochondrial extracts prepared in the absence and presence of exogenous FAD. These activities were low in livers from riboflavin-deficient rats (11, 28, 16, 6 and less than 2% of controls respectively) when prepared in the absence of exogenous FAD, and were not restored to control values when prepared in 25 microM-FAD (29, 47, 28, 7 and 17%). Clofibrate feeding increased the activities of butyryl-CoA, octanoyl-CoA and palmitoyl-CoA dehydrogenases (by 48, 116 and 98% of controls respectively), but not, by contrast, the activities of isovaleryl-CoA and isobutyryl-CoA dehydrogenases (62 and 102% of controls respectively). The mitochondrial fractions from riboflavin-deficient and from clofibrate-fed rats oxidized palmitoylcarnitine in State 3 at rates of 32 and 163% respectively of those from control rats.

Enhanced cellular uptake and in vitro antitumor activity of short-chain fatty acid acylated daunorubicin-GnRH-III bioconjugates.

PubMed

Hegedüs, Rózsa; Manea, Marilena; Orbán, Erika; Szabó, Ildikó; Kiss, Eva; Sipos, Eva; Halmos, Gábor; Mező, Gábor

2012-10-01

Here we report on the synthesis and biochemical characterization (enzymatic stability, cellular uptake, in vitro antitumor activity, membrane interaction and GnRH-receptor binding affinity) of novel short-chain fatty acid (SCFA) acylated daunorubicin-GnRH-III bioconjugates, which may serve as drug delivery systems for targeted cancer chemotherapy. Ser in position 4 of GnRH-III was replaced by Lys, followed by the acylation of its ε-amino group with various fatty acids. SCFAs are potentially chemoprotective agents by suppressing the growth of cancer cells and therefore may enhance the antitumor activity of the bioconjugates. We found that all synthesized bioconjugates had high cytostatic effect in vitro, were stable in cell culture medium for 6 h and degraded in the presence of rat liver lysosomal homogenate leading to the formation of an oxime bond-linked daunorubicin-Lys as the smallest active metabolite. In the presence of α-chymotrypsin, all compounds were digested, the degradation rate strongly depending on the type of fatty acid. The bioconjugate containing Lys(nBu) in position 4 was taken up most efficiently by the cancer cells and exerted higher in vitro cytostatic effect than the previously developed GnRH-III((4)Lys(Ac), (8)Lys(Dau = Aoa)) or the parent GnRH-III(Dau = Aoa) bioconjugate. Our results could be explained by the increased binding affinity of the newly developed compound containing Lys(nBu) to the GnRH receptors. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Long-Chain Fatty Acid Oxidation Disorders (LC-FAOD) Extension Study for Subjects Previously Enrolled in Triheptanoin Studies.

ClinicalTrials.gov

2018-06-19

Carnitine Palmitoyltransferase (CPT I or CPT II) Deficiency; Very Long Chain Acyl-CoA Dehydrogenase (VLCAD) Deficiency; Long-chain 3-hydroxy-acyl-CoA Dehydrogenase (LCHAD) Deficiency; Trifunctional Protein (TFP) Deficiency; Carnitine-acylcarnitine Translocase (CACT) Deficiency

Tuning of acyl-ACP thioesterase activity directed for tailored fatty acid synthesis.

PubMed

Feng, Yanbin; Zhang, Yunxiu; Wang, Yayue; Liu, Jiao; Liu, Yinghui; Cao, Xupeng; Xue, Song

2018-04-01

Medium-chain fatty acids have attracted significant attention as sources of biofuels in recent years. Acyl-ACP thioesterase, which is considered as the key enzyme to determine the carbon chain length, catalyzes the termination of de novo fatty acid synthesis. Although recombinant medium-chain acyl-ACP thioesterase (TE) affects the fatty acid profile in heterologous cells, tailoring of the fatty acid composition merely by engineering a specific TE is still intractable. In this study, the activity of a C8-C10-specific thioesterase FatB2 from Cuphea hookeriana on C10-ACP was quantified twice as high as that on C8-ACP based on a synthetic C8-C16 acyl-ACP pool in vitro. Whereas in vivo, it was demonstrated that ChFatB2 preferred to accumulate C8 fatty acids with 84.9% composition in the ChFatB2-engineered E. coli strain. To achieve C10 fatty acid production, ChFatB2 was rationally tuned based on structural investigation and enzymatic analysis. An I198E mutant was identified to redistribute the C8-ACP flow, resulting in C10 fatty acid being produced as the principal component at 57.6% of total fatty acids in vivo. It was demonstrated that the activity of TE relative to β-ketoacyl-ACP synthases (KAS) directly determined the fatty acid composition. Our results provide a prospective strategy in tailoring fatty acid synthesis by tuning of TE activities based on TE-ACP interaction.

Natural separation of the acyl-CoA ligase reaction results in a non-adenylating enzyme.

PubMed

Wang, Nan; Rudolf, Jeffrey D; Dong, Liao-Bin; Osipiuk, Jerzy; Hatzos-Skintges, Catherine; Endres, Michael; Chang, Chin-Yuan; Babnigg, Gyorgy; Joachimiak, Andrzej; Phillips, George N; Shen, Ben

2018-06-04

Acyl-coenzyme A (CoA) ligases catalyze the activation of carboxylic acids via a two-step reaction of adenylation followed by thioesterification. Here, we report the discovery of a non-adenylating acyl-CoA ligase PtmA2 and the functional separation of an acyl-CoA ligase reaction. Both PtmA1 and PtmA2, two acyl-CoA ligases from the biosynthetic pathway of platensimycin and platencin, are necessary for the two steps of CoA activation. Gene inactivation of ptmA1 and ptmA2 resulted in the accumulation of free acid and adenylate intermediates, respectively. Enzymatic and structural characterization of PtmA2 confirmed its ability to only catalyze thioesterification. Structural characterization of PtmA2 revealed it binds both free acid and adenylate substrates and undergoes the established mechanism of domain alternation. Finally, site-directed mutagenesis restored both the adenylation and complete CoA activation reactions. This study challenges the currently accepted paradigm of adenylating enzymes and inspires future investigations on functionally separated acyl-CoA ligases and their ramifications in biology.

Identification of amino acid residues involved in substrate specificity of plant acyl-ACP thioesterases using a bioinformatics-guided approach

PubMed Central

Mayer, Kimberly M; Shanklin, John

2007-01-01

Background The large amount of available sequence information for the plant acyl-ACP thioesterases (TEs) made it possible to use a bioinformatics-guided approach to identify amino acid residues involved in substrate specificity. The Conserved Property Difference Locator (CPDL) program allowed the identification of putative specificity-determining residues that differ between the FatA and FatB TE classes. Six of the FatA residue differences identified by CPDL were incorporated into the FatB-like parent via site-directed mutagenesis and the effect of each on TE activity was determined. Variants were expressed in E. coli strain K27 that allows determination of enzyme activity by GCMS analysis of fatty acids released into the medium. Results Substitutions at four of the positions (74, 86, 141, and 174) changed substrate specificity to varying degrees while changes at the remaining two positions, 110 and 221, essentially inactivated the thioesterase. The effects of substitutions at positions 74, 141, and 174 (3-MUT) or 74, 86, 141, 174 (4-MUT) were not additive with respect to specificity. Conclusion Four of six putative specificity determining positions in plant TEs, identified with the use of CPDL, were validated experimentally; a novel colorimetric screen that discriminates between active and inactive TEs is also presented. PMID:17201914

Active-Site Protonation States in an Acyl-Enzyme Intermediate of a Class A β-Lactamase with a Monobactam Substrate.

PubMed

Vandavasi, Venu Gopal; Langan, Patricia S; Weiss, Kevin L; Parks, Jerry M; Cooper, Jonathan B; Ginell, Stephan L; Coates, Leighton

2017-01-01

The monobactam antibiotic aztreonam is used to treat cystic fibrosis patients with chronic pulmonary infections colonized by Pseudomonas aeruginosa strains expressing CTX-M extended-spectrum β-lactamases. The protonation states of active-site residues that are responsible for hydrolysis have been determined previously for the apo form of a CTX-M β-lactamase but not for a monobactam acyl-enzyme intermediate. Here we used neutron and high-resolution X-ray crystallography to probe the mechanism by which CTX-M extended-spectrum β-lactamases hydrolyze monobactam antibiotics. In these first reported structures of a class A β-lactamase in an acyl-enzyme complex with aztreonam, we directly observed most of the hydrogen atoms (as deuterium) within the active site. Although Lys 234 is fully protonated in the acyl intermediate, we found that Lys 73 is neutral. These findings are consistent with Lys 73 being able to serve as a general base during the acylation part of the catalytic mechanism, as previously proposed. Copyright © 2016 Vandavasi et al.

Unusual heme iron-lipid acyl chain coordination in Escherichia coli flavohemoglobin.

PubMed

D'Angelo, Paola; Lucarelli, Debora; della Longa, Stefano; Benfatto, Maurizio; Hazemann, Jean Louis; Feis, Alessandro; Smulevich, Giulietta; Ilari, Andrea; Bonamore, Alessandra; Boffi, Alberto

2004-06-01

Escherichia coli flavohemoglobin is endowed with the notable property of binding specifically unsaturated and/or cyclopropanated fatty acids both as free acids or incorporated into a phospholipid molecule. Unsaturated or cyclopropanated fatty acid binding to the ferric heme results in a spectral change observed in the visible absorption, resonance Raman, extended x-ray absorption fine spectroscopy (EXAFS), and x-ray absorption near edge spectroscopy (XANES) spectra. Resonance Raman spectra, measured on the flavohemoglobin heme domain, demonstrate that the lipid (linoleic acid or total lipid extracts)-induced spectral signals correspond to a transition from a five-coordinated (typical of the ligand-free protein) to a hexacoordinated, high spin heme iron. EXAFS and XANES measurements have been carried out both on the lipid-free and on the lipid-bound protein to assign the nature of ligand in the sixth coordination position of the ferric heme iron. EXAFS data analysis is consistent with the presence of a couple of atoms in the sixth coordination position at 2.7 A in the lipid-bound derivative (bonding interaction), whereas a contribution at 3.54 A (nonbonding interaction) can be singled out in the lipid-free protein. This last contribution is assigned to the CD1 carbon atoms of the distal LeuE11, in full agreement with crystallographic data on the lipid-free protein at 1.6 A resolution obtained in the present work. Thus, the contributions at 2.7 A distance from the heme iron are assigned to a couple of carbon atoms of the lipid acyl chain, possibly corresponding to the unsaturated carbons of the linoleic acid.

Enhanced separation and analysis procedure reveals production of tri-acylated mannosylerythritol lipids by Pseudozyma aphidis.

PubMed

Goossens, Eliane; Wijnants, Marc; Packet, Dirk; Lemière, Filip

2016-11-01

Mannosylerythritol lipids (MELs) are one of the most promising biosurfactants because of their high fermentation yields (>100 g l -1 ) and during the last two decades they have gained a lot of attention due to their interesting self-assembling properties and biological activities. In this study, MELs were produced by fed-batch bioreactor fermentation of rapeseed oil with Pseudozyma aphidis MUCL 27852. This high-level MEL-producing yeast secretes four conventional MEL structures, -A, -B, -C and -D, which differ in their degree of acetylation. During our research, unknown compounds synthesized by P. aphidis were detected by thin-layer chromatography. The unknown compounds were separated by flash chromatography and identified as tri-acylated MELs by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). The third fatty acid chain on the tri-acylated MELs was positioned on the primary alcohol of the erythritol moiety and comprised long-chain acids, mainly oleic and linoleic acid, which are not found in conventional di-acylated MELs. Furthermore, the LC-MS analysis time of conventional MELs was reduced to almost one-third by switching from HPLC-MS/MS to ultraperformance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Provided optimization of the fermentation yield, P. aphidis could be an interesting novel producer of tri-acylated MELs and, thereby expand the supply and applicability of biosurfactants.

Acylated and unacylated ghrelin confer neuroprotection to mesencephalic neurons.

PubMed

Wagner, Johanna; Vulinović, Franca; Grünewald, Anne; Unger, Marcus M; Möller, Jens C; Klein, Christine; Michel, Patrick P; Ries, Vincent; Oertel, Wolfgang H; Alvarez-Fischer, Daniel

2017-12-04

The polypeptide ghrelin is an endogenous ligand at the growth hormone secretagogue receptor 1a. To ghrelin multiple functions have been ascribed including promotion of gastrointestinal motility. Postprandial ghrelin levels have been reported to be reduced in patients suffering from Parkinson disease (PD). Experimental studies revealed neuroprotective effects of ghrelin in different PD models. The purpose of the present study was (i) to further elucidate the mechanism underlying the neuroprotective action of ghrelin and (ii) to determine whether these effects occur with both the acylated and the unacylated form. The study was conducted in primary mesencephalic cultures treated with mitochondrial complex I and complex II inhibitors. We show that protective effects of ghrelin against complex I inhibition with MPP + were independent of the acylation status of ghrelin, although acylated ghrelin appeared to be more potent. Protection by both forms was also observed when neurons were exposed to the complex II inhibitor 3-NP. Both forms led to higher oxygen consumption rates upon electron transport chain uncoupling, indicating that the two peptides may exert uncoupling effects themselves. We demonstrate that the rescue provided by ghrelin required calcium influx through L-type voltage-gated calcium channels. Whereas the protective effects of acylated ghrelin required receptor binding, effects of the unacylated form remained unaffected by treatment with a ghrelin receptor antagonist. Importantly, inhibition of ghrelin O-acyltransferase failed to reduce the activity of unacylated ghrelin. Overall, our data suggest that both acylated and unacylated ghrelin afford protection to dopamine neurons but through mechanisms that only partially overlap. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

Head-group acylation of monogalactosyldiacylglycerol is a common stress response, and the acyl-galactose acyl composition varies with the plant species and applied stress

PubMed Central

Vu, Hieu Sy; Roth, Mary R.; Tamura, Pamela; Samarakoon, Thilani; Shiva, Sunitha; Honey, Samuel; Lowe, Kaleb; Schmelz, Eric A.; Williams, Todd D.; Welti, Ruth

2014-01-01

Formation of galactose-acylated monogalactosyldiacylglycerols has been shown to be induced by leaf homogenization, mechanical wounding, avirulent bacterial infection, and thawing after snap-freezing. Here, lipidomic analysis using mass spectrometry showed that galactose-acylated monogalactosyldiacylglycerols, formed in wheat (Triticum aestivum) and tomato (Solanum lycopersicum) leaves upon wounding, have acyl-galactose profiles that differ from those of wounded Arabidopsis thaliana, indicating that different plant species accumulate different acyl-galactose components in response to the same stress. Additionally, the composition of the acyl-galactose component of Arabidopsis acMGDG depends on the stress treatment. After sub-lethal freezing treatment, acMGDG contained mainly non-oxidized fatty acids esterified to galactose, whereas mostly oxidized fatty acids accumulated on galactose after wounding or bacterial infection. Compositional data are consistent with acMGDG being formed in vivo by transacylation with fatty acids from digalactosyldiacylglycerols. Oxophytodienoic acid, an oxidized fatty acid, was more concentrated on the galactosyl ring of acylated monogalactosyldiacylglycerols than in galactolipids in general. Also, oxidized fatty acid-containing acylated monogalactosyldiacylglycerols increased cumulatively when wounded Arabidopsis leaves were wounded again. These findings suggest that, in Arabidopsis, the pool of galactose-acylated monogalactosyldiacylglycerols may serve to sequester oxidized fatty acids during stress responses. PMID:24286212

The Acyl Desaturase CER17 Is Involved in Producing Wax Unsaturated Primary Alcohols and Cutin Monomers1[OPEN

PubMed Central

Yang, Xianpeng; Zhao, Huayan; Kosma, Dylan K.; Dyer, John M.; Li, Rongjun; Liu, Xiulin; Wang, Zhouya; Jenks, Matthew A.

2017-01-01

We report n-6 monounsaturated primary alcohols (C26:1, C28:1, and C30:1 homologs) in the cuticular waxes of Arabidopsis (Arabidopsis thaliana) inflorescence stem, a class of wax not previously reported in Arabidopsis. The Arabidopsis cer17 mutant was completely deficient in these monounsaturated alcohols, and CER17 was found to encode a predicted ACYL-COENZYME A DESATURASE LIKE4 (ADS4). Studies of the Arabidopsis cer4 mutant and yeast variously expressing CER4 (a predicted fatty acyl-CoA reductase) with CER17/ADS4, demonstrated CER4’s principal role in synthesis of these monounsaturated alcohols. Besides unsaturated alcohol deficiency, cer17 mutants exhibited a thickened and irregular cuticle ultrastructure and increased amounts of cutin monomers. Although unsaturated alcohols were absent throughout the cer17 stem, the mutation’s effects on cutin monomers and cuticle ultrastructure were much more severe in distal than basal stems, consistent with observations that the CER17/ADS4 transcript was much more abundant in distal than basal stems. Furthermore, distal but not basal stems of a double mutant deficient for both CER17/ADS4 and LONG-CHAIN ACYL-COA SYNTHETASE1 produced even more cutin monomers and a thicker and more disorganized cuticle ultrastructure and higher cuticle permeability than observed for wild type or either mutant parent, indicating a dramatic genetic interaction on conversion of very long chain acyl-CoA precursors. These results provide evidence that CER17/ADS4 performs n-6 desaturation of very long chain acyl-CoAs in both distal and basal stems and has a major function associated with governing cutin monomer amounts primarily in the distal segments of the inflorescence stem. PMID:28069670

Identification of a Δ5-like fatty acyl desaturase from the cephalopod Octopus vulgaris (Cuvier 1797) involved in the biosynthesis of essential fatty acids.

PubMed

Monroig, Oscar; Navarro, Juan C; Dick, James R; Alemany, Frederic; Tocher, Douglas R

2012-08-01

Long-chain polyunsaturated fatty acids (LC-PUFA) have been identified as essential compounds for common octopus (Octopus vulgaris), but precise dietary requirements have not been determined due, in part, to the inherent difficulties of performing feeding trials on paralarvae. Our objective is to establish the essential fatty acid (EFA) requirements for paralarval stages of the common octopus through characterisation of the enzymes of endogenous LC-PUFA biosynthetic pathways. In this study, we isolated a cDNA with high homology to fatty acyl desaturases (Fad). Functional characterisation in recombinant yeast showed that the octopus Fad exhibited Δ5-desaturation activity towards saturated and polyunsaturated fatty acyl substrates. Thus, it efficiently converted the yeast's endogenous 16:0 and 18:0 to 16:1n-11 and 18:1n-13, respectively, and desaturated exogenously added PUFA substrates 20:4n-3 and 20:3n-6 to 20:5n-3 (EPA) and 20:4n-6 (ARA), respectively. Although the Δ5 Fad enables common octopus to produce EPA and ARA, the low availability of its adequate substrates 20:4n-3 and 20:3n-6, either in the diet or by limited endogenous synthesis from C(18) PUFA, might indicate that EPA and ARA are indeed EFA for this species. Interestingly, the octopus Δ5 Fad can also participate in the biosynthesis of non-methylene-interrupted FA, PUFA that are generally uncommon in vertebrates but have been found previously in marine invertebrates, including molluscs, and now also confirmed to be present in specific tissues of common octopus.

Influence of the side chain and substrate on polythiophene thin film surface, bulk, and buried interfacial structures.

PubMed

Xiao, Minyu; Jasensky, Joshua; Zhang, Xiaoxian; Li, Yaoxin; Pichan, Cayla; Lu, Xiaolin; Chen, Zhan

2016-08-10

The molecular structures of organic semiconducting thin films mediate the performance of various devices composed of such materials. To fully understand how the structures of organic semiconductors alter on substrates due to different polymer side chains and different interfacial interactions, thin films of two kinds of polythiophene derivatives with different side-chains, poly(3-hexylthiophene) (P3HT) and poly(3-potassium-6-hexanoate thiophene) (P3KHT), were deposited and compared on various surfaces. A combination of analytical tools was applied in this research: contact angle goniometry and X-ray photoelectron spectroscopy (XPS) were used to characterize substrate dielectric surfaces with varied hydrophobicity for polymer film deposition; X-ray diffraction and UV-vis spectroscopy were used to examine the polythiophene film bulk structure; sum frequency generation (SFG) vibrational spectroscopy was utilized to probe the molecular structures of polymer film surfaces in air and buried solid/solid interfaces. Both side-chain hydrophobicity and substrate hydrophobicity were found to mediate the crystallinity of the polythiophene film, as well as the orientation of the thiophene ring within the polymer backbone at the buried polymer/substrate interface and the polymer thin film surface in air. For the same type of polythiophene film deposited on different substrates, a more hydrophobic substrate surface induced thiophene ring alignment with the surface normal at both the buried interface and on the surface in air. For different films (P3HT vs. P3KHT) deposited on the same dielectric substrate, a more hydrophobic polythiophene side chain caused the thiophene ring to align more towards the surface at the buried polymer/substrate interface and on the surface in air. We believe that the polythiophene surface, bulk, and buried interfacial molecular structures all influence the hole mobility within the polythiophene film. Successful characterization of an organic conducting

Production of long chain alcohols and alkanes upon coexpression of an acyl-ACP reductase and aldehyde-deformylating oxgenase with a bacterial type-I fatty acid synthase in E. coli

DOE PAGES

Coursolle, Dan; Shanklin, John; Lian, Jiazhang; ...

2015-06-23

Microbial long chain alcohols and alkanes are renewable biofuels that could one day replace petroleum-derived fuels. Here we report a novel pathway for high efficiency production of these products in Escherichia coli strain BL21(DE3). We first identified the acyl-ACP reductase/aldehyde deformylase combinations with the highest activity in this strain. Next, we used catalase coexpression to remove toxic byproducts and increase the overall titer. Finally, by introducing the type-I fatty acid synthase from Corynebacterium ammoniagenes, we were able to bypass host regulatory mechanisms of fatty acid synthesis that have thus far hampered efforts to optimize the yield of acyl-ACP-derived products inmore » BL21(DE3). When all these engineering strategies were combined with subsequent optimization of fermentation conditions, we were able to achieve a final titer around 100 mg/L long chain alcohol/alkane products including a 57 mg/L titer of pentadecane, the highest titer reported in E. coli BL21(DE3) to date. The expression of prokaryotic type-I fatty acid synthases offer a unique strategy to produce fatty acid-derived products in E. coli that does not rely exclusively on the endogenous type-II fatty acid synthase system.« less

Overexpression of human fatty acid transport protein 2/very long chain acyl-CoA synthetase 1 (FATP2/Acsvl1) reveals distinct patterns of trafficking of exogenous fatty acids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Melton, Elaina M.; Center for Cardiovascular Sciences, Albany Medical College, Albany, NY; Cerny, Ronald L.

Highlights: •Roles of FATP2 in fatty acid transport/activation contribute to lipid homeostasis. •Use of 13C- and D-labeled fatty acids provide novel insights into FATP2 function. •FATP2-dependent trafficking of FA into phospholipids results in distinctive profiles. •FATP2 functions in the transport and activation pathways for exogenous fatty acids. -- Abstract: In mammals, the fatty acid transport proteins (FATP1 through FATP6) are members of a highly conserved family of proteins, which function in fatty acid transport proceeding through vectorial acylation and in the activation of very long chain fatty acids, branched chain fatty acids and secondary bile acids. FATP1, 2 and 4,more » for example directly function in fatty acid transport and very long chain fatty acids activation while FATP5 does not function in fatty acid transport but activates secondary bile acids. In the present work, we have used stable isotopically labeled fatty acids differing in carbon length and saturation in cells expressing FATP2 to gain further insights into how this protein functions in fatty acid transport and intracellular fatty acid trafficking. Our previous studies showed the expression of FATP2 modestly increased C16:0-CoA and C20:4-CoA and significantly increased C18:3-CoA and C22:6-CoA after 4 h. The increases in C16:0-CoA and C18:3-CoA suggest FATP2 must necessarily partner with a long chain acyl CoA synthetase (Acsl) to generate C16:0-CoA and C18:3-CoA through vectorial acylation. The very long chain acyl CoA synthetase activity of FATP2 is consistent in the generation of C20:4-CoA and C22:6-CoA coincident with transport from their respective exogenous fatty acids. The trafficking of exogenous fatty acids into phosphatidic acid (PA) and into the major classes of phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidyserine (PS)) resulted in distinctive profiles, which changed with the expression of FATP2. The

Crystal structure and substrate specificity of the [beta]-ketoacyl-acyl carrier protein synthase III (FabH) from Staphylococcus aureus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qiu, Xiayang; Choudhry, Anthony E.; Janson, Cheryl A.

{beta}-Ketoacyl-ACP synthase III (FabH), an essential enzyme for bacterial viability, catalyzes the initiation of fatty acid elongation by condensing malonyl-ACP with acetyl-CoA. We have determined the crystal structure of FabH from Staphylococcus aureus, a Gram-positive human pathogen, to 2 {angstrom} resolution. Although the overall structure of S. aureus FabH is similar to that of Escherichia coli FabH, the primer binding pocket in S. aureus FabH is significantly larger than that present in E. coli FabH. The structural differences, which agree with kinetic parameters, provide explanation for the observed varying substrate specificity for E. coli and S. aureus FabH. The rankmore » order of activity of S. aureus FabH with various acyl-CoA primers was as follows: isobutyryl- > hexanoyl- > butyryl- > isovaleryl- >> acetyl-CoA. The availability of crystal structure may aid in designing potent, selective inhibitors of S. aureus FabH.« less

Chemoselective O-acylation of hydroxyamino acids and amino alcohols under acidic reaction conditions: History, scope and applications

PubMed Central

2015-01-01

Summary Amino acids, whether natural, semisynthetic or synthetic, are among the most important and useful chiral building blocks available for organic chemical synthesis. In principle, they can function as inexpensive, chiral and densely functionalized starting materials. On the other hand, the use of amino acid starting materials routinely necessitates protective group chemistry, and in reality, large-scale preparations of even the simplest side-chain derivatives of many amino acids often become annoyingly strenuous due to the necessity of employing protecting groups, on one or more of the amino acid functionalities, during the synthetic sequence. However, in the case of hydroxyamino acids such as hydroxyproline, serine, threonine, tyrosine and 3,4-dihydroxyphenylalanine (DOPA), many O-acyl side-chain derivatives are directly accessible via a particularly expedient and scalable method not commonly applied until recently. Direct acylation of unprotected hydroxyamino acids with acyl halides or carboxylic anhydrides under appropriately acidic reaction conditions renders possible chemoselective O-acylation, furnishing the corresponding side-chain esters directly, on multigram-scale, in a single step, and without chromatographic purification. Assuming a certain degree of stability under acidic reaction conditions, the method is also applicable for a number of related compounds, such as various amino alcohols and the thiol-functional amino acid cysteine. While the basic methodology underlying this approach has been known for decades, it has evolved through recent developments connected to amino acid-derived chiral organocatalysts to become a more widely recognized procedure for large-scale preparation of many useful side-chain derivatives of hydroxyamino acids and related compounds. Such derivatives are useful in peptide chemistry and drug development, as amino acid amphiphiles for asymmetric catalysis, and as amino acid acrylic precursors for preparation of

Acylation of Ferrocene: A Greener Approach

ERIC Educational Resources Information Center

Birdwhistell, Kurt R.; Nguyen, Andy; Ramos, Eric J.; Kobelja, Robert

2008-01-01

The acylation of ferrocene is a common reaction used in organic laboratories to demonstrate Friedel-Crafts acylation and the purification of compounds using column chromatography. This article describes an acylation of ferrocene experiment that is more eco-friendly than the conventional acylation experiment. The traditional experiment was modified…

Structurally divergent lysophosphatidic acid acyltransferases with high selectivity for saturated medium chain fatty acids from Cuphea seeds.

PubMed

Kim, Hae Jin; Silva, Jillian E; Iskandarov, Umidjon; Andersson, Mariette; Cahoon, Rebecca E; Mockaitis, Keithanne; Cahoon, Edgar B

2015-12-01

Lysophosphatidic acid acyltransferase (LPAT) catalyzes acylation of the sn-2 position on lysophosphatidic acid by an acyl CoA substrate to produce the phosphatidic acid precursor of polar glycerolipids and triacylglycerols (TAGs). In the case of TAGs, this reaction is typically catalyzed by an LPAT2 from microsomal LPAT class A that has high specificity for C18 fatty acids containing Δ9 unsaturation. Because of this specificity, the occurrence of saturated fatty acids in the TAG sn-2 position is infrequent in seed oils. To identify LPATs with variant substrate specificities, deep transcriptomic mining was performed on seeds of two Cuphea species producing TAGs that are highly enriched in saturated C8 and C10 fatty acids. From these analyses, cDNAs for seven previously unreported LPATs were identified, including cDNAs from Cuphea viscosissima (CvLPAT2) and Cuphea avigera var. pulcherrima (CpuLPAT2a) encoding microsomal, seed-specific class A LPAT2s and a cDNA from C. avigera var. pulcherrima (CpuLPATB) encoding a microsomal, seed-specific LPAT from the bacterial-type class B. The activities of these enzymes were characterized in Camelina sativa by seed-specific co-expression with cDNAs for various Cuphea FatB acyl-acyl carrier protein thioesterases (FatB) that produce a variety of saturated medium-chain fatty acids. CvLPAT2 and CpuLPAT2a expression resulted in accumulation of 10:0 fatty acids in the Camelina sativa TAG sn-2 position, indicating a 10:0 CoA specificity that has not been previously described for plant LPATs. CpuLPATB expression generated TAGs with 14:0 at the sn-2 position, but not 10:0. Identification of these LPATs provides tools for understanding the structural basis of LPAT substrate specificity and for generating altered oil functionalities. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

High-level accumulation of oleyl oleate in plant seed oil by abundant supply of oleic acid substrates to efficient wax ester synthesis enzymes.

PubMed

Yu, Dan; Hornung, Ellen; Iven, Tim; Feussner, Ivo

2018-01-01

Biotechnology enables the production of high-valued industrial feedstocks from plant seed oil. The plant-derived wax esters with long-chain monounsaturated acyl moieties, like oleyl oleate, have favorite properties for lubrication. For biosynthesis of wax esters using acyl-CoA substrates, expressions of a fatty acyl reductase (FAR) and a wax synthase (WS) in seeds are sufficient. For optimization of the enzymatic activity and subcellular localization of wax ester synthesis enzymes, two fusion proteins were created, which showed wax ester-forming activities in Saccharomyces cerevisiae . To promote the formation of oleyl oleate in seed oil, WSs from Acinetobactor baylyi ( Ab WSD1) and Marinobacter aquaeolei ( Ma WS2), as well as the two created fusion proteins were tested in Arabidopsis to evaluate their abilities and substrate preference for wax ester production. The tested seven enzyme combinations resulted in different yields and compositions of wax esters. Expression of a FAR of Marinobacter aquaeolei ( Ma FAR) with Ab WSD1 or Ma WS2 led to a high incorporation of C 18 substrates in wax esters. The Ma FAR/TM Mm AWAT2- Ab WSD1 combination resulted in the incorporation of more C 18:1 alcohol and C 18:0 acyl moieties into wax esters compared with Ma FAR/ Ab WSD1. The fusion protein of a WS from Simmondsia chinensis ( Sc WS) with MaFAR exhibited higher specificity toward C 20:1 substrates in preference to C 18:1 substrates. Expression of Ma FAR/ Ab WSD1 in the Arabidopsis fad2 fae1 double mutant resulted in the accumulation of oleyl oleate (18:1/18:1) in up to 62 mol% of total wax esters in seed oil, which was much higher than the 15 mol% reached by Ma FAR/ Ab WSD1 in Arabidopsis Col-0 background. In order to increase the level of oleyl oleate in seed oil of Camelina , lines expressing Ma FAR/ Sc WS were crossed with a transgenic high oleate line. The resulting plants accumulated up to >40 mg g seed -1 of wax esters, containing 27-34 mol% oleyl oleate. The

Short- and medium-chain fatty acids in energy metabolism: the cellular perspective

PubMed Central

Schönfeld, Peter; Wojtczak, Lech

2016-01-01

Short- and medium-chain fatty acids (SCFAs and MCFAs), independently of their cellular signaling functions, are important substrates of the energy metabolism and anabolic processes in mammals. SCFAs are mostly generated by colonic bacteria and are predominantly metabolized by enterocytes and liver, whereas MCFAs arise mostly from dietary triglycerides, among them milk and dairy products. A common feature of SCFAs and MCFAs is their carnitine-independent uptake and intramitochondrial activation to acyl-CoA thioesters. Contrary to long-chain fatty acids, the cellular metabolism of SCFAs and MCFAs depends to a lesser extent on fatty acid-binding proteins. SCFAs and MCFAs modulate tissue metabolism of carbohydrates and lipids, as manifested by a mostly inhibitory effect on glycolysis and stimulation of lipogenesis or gluconeogenesis. SCFAs and MCFAs exert no or only weak protonophoric and lytic activities in mitochondria and do not significantly impair the electron transport in the respiratory chain. SCFAs and MCFAs modulate mitochondrial energy production by two mechanisms: they provide reducing equivalents to the respiratory chain and partly decrease efficacy of oxidative ATP synthesis. PMID:27080715

Localization of acyl ghrelin- and des-acyl ghrelin-immunoreactive cells in the rat stomach and their responses to intragastric pH.

PubMed

Mizutani, Makoto; Atsuchi, Kaori; Asakawa, Akihiro; Matsuda, Norifumi; Fujimura, Masaki; Inui, Akio; Kato, Ikuo; Fujimiya, Mineko

2009-11-01

Acyl ghrelin has a 28-amino acid sequence with O-n-octanoyl acid modification at the serine 3 position, whereas des-acyl ghrelin has no octanoyl acid modification. Although these peptides exert different physiological functions, no previous studies have shown the different localization of acyl ghrelin and des-acyl ghrelin in the stomach. Here we have developed an antibody specific for des-acyl ghrelin that does not crossreact with acyl ghrelin. Both acyl ghrelin- and des-acyl ghrelin-immunoreactive cells were distributed in the oxyntic and antral mucosa of the rat stomach, with higher density in the antral mucosa than oxyntic mucosa. Immunofluorescence double staining showed that acyl ghrelin- and des-acyl ghrelin-positive reactions overlapped in closed-type round cells, whereas des-acyl ghrelin-positive reaction was found in open-type cells in which acyl ghrelin was negative. Acyl ghrelin-/des-acyl ghrelin-positive closed-type cells contain obestatin; on the other hand, des-acyl ghrelin-positive open-type cells contain somatostatin. We measured the release of acyl ghrelin and des-acyl ghrelin in vascularly perfused rat stomach by ELISA, and the effects of different intragastric pH levels on the release of each peptide were examined. The release of des-acyl ghrelin from the perfused stomach was greater at pH 2 than at pH 4; however, the release of acyl ghrelin was not affected by intragastric pH. The present study demonstrated the differential localization of acyl ghrelin and des-acyl ghrelin in the rat stomach and their different responses to the intragastric pH.

An Open-label Phase 2 Study of UX007 (Triheptanoin) in Subjects With Long-Chain Fatty Acid Oxidation Disorders (LC-FAOD)

ClinicalTrials.gov

2018-06-01

Long-chain Fatty Acid Oxidation Disorders (LC-FAOD); Carnitine Palmitoyltransferase (CPT II) Deficiency; Very Long Chain Acyl-CoA Dehydrogenase (VLCAD) Deficiency; Longchain 3-hydroxy-acyl-CoA Dehydrogenase (LCHAD) Deficiency; Trifunctional Protein (TFP) Deficiency

Arabidopsis membrane-associated acyl-CoA-binding protein ACBP1 is involved in stem cuticle formation

PubMed Central

Xue, Yan; Xiao, Shi; Kim, Juyoung; Lung, Shiu-Cheung; Chen, Liang; Tanner, Julian A.; Suh, Mi Chung; Chye, Mee-Len

2014-01-01

The membrane-anchored Arabidopsis thaliana ACYL-COA-BINDING PROTEIN1 (AtACBP1) plays important roles in embryogenesis and abiotic stress responses, and interacts with long-chain (LC) acyl-CoA esters. Here, AtACBP1 function in stem cuticle formation was investigated. Transgenic Arabidopsis transformed with an AtACBP1pro::GUS construct revealed β-glucuronidase (GUS) expression on the stem (but not leaf) surface, suggesting a specific role in stem cuticle formation. Isothermal titration calorimetry results revealed that (His)6-tagged recombinant AtACBP1 interacts with LC acyl-CoA esters (18:1-, 18:2-, and 18:3-CoAs) and very-long-chain (VLC) acyl-CoA esters (24:0-, 25:0-, and 26:0-CoAs). VLC fatty acids have been previously demonstrated to act as precursors in wax biosynthesis. Gas chromatography (GC)–flame ionization detector (FID) and GC–mass spectrometry (MS) analyses revealed that an acbp1 mutant showed a reduction in stem and leaf cuticular wax and stem cutin monomer composition in comparison with the wild type (Col-0). Consequently, the acbp1 mutant showed fewer wax crystals on the stem surface in scanning electron microscopy and an irregular stem cuticle layer in transmission electron microscopy in comparison with the wild type. Also, the mutant stems consistently showed a decline in expression of cuticular wax and cutin biosynthetic genes in comparison with the wild type, and the mutant leaves were more susceptible to infection by the necrotrophic pathogen Botrytis cinerea. Taken together, these findings suggest that AtACBP1 participates in Arabidopsis stem cuticle formation by trafficking VLC acyl-CoAs. PMID:25053648

Correcting false positive medium-chain acyl-CoA dehydrogenase deficiency results from newborn screening; synthesis, purification, and standardization of branched-chain C8 acylcarnitines for use in their selective and accurate absolute quantitation by UHPLC-MS/MS.

PubMed

Minkler, Paul E; Stoll, Maria S K; Ingalls, Stephen T; Hoppel, Charles L

2017-04-01

While selectively quantifying acylcarnitines in thousands of patient samples using UHPLC-MS/MS, we have occasionally observed unidentified branched-chain C8 acylcarnitines. Such observations are not possible using tandem MS methods, which generate pseudo-quantitative acylcarnitine "profiles". Since these "profiles" select for mass alone, they cannot distinguish authentic signal from isobaric and isomeric interferences. For example, some of the samples containing branched-chain C8 acylcarnitines were, in fact, expanded newborn screening false positive "profiles" for medium-chain acyl-CoA dehydrogenase deficiency (MCADD). Using our fast, highly selective, and quantitatively accurate UHPLC-MS/MS acylcarnitine determination method, we corrected the false positive tandem MS results and reported the sample results as normal for octanoylcarnitine (the marker for MCADD). From instances such as these, we decided to further investigate the presence of branched-chain C8 acylcarnitines in patient samples. To accomplish this, we synthesized and chromatographically characterized several branched-chain C8 acylcarnitines (in addition to valproylcarnitine): 2-methylheptanoylcarnitine, 6-methylheptanoylcarnitine, 2,2-dimethylhexanoylcarnitine, 3,3-dimethylhexanoylcarnitine, 3,5-dimethylhexanoylcarnitine, 2-ethylhexanoylcarnitine, and 2,4,4-trimethylpentanoylcarnitine. We then compared their behavior with branched-chain C8 acylcarnitines observed in patient samples and demonstrated our ability to chromographically resolve, and thus distinguish, octanoylcarnitine from branched-chain C8 acylcarnitines, correcting false positive MCADD results from expanded newborn screening. Copyright © 2017 Elsevier Inc. All rights reserved.

Recombinant expression, purification, and characterization of an acyl-CoA binding protein from Aspergillus oryzae.

PubMed

Hao, Qing; Liu, Xiaoguang; Zhao, Guozhong; Jiang, Lu; Li, Ming; Zeng, Bin

2016-03-01

To characterize biochemically the lipid metabolism-regulating acyl-CoA binding protein (ACBP) from the industrially-important fungus Aspergillus oryzae. A full-length cDNA encoding a candidate ACBP from A. oryzae (AoACBP) was cloned and expressed in Escherichia coli as a maltose-binding protein (MBP) fusion protein. The MBP-AoACBP protein was purified by an amylose resin chromatography column. SDS-PAGE showed that MBP-AoACBP has an estimated molecular weight of 82 kDa. Microscale thermophoresis binding assay showed that the recombinant AoACBP displayed much greater affinity for palmitoyl-CoA (K d = 80 nM) than for myristoyl-CoA (K d = 510 nM), thus demonstrating the preference of AoACBP for long-chain acyl-CoA. The data support the identification of AoACBP as a long-chain ACBP in A. oryzae.

Production of medium chain fatty acid rich mustard oil using packed bed bioreactor.

PubMed

Sengupta, Avery; Roy, Susmita; Mukherjee, Sohini; Ghosh, Mahua

2015-01-01

A comparative study was done on the production of different medium chain fatty acid (MCFA) rich mustard oil using a stirred tank batchreactor (STBR) and packed bed bio reactor (PBBR) using three commercially available immobilised lipases viz. Thermomyces lanuginosus, Candida antarctica and Rhizomucor meihe. Three different MCFAs capric, caprylic and lauric acids were incorporated in the mustard oil. Reaction parameters, such as substrate molar ratio, reaction temperature and enzyme concentration were standardized in the STBR and maintained in the PBBR. To provide equal time of residence between the substrate and enzyme in both the reactors for the same amount of substrates, the substrate flow rate in the PBBR was maintainedat 0.27 ml/min. Gas liquid chromatography was used to monitor the incorporation of MCFA in mustard oil. The study showed that the PBBR was more efficient than the STBR in the synthesis of structured lipids with less migration of acyl groups. The physico-chemical parameters of the product along with fatty acid composition in all positions and sn-2 positions were also determined.

Acyl CoA profiles of transgenic plants that accumulate medium-chain fatty acids indicate inefficient storage lipid synthesis in developing oilseeds.

PubMed

Larson, Tony R; Edgell, Teresa; Byrne, James; Dehesh, Katayoon; Graham, Ian A

2002-11-01

Several Brassica napus lines transformed with genes responsible for the synthesis of medium- or long-chain fatty acids were examined to determine limiting factor(s) for the subsequent accumulation of these fatty acids in seed lipids. Examination of a decanoic acid (10:0) accumulating line revealed a disproportionately high concentration of 10:0 CoA during seed development compared to long-chain acyl CoAs isolated from the same tissues, suggesting that poor incorporation of 10:0 CoA into seed lipids limits 10:0 fatty acid accumulation. This relationship was also seen for dodecanoyl (12:0) CoA and fatty acid in a high 12:0 line, but not for octadecanoic (18:0) CoA and fatty acid in a high 18:0 line. Comparison of 10:0 CoA and fatty acid proportions from seeds at different developmental stages for transgenic B. napus and Cuphea hookeriana, the source plant for the medium-chain thioesterase and 3-ketoacyl-ACP synthase transgenes, revealed that C. hookeriana incorporates 10:0 CoA into seed lipids more efficiently than transgenic B. napus. Furthermore, beta-oxidation and glyoxylate cycle activities were not increased above wild type levels during seed development in the 8:0/10:0 line, suggesting that lipid catabolism was not being induced in response to the elevated 10:0 CoA concentrations. Taken together, these data suggest that transgenic plants that are engineered to synthesize medium-chain fatty acids may lack the necessary mechanisms, such as specific acyltransferases, to incorporate these fatty acids efficiently into seed lipids.

Thioesterase Superfamily Member 2/Acyl-CoA Thioesterase 13 (Them2/Acot13) Regulates Adaptive Thermogenesis in Mice*

PubMed Central

Kang, Hye Won; Ozdemir, Cafer; Kawano, Yuki; LeClair, Katherine B.; Vernochet, Cecile; Kahn, C. Ronald; Hagen, Susan J.; Cohen, David E.

2013-01-01

Members of the acyl-CoA thioesterase (Acot) gene family hydrolyze fatty acyl-CoAs, but their biological functions remain incompletely understood. Thioesterase superfamily member 2 (Them2; synonym Acot13) is enriched in oxidative tissues, associated with mitochondria, and relatively specific for long chain fatty acyl-CoA substrates. Using Them2−/− mice, we have demonstrated key roles for Them2 in regulating hepatic glucose and lipid metabolism. However, reduced body weights and decreased adiposity in Them2−/− mice observed despite increased food consumption were not well explained. To explore a role in thermogenesis, mice were exposed to ambient temperatures ranging from thermoneutrality (30 °C) to cold (4 °C). In response to short term (24-h) exposures to decreasing ambient temperatures, Them2−/− mice exhibited increased adaptive responses in physical activity, food consumption, and energy expenditure when compared with Them2+/+ mice. By contrast, genotype-dependent differences were not observed in mice that were equilibrated (96 h) at each ambient temperature. In brown adipose tissue, the absence of Them2 was associated with reduced lipid droplets, alterations in the ultrastructure of mitochondria, and increased expression of thermogenic genes. Indicative of a direct regulatory role for Them2 in heat production, cultured primary brown adipocytes from Them2−/− mice exhibited increased norepinephrine-mediated triglyceride hydrolysis and increased rates of O2 consumption, together with elevated expression of thermogenic genes. At least in part by regulating intracellular fatty acid channeling, Them2 functions in brown adipose tissue to suppress adaptive increases in energy expenditure. PMID:24072708

A Single Acyl-CoA Dehydrogenase Is Required For Catabolism Of Isoleucine, Valine And Short-Chain Fatty Acids In Aspergillus nidulans

PubMed Central

Maggio-Hall, Lori A.; Lyne, Paul; Wolff, Jon A.; Keller, Nancy P.

2010-01-01

An acyl-CoA dehydrogenase has been identified as part of the mitochondrial β-oxidation pathway in the ascomycete fungus Aspergillus nidulans. Disruption of the scdA gene prevented use of butyric acid (C4) and hexanoic acid (C6) as carbon sources and reduced cellular butyryl-CoA dehydrogenase activity by 7.5-fold. While the mutant strain exhibited wild-type levels of growth on erucic acid (C22:1) and oleic acid (C18:1), some reduction in growth was observed with myristic acid (C14). The ΔscdA mutation was found to be epistatic to a mutation downstream in the β-oxidation pathway (disruption of enoyl-CoA hydratase). The ΔscdA mutant was also unable to use isoleucine or valine as a carbon source. Transcription of scdA was observed in the presence of either fatty acids or amino acids. When the mutant was grown in medium containing either isoleucine or valine, organic acid analysis of culture supernatants showed accumulation of 2-oxo acid intermediates of branched chain amino acid catabolism, suggesting feedback inhibition of the upstream branched-chain α-keto acid dehydrogenase. PMID:17656140

Compared effects of missense mutations in Very-Long-Chain Acyl-CoA Dehydrogenase deficiency: Combined analysis by structural, functional and pharmacological approaches.

PubMed

Gobin-Limballe, Stéphanie; McAndrew, Ryan P; Djouadi, Fatima; Kim, Jung-Ja; Bastin, Jean

2010-05-01

Very-Long-Chain Acyl-CoA Dehydrogenase deficiency (VLCADD) is an autosomal recessive disorder considered as one of the more common ss-oxidation defects, possibly associated with neonatal cardiomyopathy, infantile hepatic coma, or adult-onset myopathy. Numerous gene missense mutations have been described in these VLCADD phenotypes, but only few of them have been structurally and functionally analyzed, and the molecular basis of disease variability is still poorly understood. To address this question, we first analyzed fourteen disease-causing amino acid changes using the recently described crystal structure of VLCAD. The predicted effects varied from the replacement of amino acid residues lining the substrate binding cavity, involved in holoenzyme-FAD interactions or in enzyme dimerisation, predicted to have severe functional consequences, up to amino acid substitutions outside key enzyme domains or lying on near enzyme surface, with predicted milder consequences. These data were combined with functional analysis of residual fatty acid oxidation (FAO) and VLCAD protein levels in patient cells harboring these mutations, before and after pharmacological stimulation by bezafibrate. Mutations identified as detrimental to the protein structure in the 3-D model were generally associated to profound FAO and VLCAD protein deficiencies in the patient cells, however, some mutations affecting FAD binding or monomer-monomer interactions allowed a partial response to bezafibrate. On the other hand, bezafibrate restored near-normal FAO rates in some mutations predicted to have milder consequences on enzyme structure. Overall, combination of structural, biochemical, and pharmacological analysis allowed assessment of the relative severity of individual mutations, with possible applications for disease management and therapeutic approach. Copyright 2010 Elsevier B.V. All rights reserved.

A Cuphea beta-ketoacyl-ACP synthase shifts the synthesis of fatty acids towards shorter chains in Arabidopsis seeds expressing Cuphea FatB thioesterases.

PubMed

Leonard, J M; Knapp, S J; Slabaugh, M B

1998-03-01

Acyl-acyl carrier protein (ACP) thioesterases with specificities on medium chain substrates (C8-C14) are requisite enzymes in plants that produce 8:0, 10:0, 12:0 and 14:0 seed oils, but they may not be the sole enzymatic determinants of chain length. The contribution to chain length regulation of a beta-ketoacyl-ACP synthase, Cw KAS A1, derived from Cuphea wrightii, a species that accumulates 30% 10:0 and 54% 12:0 in seed oils, was investigated. Expression of Cw KAS A1 in Arabidopsis seeds reduced 16:0 from 8.2 to 6.2 mol%, suggesting a KAS II-type activity. In the presence of the KAS I inhibitor cerulenin, however, transgenic seed extracts extended 6:0- and 8:0-ACP at a rate four- to fivefold greater than extracts from untransformed plants, whereas no difference was observed in extension of 14:0- and 16:0-ACP. The effect of KAS A1 on seed oils was tested by combining it with the C. wrightii medium chain-specific thioesterases, Cw FatB1 and Cw FatB2, in crosses of transformed plants. Fatty acid synthesis thesis shifted towards shorter chains in progeny expressing both classes of enzymes. KasA1/FatB1 homozygotes produced threefold more 12:0 than the FatB1 parent while 14:0 and 16:0 were reduced by one-third and one-half, respectively. F2 progeny expressing KasA1 and FatB2 produced twofold more 10:0 and 1.4-fold more 12:0 than the FatB2 parent, and the double-transgenic progeny produced one-quarter less 14:0 and one-half less 16:0 than the FatB2 parent. It is hypothesized that the shift towards production of shorter chains resulted from increased pools of medium chain acyl-ACP resulting from KAS A1 activity. The combined activities of KAS A1 and FatB thioesterases appear to determine the C. wrightii phenotype.

Substrate promiscuity of a rosmarinic acid synthase from lavender (Lavandula angustifolia L.).

PubMed

Landmann, Christian; Hücherig, Stefanie; Fink, Barbara; Hoffmann, Thomas; Dittlein, Daniela; Coiner, Heather A; Schwab, Wilfried

2011-08-01

One of the most common types of modification of secondary metabolites is the acylation of oxygen- and nitrogen-containing substrates to produce esters and amides, respectively. Among the known acyltransferases, the members of the plant BAHD family are capable of acylating a wide variety of substrates. Two full-length acyltransferase cDNAs (LaAT1 and 2) were isolated from lavender flowers (Lavandula angustifolia L.) by reverse transcriptase-PCR using degenerate primers based on BAHD sequences. Recombinant LaAT1 exhibited a broad substrate tolerance accepting (hydroxy)cinnamoyl-CoAs as acyl donors and not only tyramine, tryptamine, phenylethylamine and anthranilic acid but also shikimic acid and 4-hydroxyphenyllactic acid as acceptors. Thus, LaLT1 forms esters and amides like its phylogenetic neighbors. In planta LaAT1 might be involved in the biosynthesis of rosmarinic acid, the ester of caffeic acid and 3,4-dihydroxyphenyllactic acid, a major constituent of lavender flowers. LaAT2 is one of three members of clade VI with unknown function.

Characterization of Acyl-CoA Synthetase Isoforms In Pancreatic Beta Cells: Gene Silencing Shows Participation of ACSL3 and ACSL4 In Insulin Secretion

PubMed Central

Ansari, Israr-ul H.; Longacre, Melissa J.; Stoker, Scott W.; Kendrick, Mindy A.; O’Neill, Lucas M.; Zitur, Laura J.; Fernandez, Luis A.; Ntambi, James M.; MacDonald, Michael J.

2017-01-01

Long-chain acyl-CoA synthetases (ACSLs) convert fatty acids to fatty acyl-CoAs to regulate various physiologic processes. We characterized the ACSL isoforms in a cell line of homogeneous rat beta cells (INS-1 832/13 cells) and human pancreatic islets. ACSL4 and ACSL3 proteins were present in the beta cells and human and rat pancreatic islets and concentrated in insulin secretory granules and less in mitochondria and negligible in other intracellular organelles. ACSL1 and ACSL6 proteins were not seen in INS-1 832/13 cells or pancreatic islets. ACSL5 protein was seen only in INS-1 832/13 cells. With shRNA-mediated gene silencing we developed stable ACSL knockdown cell lines from INS-1 832/13 cells. Glucose-stimulated insulin release was inhibited ~ 50% with ACSL4 and ACSL3 knockdown and unaffected in cell lines with knockdown of ACSL5, ACLS6 and ACSL1. Lentivirus shRNA-mediated gene silencing of ACSL4 and ACSL3 in human pancreatic islets inhibited glucose-stimulated insulin release. ACSL4 and ACSL3 knockdown cells showed inhibition of ACSL enzyme activity more with arachidonate than with palmitate as a substrate, consistent with their preference for unsaturated fatty acids as substrates. ACSL4 knockdown changed the patterns of fatty acids in phosphatidylserines and phosphatidylethanolamines. The results show the involvement of ACLS4 and ACLS3 in insulin secretion. PMID:28193492

Novel Structural Components Contribute to the High Thermal Stability of Acyl Carrier Protein from Enterococcus faecalis*

PubMed Central

Park, Young-Guen; Jung, Min-Cheol; Song, Heesang; Jeong, Ki-Woong; Bang, Eunjung; Hwang, Geum-Sook; Kim, Yangmee

2016-01-01

Enterococcus faecalis is a Gram-positive, commensal bacterium that lives in the gastrointestinal tracts of humans and other mammals. It causes severe infections because of high antibiotic resistance. E. faecalis can endure extremes of temperature and pH. Acyl carrier protein (ACP) is a key element in the biosynthesis of fatty acids responsible for acyl group shuttling and delivery. In this study, to understand the origin of high thermal stabilities of E. faecalis ACP (Ef-ACP), its solution structure was investigated for the first time. CD experiments showed that the melting temperature of Ef-ACP is 78.8 °C, which is much higher than that of Escherichia coli ACP (67.2 °C). The overall structure of Ef-ACP shows the common ACP folding pattern consisting of four α-helices (helix I (residues 3–17), helix II (residues 39–53), helix III (residues 60–64), and helix IV (residues 68–78)) connected by three loops. Unique Ef-ACP structural features include a hydrophobic interaction between Phe45 in helix II and Phe18 in the α1α2 loop and a hydrogen bonding between Ser15 in helix I and Ile20 in the α1α2 loop, resulting in its high thermal stability. Phe45-mediated hydrophobic packing may block acyl chain binding subpocket II entry. Furthermore, Ser58 in the α2α3 loop in Ef-ACP, which usually constitutes a proline in other ACPs, exhibited slow conformational exchanges, resulting in the movement of the helix III outside the structure to accommodate a longer acyl chain in the acyl binding cavity. These results might provide insights into the development of antibiotics against pathogenic drug-resistant E. faecalis strains. PMID:26631734

Study of Triheptanoin for Treatment of Long-Chain Fatty Acid Oxidation Disorder

ClinicalTrials.gov

2017-03-21

Very Long-chain acylCoA Dehydrogenase (VLCAD) Deficiency; Carnitine Palmitoyltransferase 2 (CPT2) Deficiency; Mitochondrial Trifunctional Protein (TFP) Deficiency; Long-chain 3 hydroxyacylCoA Dehydrogenase (LCHAD) Deficiency

Trapping of the Enoyl-Acyl Carrier Protein Reductase–Acyl Carrier Protein Interaction

PubMed Central

Tallorin, Lorillee; Finzel, Kara; Nguyen, Quynh G.; Beld, Joris; La Clair, James J.; Burkart, Michael D.

2016-01-01

An ideal target for metabolic engineering, fatty acid biosynthesis remains poorly understood on a molecular level. These carrier protein-dependent pathways require fundamental protein–protein interactions to guide reactivity and processivity, and their control has become one of the major hurdles in successfully adapting these biological machines. Our laboratory has developed methods to prepare acyl carrier proteins (ACPs) loaded with substrate mimetics and cross-linkers to visualize and trap interactions with partner enzymes, and we continue to expand the tools for studying these pathways. We now describe application of the slow-onset, tight-binding inhibitor triclosan to explore the interactions between the type II fatty acid ACP from Escherichia coli, AcpP, and its corresponding enoyl-ACP reductase, FabI. We show that the AcpP–triclosan complex demonstrates nM binding, inhibits in vitro activity, and can be used to isolate FabI in complex proteomes. PMID:26938266

Structure, High Affinity, and Negative Cooperativity of the Escherichia coli Holo-(Acyl Carrier Protein):Holo-(Acyl Carrier Protein) Synthase Complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marcella, Aaron M.; Culbertson, Sannie J.; Shogren-Knaak, Michael A.

The Escherichia coli holo-(acyl carrier protein) synthase (ACPS) catalyzes the coenzyme A-dependent activation of apo-ACPP to generate holo-(acyl carrier protein) (holo-ACPP) in an early step of fatty acid biosynthesis. E. coli ACPS is sufficiently different from the human fatty acid synthase to justify the development of novel ACPS-targeting antibiotics. Models of E. coli ACPS in unliganded and holo-ACPP-bound forms solved by X-ray crystallography to 2.05 and 4.10 Å, respectively, revealed that ACPS bound three product holo-ACPP molecules to form a 3:3 hexamer. Solution NMR spectroscopy experiments validated the ACPS binding interface on holo-ACPP using chemical shift perturbations and by determiningmore » the relative orientation of holo-ACPP to ACPS by fitting residual dipolar couplings. The binding interface is organized to arrange contacts between positively charged ACPS residues and the holo-ACPP phosphopantetheine moiety, indicating product contains more stabilizing interactions than expected in the enzyme:substrate complex. Indeed, holo-ACPP bound the enzyme with greater affinity than the substrate, apo-ACPP, and with negative cooperativity. The first equivalent of holo-ACPP bound with a KD = 62 ± 13 nM, followed by the binding of two more equivalents of holo-ACPP with KD = 1.2 ± 0.2 μM. Cooperativity was not observed for apo-ACPP which bound with KD = 2.4 ± 0.1 μM. Strong product binding and high levels of holo-ACPP in the cell identify a potential regulatory role of ACPS in fatty acid biosynthesis.« less

Delineating Substrate Diversity of Disparate Short-Chain Dehydrogenase Reductase from Debaryomyces hansenii.

PubMed

Ghatak, Arindam; Bharatham, Nagakumar; Shanbhag, Anirudh P; Datta, Santanu; Venkatraman, Janani

2017-01-01

Short-chain dehydrogenase reductases (SDRs) have been utilized for catalyzing the reduction of many aromatic/aliphatic prochiral ketones to their respective alcohols. However, there is a paucity of data that elucidates their innate biological role and diverse substrate space. In this study, we executed an in-depth biochemical characterization and substrate space mapping (with 278 prochiral ketones) of an unannotated SDR (DHK) from Debaryomyces hansenii and compared it with structurally and functionally characterized SDR Synechococcus elongatus. PCC 7942 FabG to delineate its industrial significance. It was observed that DHK was significantly more efficient than FabG, reducing a diverse set of ketones albeit at higher conversion rates. Comparison of the FabG structure with a homology model of DHK and a docking of substrate to both structures revealed the presence of additional flexible loops near the substrate binding site of DHK. The comparative elasticity of the cofactor and substrate binding site of FabG and DHK was experimentally substantiated using differential scanning fluorimetry. It is postulated that the loop flexibility may account for the superior catalytic efficiency of DHK although the positioning of the catalytic triad is conserved.

Delineating Substrate Diversity of Disparate Short-Chain Dehydrogenase Reductase from Debaryomyces hansenii

PubMed Central

Ghatak, Arindam; Bharatham, Nagakumar; Shanbhag, Anirudh P.; Datta, Santanu; Venkatraman, Janani

2017-01-01

Short-chain dehydrogenase reductases (SDRs) have been utilized for catalyzing the reduction of many aromatic/aliphatic prochiral ketones to their respective alcohols. However, there is a paucity of data that elucidates their innate biological role and diverse substrate space. In this study, we executed an in-depth biochemical characterization and substrate space mapping (with 278 prochiral ketones) of an unannotated SDR (DHK) from Debaryomyces hansenii and compared it with structurally and functionally characterized SDR Synechococcus elongatus. PCC 7942 FabG to delineate its industrial significance. It was observed that DHK was significantly more efficient than FabG, reducing a diverse set of ketones albeit at higher conversion rates. Comparison of the FabG structure with a homology model of DHK and a docking of substrate to both structures revealed the presence of additional flexible loops near the substrate binding site of DHK. The comparative elasticity of the cofactor and substrate binding site of FabG and DHK was experimentally substantiated using differential scanning fluorimetry. It is postulated that the loop flexibility may account for the superior catalytic efficiency of DHK although the positioning of the catalytic triad is conserved. PMID:28107498

Alcohol-to-acid ratio and substrate concentration affect product structure in chain elongation reactions initiated by unacclimatized inoculum.

PubMed

Liu, Yuhao; Lü, Fan; Shao, Liming; He, Pinjing

2016-10-01

The objective of the study was to investigate whether the ratio of ethanol to acetate affects yield and product structure in chain elongation initiated by unacclimatized mixed cultures. The effect of varying the substrate concentration, while maintaining the same ratio of alcohol to acid, was also investigated. With a high substrate concentration, an alcohol to acid ratio >2:1 provided sufficient electron donor capacity for the chain elongation reaction. With an ethanol to acetate ratio of 3:1 (300mM total carbon), the highest n-caproate concentration (3033±98mg/L) was achieved during the stable phase of the reaction. A lower substrate concentration (150mM total carbon) gave a lower yield of products and led to reduced carbon transformation efficiency compared with other reaction conditions. The use of unacclimatized inoculum in chain elongation can produce significant amounts of odd-carbon-number carboxylates as a result of protein hydrolysis. Copyright © 2016 Elsevier Ltd. All rights reserved.

A Class of Reactive Acyl-CoA Species Reveals the Non-Enzymatic Origins of Protein Acylation

PubMed Central

Wagner, Gregory R.; Bhatt, Dhaval P.; O’Connell, Thomas M.; Thompson, J. Will; Dubois, Laura G.; Backos, Donald S.; Yang, Hao; Mitchell, Grant A.; Ilkayeva, Olga R.; Stevens, Robert D.; Grimsrud, Paul A.; Hirschey, Matthew D.

2017-01-01

SUMMARY The mechanisms underlying the formation of acyl protein modifications remain poorly understood. By investigating the reactivity of endogenous acyl-CoA metabolites, we found a class of acyl-CoAs that undergoes intramolecular catalysis to form reactive intermediates which non-enzymatically modify proteins. Based on this mechanism, we predicted, validated, and characterized a protein modification: 3-hydroxy-3-methylglutaryl(HMG)-lysine. In a model of altered HMG-CoA metabolism, we found evidence of two additional protein modifications: 3-methylglutaconyl(MGc)-lysine and 3-methylglutaryl(MG)-lysine. Using quantitative proteomics, we compared the ‘acylomes’ of two reactive acyl-CoA species, namely HMG-CoA and glutaryl-CoA, which are generated in different pathways. We found proteins that are uniquely modified by each reactive metabolite, as well as common proteins and pathways. We identified the tricarboxylic acid cycle as a pathway commonly regulated by acylation, and validated malate dehydrogenase as a key target. These data uncover a fundamental relationship between reactive acyl-CoA species and proteins, and define a new regulatory paradigm in metabolism. PMID:28380375

Acyl silicates and acyl aluminates as activated intermediates in peptide formation on clays

NASA Technical Reports Server (NTRS)

White, D. H.; Kennedy, R. M.; Macklin, J.

1984-01-01

Glycine reacts with heating on dried clays and other minerals to give peptides in much better yield than in the absence of mineral. This reaction was proposed to occur by way of an activated intermediate such as an acyl silicate or acyl aluminate analogous to acyl phosphates involved in several biochemical reactions including peptide bond synthesis. The proposed mechanism has been confirmed by trapping the intermediate, as well as by direct spectroscopic observation of a related intermediate. The reaction of amino acids on periodically dried mineral surfaces represents a widespead, geologically realistic setting for prebiotic peptide formation via in situ activation.

Analogs of palmitoyl-CoA that are substrates for myristoyl-CoA:protein N-myristoyltransferase.

PubMed

Rudnick, D A; Lu, T; Jackson-Machelski, E; Hernandez, J C; Li, Q; Gokel, G W; Gordon, J I

1992-11-01

Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (Nmt1p; EC 2.3.1.97) is an essential enzyme that is highly selective for myristoyl-CoA in vivo. It is unclear why myristate (C14:0), a rare cellular fatty acid, has been selected for this covalent protein modification over more abundant fatty acids such as palmitate (C16:0), nor is it obvious how the enzyme's acyl-CoA binding site is able to discriminate between these two fatty acids. Introduction of a cis double bond between C5 and C6 of palmitate [(Z)-5-hexadecenoic acid] or a triple bond between C4 and C5 or C6 and C7 (Y4- and Y6-hexadecenoic acids) yields compounds that, when converted to their CoA derivatives, approach the activity of myristoyl-CoA as Nmt1p substrates in vitro. Kinetic studies of 42 C12-C18 fatty acids containing triple bonds, para-phenylene, or a 2,5-furyl group, as well as cis and trans double bonds, suggest that the geometry of the enzyme's acyl-CoA binding site requires that the acyl chain of active substrates assume a bent conformation in the vicinity of C5. Moreover, the distance between C1 and the bend appears to be a critical determinant for optimal positioning of the acyl-CoA in this binding site so that peptide substrates can subsequently bind in the sequential ordered bi-bi reaction mechanism. Identification of active, conformationally restricted analogs of palmitate offers an opportunity to "convert" wild-type or mutant Nmts to palmitoyltransferases so that they can deliver these C16 fatty acids to critical N-myristoylproteins in vivo. nmt181p contains a Gly-451-->Asp mutation, which causes a marked reduction in the enzyme's affinity for myristoyl-CoA. Strains of S. cerevisiae containing nmt1-181 exhibit temperature-sensitive myristic acid auxotrophy: their complete growth arrest at 37 degrees C is relieved when the medium is supplemented with 500 microM C14:0 but not with C16:0. The CoA derivatives of (Z)-5-hexadecenoic and Y6-hexadecynoic acids are as active

Analogs of palmitoyl-CoA that are substrates for myristoyl-CoA:protein N-myristoyltransferase.

PubMed Central

Rudnick, D A; Lu, T; Jackson-Machelski, E; Hernandez, J C; Li, Q; Gokel, G W; Gordon, J I

1992-01-01

Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (Nmt1p; EC 2.3.1.97) is an essential enzyme that is highly selective for myristoyl-CoA in vivo. It is unclear why myristate (C14:0), a rare cellular fatty acid, has been selected for this covalent protein modification over more abundant fatty acids such as palmitate (C16:0), nor is it obvious how the enzyme's acyl-CoA binding site is able to discriminate between these two fatty acids. Introduction of a cis double bond between C5 and C6 of palmitate [(Z)-5-hexadecenoic acid] or a triple bond between C4 and C5 or C6 and C7 (Y4- and Y6-hexadecenoic acids) yields compounds that, when converted to their CoA derivatives, approach the activity of myristoyl-CoA as Nmt1p substrates in vitro. Kinetic studies of 42 C12-C18 fatty acids containing triple bonds, para-phenylene, or a 2,5-furyl group, as well as cis and trans double bonds, suggest that the geometry of the enzyme's acyl-CoA binding site requires that the acyl chain of active substrates assume a bent conformation in the vicinity of C5. Moreover, the distance between C1 and the bend appears to be a critical determinant for optimal positioning of the acyl-CoA in this binding site so that peptide substrates can subsequently bind in the sequential ordered bi-bi reaction mechanism. Identification of active, conformationally restricted analogs of palmitate offers an opportunity to "convert" wild-type or mutant Nmts to palmitoyltransferases so that they can deliver these C16 fatty acids to critical N-myristoylproteins in vivo. nmt181p contains a Gly-451-->Asp mutation, which causes a marked reduction in the enzyme's affinity for myristoyl-CoA. Strains of S. cerevisiae containing nmt1-181 exhibit temperature-sensitive myristic acid auxotrophy: their complete growth arrest at 37 degrees C is relieved when the medium is supplemented with 500 microM C14:0 but not with C16:0. The CoA derivatives of (Z)-5-hexadecenoic and Y6-hexadecynoic acids are as active

Short- and medium-chain fatty acids in energy metabolism: the cellular perspective.

PubMed

Schönfeld, Peter; Wojtczak, Lech

2016-06-01

Short- and medium-chain fatty acids (SCFAs and MCFAs), independently of their cellular signaling functions, are important substrates of the energy metabolism and anabolic processes in mammals. SCFAs are mostly generated by colonic bacteria and are predominantly metabolized by enterocytes and liver, whereas MCFAs arise mostly from dietary triglycerides, among them milk and dairy products. A common feature of SCFAs and MCFAs is their carnitine-independent uptake and intramitochondrial activation to acyl-CoA thioesters. Contrary to long-chain fatty acids, the cellular metabolism of SCFAs and MCFAs depends to a lesser extent on fatty acid-binding proteins. SCFAs and MCFAs modulate tissue metabolism of carbohydrates and lipids, as manifested by a mostly inhibitory effect on glycolysis and stimulation of lipogenesis or gluconeogenesis. SCFAs and MCFAs exert no or only weak protonophoric and lytic activities in mitochondria and do not significantly impair the electron transport in the respiratory chain. SCFAs and MCFAs modulate mitochondrial energy production by two mechanisms: they provide reducing equivalents to the respiratory chain and partly decrease efficacy of oxidative ATP synthesis. Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.

The gut hormone ghrelin partially reverses energy substrate metabolic alterations in the failing heart.

PubMed

Mitacchione, Gianfranco; Powers, Jeffrey C; Grifoni, Gino; Woitek, Felix; Lam, Amy; Ly, Lien; Settanni, Fabio; Makarewich, Catherine A; McCormick, Ryan; Trovato, Letizia; Houser, Steven R; Granata, Riccarda; Recchia, Fabio A

2014-07-01

The gut-derived hormone ghrelin, especially its acylated form, plays a major role in the regulation of systemic metabolism and exerts also relevant cardioprotective effects; hence, it has been proposed for the treatment of heart failure (HF). We tested the hypothesis that ghrelin can directly modulate cardiac energy substrate metabolism. We used chronically instrumented dogs, 8 with pacing-induced HF and 6 normal controls. Human des-acyl ghrelin [1.2 nmol/kg per hour] was infused intravenously for 15 minutes, followed by washout (rebaseline) and infusion of acyl ghrelin at the same dose. (3)H-oleate and (14)C-glucose were coinfused and arterial and coronary sinus blood sampled to measure cardiac free fatty acid and glucose oxidation and lactate uptake. As expected, cardiac substrate metabolism was profoundly altered in HF because baseline oxidation levels of free fatty acids and glucose were, respectively, >70% lower and >160% higher compared with control. Neither des-acyl ghrelin nor acyl ghrelin significantly affected function and metabolism in normal hearts. However, in HF, des-acyl and acyl ghrelin enhanced myocardial oxygen consumption by 10.2±3.5% and 9.9±3.7%, respectively (P<0.05), and cardiac mechanical efficiency was not significantly altered. This was associated, respectively, with a 41.3±6.7% and 32.5±10.9% increase in free fatty acid oxidation and a 31.3±9.2% and 41.4±8.9% decrease in glucose oxidation (all P<0.05). Acute increases in des-acyl or acyl ghrelin do not interfere with cardiac metabolism in normal dogs, whereas they enhance free fatty acid oxidation and reduce glucose oxidation in HF dogs, thus partially correcting metabolic alterations in HF. This novel mechanism might contribute to the cardioprotective effects of ghrelin in HF. © 2014 American Heart Association, Inc.

Two bifunctional enzymes from the marine protist Thraustochytrium roseum: biochemical characterization of wax ester synthase/acyl-CoA:diacylglycerol acyltransferase activity catalyzing wax ester and triacylglycerol synthesis.

PubMed

Zhang, Nannan; Mao, Zejing; Luo, Ling; Wan, Xia; Huang, Fenghong; Gong, Yangmin

2017-01-01

�°C, and WS activity was greatly decreased when temperature exceeds 47 °C. TrWSD4 and TrWSD5 are insensitive to ionic strength and reduced WS activity was observed when salt concentration exceeded 800 mM. The potential of T. roseum WS/DGATs to establish novel process for biotechnological production of WEs was demonstrated by heterologous expression in recombinant yeast. Expression of either TrWSD4 or TrWSD5 in Saccharomyces cerevisiae quadruple mutant H1246, which is devoid of storage lipids, resulted in the accumulation of WEs, but not any detectable TAGs, indicating a predominant WS activity in yeast. This study demonstrates both in vitro WS and DGAT activity of two T. roseum WS/DGATs, which were characterized as unspecific acyltransferases accepting a broad range of acyl-CoAs and fatty alcohols as substrates for WS activity but displaying substrate preference for medium-chain acyl-CoAs. In vivo characterization shows that these two WS/DGATs predominantly function as wax synthase and presents the feasibility for production of WEs by heterologous hosts.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brown, J.H.; Lynch, D.V.; Thompson, J.E.

During senescence of cut carnation flowers, there is extensive breakdown of microsomal phospholipid. This is attributable, at least in part, to lipolytic activity associated directly with the microsomal membranes. Evidence indicating that one or more of the lipid-degrading enzymes in these membranes preferentially degrade phospholipid molecular species containing two diunsaturated acyl chains or at least one polyunsaturated acyl chain has been obtained by using radiolabeled phosphatidylcholine substrates. 16:0/sup *//16:0/sup */; 16:0/18:2/sup */, and 18:1/sup *//18:1/sup */ phosphatidylcholine were degraded only minimally over a 3 hour period by microsomes isolated from senescing flowers. By contrast, (U-/sup 14/C)phosphatidylcholine, which comprises various molecularmore » species including those containing polyunsaturated acyl chains, and 18:0/20:4/sup */ phosphatidylcholine were extensively degraded. Under identical conditions, but in the absence of added radiolabeled substrate, endogenous 18:2/18:2, 18:1/18:3, and 18:2/18:3 phosphatidylcholine were selectively depleted from the membranes. During natural senescence of the flowers, there was a sharp decline in microsomal 16:0/18:1 and 18:1/18:2 phosphatidylcholine, whereas molecular species containing two diunsaturated acyl chains or at least one polyunsaturated acyl chain remained unchanged or decreased only slightly. The data have been interpreted as indicating that provision of particular molecular species susceptible to lipase attack is a prerequisite to phospholipid catabolism in senescing membranes.« less

Synthesis of Polyheterocyclic Pyrrolo[3,4-b]pyridin-5-ones via a One-Pot (Ugi-3CR/aza Diels-Alder/N-acylation/aromatization/SN2) Process. A Suitable Alternative towards Novel Aza-Analogues of Falipamil.

PubMed

Zamudio-Medina, Angel; García-González, Ailyn N; Herrera-Carrillo, Genesis K; Zárate-Zárate, Daniel; Benavides-Macías, Adriana; Tamariz, Joaquín; Ibarra, Ilich A; Islas-Jácome, Alejandro; González-Zamora, Eduardo

2018-03-27

We describe the one-pot synthesis of twenty polyheterocyclic pyrrolo[3,4- b ]pyridin-5-ones via a cascade process (Ugi-3CR/aza Diels-Alder/ N -acylation/aromatization) in 20 to 95% overall yields, as well as four pharmacologically promising analogues via an improved cascade process (Ugi-3CR/aza Diels-Alder/ N -acylation/aromatization/S N 2): two piperazine-linked pyrrolo[3,4- b ]pyridin-5-ones in 33 and 34%, and a couple of Falipamil aza-analogues in 30 and 35% overall yields. It is worth highlighting the good substrate scope found, because final products are furnished with alkyl, aryl, and heterocyclic substituents. The use of chain-ring tautomerizable isocyanides (as key reagents for the Ugi-type three component reaction) allowed for a rapid and efficient assembly of the polysubstituted oxindoles, which were used in situ toward the complex products, conferring features like robustness, sustainability, and the one-pot approach to this synthetic methodology.

Effect of Acylglycerol Composition and Fatty Acyl Chain Length on Lipid Digestion in pH-Stat Digestion Model and Simulated In Vitro Digestion Model.

PubMed

Qi, Jin F; Jia, Cai H; Shin, Jung A; Woo, Jeong M; Wang, Xiang Y; Park, Jong T; Hong, Soon T; Lee, K-T

2016-02-01

In this study, a pH-stat digestion model and a simulated in vitro digestion model were employed to evaluate the digestion degree of lipids depending on different acylglycerols and acyl chain length (that is, diacylglycerol [DAG] compared with soybean oil representing long-chain triacylglycerol compared with medium-chain triacylglycerol [MCT]). In the pH-stat digestion model, differences were observed among the digestion degrees of 3 oils using digestion rate (k), digestion half-time (t1/2 ), and digestion extent (Φmax). The results showed the digestion rate order was MCT > soybean oil > DAG. Accordingly, the order of digestion half-times was MCT < soybean oil < DAG. In simulated in vitro digestion model, digestion rates (k') and digestion half-times (t'1/2 ) were also obtained and the results showed a digestion rate order of MCT (k' = 0.068 min(-1) ) > soybean oil (k' = 0.037 min(-1) ) > DAG (k' = 0.024 min(-1) ). Consequently, the order of digestion half-times was MCT (t'1/2 = 10.20 min) < soybean oil (t'1/2 = 18.74 min) < DAG (t'1/2 = 29.08 min). The parameters obtained using the 2 models showed MCT was digested faster than soybean oil, and that soybean oil was digested faster than DAG. © 2015 Institute of Food Technologists®

Biological and surface-active properties of double-chain cationic amino acid-based surfactants.

PubMed

Greber, Katarzyna E; Dawgul, Małgorzata; Kamysz, Wojciech; Sawicki, Wiesław; Łukasiak, Jerzy

2014-08-01

Cationic amino acid-based surfactants were synthesized via solid phase peptide synthesis and terminal acylation of their α and ε positions with saturated fatty acids. Five new lipopeptides, N-α-acyl-N-ε-acyl lysine analogues, were obtained. Minimum inhibitory concentration and minimum bactericidal (fungicidal) concentration were determined on reference strains of bacteria and fungi to evaluate the antimicrobial activity of the lipopeptides. Toxicity to eukaryotic cells was examined via determination of the haemolytic activities. The surface-active properties of these compounds were evaluated by measuring the surface tension and formation of micelles as a function of concentration in aqueous solution. The cationic surfactants demonstrated diverse antibacterial activities dependent on the length of the fatty acid chain. Gram-negative bacteria and fungi showed a higher resistance than Gram-positive bacterial strains. It was found that the haemolytic activities were also chain length-dependent values. The surface-active properties showed a linear correlation between the alkyl chain length and the critical micelle concentration.

Regulation of C. elegans fat uptake and storage by acyl-CoA synthase-3 is dependent on NR5A family nuclear hormone receptor nhr-25

PubMed Central

Mullaney, Brendan; Ashrafi, Kaveh

2010-01-01

Summary Acyl-CoA synthases are important for lipid synthesis and breakdown, generation of signaling molecules and lipid modification of proteins, highlighting the challenge of understanding metabolic pathways within intact organisms. From a C. elegans mutagenesis screen, we found that loss of ACS-3, a long-chain acyl-CoA synthase, causes enhanced intestinal lipid uptake, de novo fat synthesis, and accumulation of enlarged, neutral lipid rich intestinal depots. Here, we show that ACS-3 functions in seam cells, epidermal cells anatomically distinct from sites of fat uptake and storage, and that acs-3 mutant phenotypes require the nuclear hormone receptor NHR-25, a key regulator of C. elegans molting. Our findings suggest that ACS-3 derived long chain fatty acyl-CoAs, perhaps incorporated into complex ligands such as phosphoinositides, modulate NHR-25 function, which in turn regulates an endocrine program of lipid uptake and synthesis. These results reveal a link between acyl-CoA synthase function and an NR5A family nuclear receptor in C. elegans. PMID:20889131

Regioselective lipase-catalyzed synthesis of 3-o-acyl derivatives of resveratrol and study of their antioxidant properties.

PubMed

Torres, Pamela; Poveda, Ana; Jimenez-Barbero, Jesús; Ballesteros, Antonio; Plou, Francisco J

2010-01-27

One of the approaches to increasing the bioavailability of resveratrol is to protect its 3-OH phenolic group. In this work, regioselective acylation of resveratrol at 3-OH was achieved by transesterification with vinyl acetate catalyzed by immobilized lipase from Alcaligenes sp. (lipase QLG). The maximum yield of 3-O-acetylresveratrol was approximately 75%, as the lipase also catalyzes its further acetylation affording the diester 3,4'-di-O-acetylresveratrol and finally the peracetylated derivative. Long saturated and unsaturated fatty acid vinyl esters were also effective as acyl donors with similar regioselectivity. In contrast, lipase B from Candida antarctica catalyzes the acylation of the phenolic group 4'-OH with 80% yield and negligible formation of higher esters. The analysis of the antioxidant properties showed that the Trolox equivalent antioxidant capability (TEAC) values for the acetyl and stearoyl derivatives at 3-OH were, respectively, 40% and 25% referred to resveratrol. The addition of an acyl chain in the 3-OH position caused a higher loss of activity compared with that at the 4'-OH.

Enzymes involved in branched-chain amino acid metabolism in humans.

PubMed

Adeva-Andany, María M; López-Maside, Laura; Donapetry-García, Cristóbal; Fernández-Fernández, Carlos; Sixto-Leal, Cristina

2017-06-01

Branched-chain amino acids (leucine, isoleucine and valine) are structurally related to branched-chain fatty acids. Leucine is 2-amino-4-methyl-pentanoic acid, isoleucine is 2-amino-3-methyl-pentanoic acid, and valine is 2-amino-3-methyl-butanoic acid. Similar to fatty acid oxidation, leucine and isoleucine produce acetyl-coA. Additionally, leucine generates acetoacetate and isoleucine yields propionyl-coA. Valine oxidation produces propionyl-coA, which is converted into methylmalonyl-coA and succinyl-coA. Branched-chain aminotransferase catalyzes the first reaction in the catabolic pathway of branched-chain amino acids, a reversible transamination that converts branched-chain amino acids into branched-chain ketoacids. Simultaneously, glutamate is converted in 2-ketoglutarate. The branched-chain ketoacid dehydrogenase complex catalyzes the irreversible oxidative decarboxylation of branched-chain ketoacids to produce branched-chain acyl-coA intermediates, which then follow separate catabolic pathways. Human tissue distribution and function of most of the enzymes involved in branched-chain amino acid catabolism is unknown. Congenital deficiencies of the enzymes involved in branched-chain amino acid metabolism are generally rare disorders. Some of them are associated with reduced pyruvate dehydrogenase complex activity and respiratory chain dysfunction that may contribute to their clinical phenotype. The biochemical phenotype is characterized by accumulation of the substrate to the deficient enzyme and its carnitine and/or glycine derivatives. It was established at the beginning of the twentieth century that the plasma level of the branched-chain amino acids is increased in conditions associated with insulin resistance such as obesity and diabetes mellitus. However, the potential clinical relevance of this elevation is uncertain.

Structure, High Affinity, and Negative Cooperativity of the Escherichia coli Holo-(Acyl Carrier Protein):Holo-(Acyl Carrier Protein) Synthase Complex.

PubMed

Marcella, Aaron M; Culbertson, Sannie J; Shogren-Knaak, Michael A; Barb, Adam W

2017-11-24

The Escherichia coli holo-(acyl carrier protein) synthase (ACPS) catalyzes the coenzyme A-dependent activation of apo-ACPP to generate holo-(acyl carrier protein) (holo-ACPP) in an early step of fatty acid biosynthesis. E. coli ACPS is sufficiently different from the human fatty acid synthase to justify the development of novel ACPS-targeting antibiotics. Models of E. coli ACPS in unliganded and holo-ACPP-bound forms solved by X-ray crystallography to 2.05and 4.10Å, respectively, revealed that ACPS bound three product holo-ACPP molecules to form a 3:3 hexamer. Solution NMR spectroscopy experiments validated the ACPS binding interface on holo-ACPP using chemical shift perturbations and by determining the relative orientation of holo-ACPP to ACPS by fitting residual dipolar couplings. The binding interface is organized to arrange contacts between positively charged ACPS residues and the holo-ACPP phosphopantetheine moiety, indicating product contains more stabilizing interactions than expected in the enzyme:substrate complex. Indeed, holo-ACPP bound the enzyme with greater affinity than the substrate, apo-ACPP, and with negative cooperativity. The first equivalent of holo-ACPP bound with a K D =62±13nM, followed by the binding of two more equivalents of holo-ACPP with K D =1.2±0.2μM. Cooperativity was not observed for apo-ACPP which bound with K D =2.4±0.1μM. Strong product binding and high levels of holo-ACPP in the cell identify a potential regulatory role of ACPS in fatty acid biosynthesis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Changes in blood carnitine and acylcarnitine profiles of very long-chain acyl-CoA dehydrogenase-deficient mice subjected to stress.

PubMed

Spiekerkoetter, U; Tokunaga, C; Wendel, U; Mayatepek, E; Exil, V; Duran, M; Wijburg, F A; Wanders, R J A; Strauss, A W

2004-03-01

In humans with deficiency of the very long-chain acyl-CoA dehydrogenase (VLCAD), C14-C18 acylcarnitines accumulate. In this paper we have used the VLCAD knockout mouse as a model to study changes in blood carnitine and acylcarnitine profiles under stress. VLCAD knockout mice exhibit stress-induced hypoglycaemia and skeletal myopathy; symptoms resembling human VLCADD. To study the extent of biochemical derangement in response to different stressors, we determined blood carnitine and acylcarnitine profiles after exercise on a treadmill, fasting, or exposure to cold. Even in a nonstressed, well-fed state, knockout mice presented twofold higher C14-C18 acylcarnitines and a lower free carnitine of 72% as compared to wild-type littermates. After 1 h of intense exercise, the C14-C18 acylcarnitines in blood significantly increased, but free carnitine remained unchanged. After 8 h of fasting at 4 degrees C, the long-chain acylcarnitines were elevated 5-fold in knockout mice in comparison with concentrations in unstressed wild-type mice (P < 0.05), and four out of 12 knockout mice died. Free carnitine decreased to 44% as compared with unstressed wild-type mice. An increase in C14-C18 acylcarnitines and a decrease of free carnitine were also observed in fasted heterozygous and wild-type mice. Long-chain acylcarnitines in blood increase in knockout mice in response to different stressors and concentrations correlate with the clinical condition. A decrease in blood free carnitine in response to severe stress is observed in knockout mice but also in wild-type littermates. Monitoring blood acylcarnitine profiles in response to different stressors may allow systematic analysis of therapeutic interventions in VLCAD knockout mice.

N-Acyl amino acids and their impact on biological processes.

PubMed

Hanuš, Lumír; Shohami, Esther; Bab, Itai; Mechoulam, Raphael

2014-01-01

Over the last two decades a large number of N-long-chain acyl amino acids have been identified in the mammalian body. The pharmacological activities of only a few of them have been investigated and some have been found to be of considerable interest. Thus arachidonoyl serine is vasodilatory and neuroprotective, arachidonoyl glycine is antinociceptive, and oleoyl serine rescues bone loss. However, the pathophysiological/biochemical roles of these amides are mostly unknown. © 2014 International Union of Biochemistry and Molecular Biology.

The hexanoyl-CoA precursor for cannabinoid biosynthesis is formed by an acyl-activating enzyme in Cannabis sativa trichomes.

PubMed

Stout, Jake M; Boubakir, Zakia; Ambrose, Stephen J; Purves, Randy W; Page, Jonathan E

2012-08-01

The psychoactive and analgesic cannabinoids (e.g. Δ(9) -tetrahydrocannabinol (THC)) in Cannabis sativa are formed from the short-chain fatty acyl-coenzyme A (CoA) precursor hexanoyl-CoA. Cannabinoids are synthesized in glandular trichomes present mainly on female flowers. We quantified hexanoyl-CoA using LC-MS/MS and found levels of 15.5 pmol g(-1) fresh weight in female hemp flowers with lower amounts in leaves, stems and roots. This pattern parallels the accumulation of the end-product cannabinoid, cannabidiolic acid (CBDA). To search for the acyl-activating enzyme (AAE) that synthesizes hexanoyl-CoA from hexanoate, we analyzed the transcriptome of isolated glandular trichomes. We identified 11 unigenes that encoded putative AAEs including CsAAE1, which shows high transcript abundance in glandular trichomes. In vitro assays showed that recombinant CsAAE1 activates hexanoate and other short- and medium-chained fatty acids. This activity and the trichome-specific expression of CsAAE1 suggest that it is the hexanoyl-CoA synthetase that supplies the cannabinoid pathway. CsAAE3 encodes a peroxisomal enzyme that activates a variety of fatty acid substrates including hexanoate. Although phylogenetic analysis showed that CsAAE1 groups with peroxisomal AAEs, it lacked a peroxisome targeting sequence 1 (PTS1) and localized to the cytoplasm. We suggest that CsAAE1 may have been recruited to the cannabinoid pathway through the loss of its PTS1, thereby redirecting it to the cytoplasm. To probe the origin of hexanoate, we analyzed the trichome expressed sequence tag (EST) dataset for enzymes of fatty acid metabolism. The high abundance of transcripts that encode desaturases and a lipoxygenase suggests that hexanoate may be formed through a pathway that involves the oxygenation and breakdown of unsaturated fatty acids. © 2012 National Research Council of Canada. The Plant Journal © 2012 Blackwell Publishing Ltd.

Purification and substrate specificity of Staphylococcus hyicus lipase.

PubMed

van Oort, M G; Deveer, A M; Dijkman, R; Tjeenk, M L; Verheij, H M; de Haas, G H; Wenzig, E; Götz, F

1989-11-28

The Staphylococcus hyicus lipase gene has been cloned and expressed in Staphylococcus carnosus. From the latter organism the enzyme was secreted into the medium as a protein with an apparent molecular mass of 86 kDa. This protein was purified, and the amino-terminal sequence showed that the primary gene product was indeed cleaved at the proposed signal peptide cleavage site. The protein was purified from large-scale preparations after tryptic digestion. This limited proteolysis reduced the molecular mass to 46 kDa and increased the specific activity about 3-fold. Although the enzyme had a low specific activity in the absence of divalent cations, the activity increased about 40-fold in the presence of Sr2+ or Ca2+ ions. The purified lipase has a broad substrate specificity. The acyl chains were removed from the primary and secondary positions of natural neutral glycerides and from a variety of synthetic glyceride analogues. Thus triglycerides were fully hydrolyzed to free fatty acid and glycerol. The enzyme hydrolyzed naturally occurring phosphatidylcholines, their synthetic short-chain analogues, and lysophospholipids to free fatty acids and water-soluble products. The enzyme had a 2-fold higher activity on micelles of short-chain D-lecithins than on micelles composed of the L-isomers. Thus the enzyme from S. hyicus has lipase activity and also high phospholipase A and lysophospholipase activity.

Fatty Acid Synthesis in Pea Root Plastids Is Inhibited by the Action of Long-Chain Acyl- Coenzyme As on Metabolite Transporters1

PubMed Central

Fox, Simon R.; Rawsthorne, Stephen; Hills, Matthew J.

2001-01-01

The uptake in vitro of glucose (Glc)-6-phosphate (Glc-6-P) into plastids from the roots of 10- to 14-d-old pea (Pisum sativum L. cv Puget) plants was inhibited by oleoyl-coenzyme A (CoA) concentrations in the low micromolar range (1–2 μm). The IC50 (the concentration of inhibitor that reduces enzyme activity by 50%) for the inhibition of Glc-6-P uptake was approximately 750 nm; inhibition was reversed by recombinant rapeseed (Brassica napus) acyl-CoA binding protein. In the presence of ATP (3 mm) and CoASH (coenzyme A; 0.3 mm), Glc-6-P uptake was inhibited by 60%, due to long-chain acyl-CoA synthesis, presumably from endogenous sources of fatty acids present in the preparations. Addition of oleoyl-CoA (1 μm) decreased carbon flux from Glc-6-P into the synthesis of starch and through the oxidative pentose phosphate (OPP) pathway by up to 73% and 40%, respectively. The incorporation of carbon from Glc-6-P into fatty acids was not detected under any conditions. Oleoyl-CoA inhibited the incorporation of acetate into fatty acids by 67%, a decrease similar to that when ATP was excluded from incubations. The oleoyl-CoA-dependent inhibition of fatty acid synthesis was attributable to a direct inhibition of the adenine nucleotide translocator by oleoyl-CoA, which indirectly reduced fatty acid synthesis by ATP deprivation. The Glc-6-P-dependent stimulation of acetate incorporation into fatty acids was reversed by the addition of oleoyl-CoA. PMID:11457976

Structural and Functional Studies of Fatty Acyl Adenylate Ligases from E. coli and L. pneumophila

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Z.; Swaminathan, S.; Zhou, R.

2011-02-18

Fatty acyl-AMP ligase (FAAL) is a new member of a family of adenylate-forming enzymes that were recently discovered in Mycobacterium tuberculosis. They are similar in sequence to fatty acyl-coenzyme A (CoA) ligases (FACLs). However, while FACLs perform a two-step catalytic reaction, AMP ligation followed by CoA ligation using ATP and CoA as cofactors, FAALs produce only the acyl adenylate and are unable to perform the second step. We report X-ray crystal structures of full-length FAAL from Escherichia coli (EcFAAL) and FAAL from Legionella pneumophila (LpFAAL) bound to acyl adenylate, determined at resolution limits of 3.0 and 1.85 {angstrom}, respectively. Themore » structures share a larger N-terminal domain and a smaller C-terminal domain, which together resemble the previously determined structures of FAAL and FACL proteins. Our two structures occur in quite different conformations. EcFAAL adopts the adenylate-forming conformation typical of FACLs, whereas LpFAAL exhibits a unique intermediate conformation. Both EcFAAL and LpFAAL have insertion motifs that distinguish them from the FACLs. Structures of EcFAAL and LpFAAL reveal detailed interactions between this insertion motif and the interdomain hinge region and with the C-terminal domain. We suggest that the insertion motifs support sufficient interdomain motions to allow substrate binding and product release during acyl adenylate formation, but they preclude CoA binding, thereby preventing CoA ligation.« less

Structural and Functional Studies of Fatty Acyl Adenylate Ligases from E. coli and L. pneumophila

DOE Office of Scientific and Technical Information (OSTI.GOV)

Z Zhang; R Zhou; J Sauder

2011-12-31

Fatty acyl-AMP ligase (FAAL) is a new member of a family of adenylate-forming enzymes that were recently discovered in Mycobacterium tuberculosis. They are similar in sequence to fatty acyl-coenzyme A (CoA) ligases (FACLs). However, while FACLs perform a two-step catalytic reaction, AMP ligation followed by CoA ligation using ATP and CoA as cofactors, FAALs produce only the acyl adenylate and are unable to perform the second step. We report X-ray crystal structures of full-length FAAL from Escherichia coli (EcFAAL) and FAAL from Legionella pneumophila (LpFAAL) bound to acyl adenylate, determined at resolution limits of 3.0 and 1.85 {angstrom}, respectively. Themore » structures share a larger N-terminal domain and a smaller C-terminal domain, which together resemble the previously determined structures of FAAL and FACL proteins. Our two structures occur in quite different conformations. EcFAAL adopts the adenylate-forming conformation typical of FACLs, whereas LpFAAL exhibits a unique intermediate conformation. Both EcFAAL and LpFAAL have insertion motifs that distinguish them from the FACLs. Structures of EcFAAL and LpFAAL reveal detailed interactions between this insertion motif and the interdomain hinge region and with the C-terminal domain. We suggest that the insertion motifs support sufficient interdomain motions to allow substrate binding and product release during acyl adenylate formation, but they preclude CoA binding, thereby preventing CoA ligation.« less

Arabidopsis thaliana GH3.5 acyl acid amido synthetase mediates metabolic crosstalk in auxin and salicylic acid homeostasis

PubMed Central

Westfall, Corey S.; Sherp, Ashley M.; Zubieta, Chloe; Alvarez, Sophie; Schraft, Evelyn; Marcellin, Romain; Ramirez, Loren; Jez, Joseph M.

2016-01-01

In Arabidopsis thaliana, the acyl acid amido synthetase Gretchen Hagen 3.5 (AtGH3.5) conjugates both indole-3-acetic acid (IAA) and salicylic acid (SA) to modulate auxin and pathogen response pathways. To understand the molecular basis for the activity of AtGH3.5, we determined the X-ray crystal structure of the enzyme in complex with IAA and AMP. Biochemical analysis demonstrates that the substrate preference of AtGH3.5 is wider than originally described and includes the natural auxin phenylacetic acid (PAA) and the potential SA precursor benzoic acid (BA). Residues that determine IAA versus BA substrate preference were identified. The dual functionality of AtGH3.5 is unique to this enzyme although multiple IAA-conjugating GH3 proteins share nearly identical acyl acid binding sites. In planta analysis of IAA, PAA, SA, and BA and their respective aspartyl conjugates were determined in wild-type and overexpressing lines of A. thaliana. This study suggests that AtGH3.5 conjugates auxins (i.e., IAA and PAA) and benzoates (i.e., SA and BA) to mediate crosstalk between different metabolic pathways, broadening the potential roles for GH3 acyl acid amido synthetases in plants. PMID:27849615

Altered Energetics of Exercise Explain Risk of Rhabdomyolysis in Very Long-Chain Acyl-CoA Dehydrogenase Deficiency

PubMed Central

Diekman, E. F.; Visser, G.; Schmitz, J. P. J.; Nievelstein, R. A. J.; de Sain-van der Velden, M.; Wardrop, M.; Van der Pol, W. L.; Houten, S. M.; van Riel, N. A. W.; Takken, T.; Jeneson, J. A. L.

2016-01-01

Rhabdomyolysis is common in very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) and other metabolic myopathies, but its pathogenic basis is poorly understood. Here, we show that prolonged bicycling exercise against a standardized moderate workload in VLCADD patients is associated with threefold bigger changes in phosphocreatine (PCr) and inorganic phosphate (Pi) concentrations in quadriceps muscle and twofold lower changes in plasma acetyl-carnitine levels than in healthy subjects. This result is consistent with the hypothesis that muscle ATP homeostasis during exercise is compromised in VLCADD. However, the measured rates of PCr and Pi recovery post-exercise showed that the mitochondrial capacity for ATP synthesis in VLCADD muscle was normal. Mathematical modeling of oxidative ATP metabolism in muscle composed of three different fiber types indicated that the observed altered energy balance during submaximal exercise in VLCADD patients may be explained by a slow-to-fast shift in quadriceps fiber-type composition corresponding to 30% of the slow-twitch fiber-type pool in healthy quadriceps muscle. This study demonstrates for the first time that quadriceps energy balance during exercise in VLCADD patients is altered but not because of failing mitochondrial function. Our findings provide new clues to understanding the risk of rhabdomyolysis following exercise in human VLCADD. PMID:26881790

Alterations by peroxisome proliferators of acyl composition of hepatic phosphatidylcholine in rats, mice and guinea-pigs. Role of stearoyl-CoA desaturase.

PubMed Central

Kawashima, Y; Hirose, A; Kozuka, H

1986-01-01

Rats, mice and guinea-pigs were administered p-chlorophenoxyisobutyric acid (clofibric acid) or 2,2'-(decamethylenedithio)diethanol (tiadenol). The treatments of rats and mice with either clofibric acid or tiadenol increased markedly the activities of stearoyl-CoA desaturase, palmitoyl-CoA chain elongation, 1-acylglycerophosphate (1-acyl-GP) acyltransferase and 1-acylglycerophosphocholine (1-acyl-GPC) acyltransferase, but not 2-acylglycerophosphocholine (2-acyl-GPC) acyltransferase in liver microsomes. The treatment of guinea-pigs with clofibric acid did not cause any change in the activities of these enzymes. The treatment of guinea-pigs with tiadenol caused a slight, but significant, increase in the activities of 1-acyl-GP acyltransferase and 1-acyl-GPC acyltransferase. The treatment of rats and mice with either clofibric acid or tiadenol increased markedly the proportion of 18:1 and decreased greatly the proportion of 18:0 in liver microsomal phosphatidylcholine. However, there is a considerable difference in the effects of the two peroxisome proliferators on the composition of polyunsaturated fatty acids in phosphatidylcholine between rats and mice. The treatment of guinea-pigs with either of the two peroxisome proliferators caused no change in acyl composition of phosphatidylcholine. The possible role of stearoyl-CoA desaturation in the regulation of acyl composition of phosphatidylcholine was discussed. PMID:2874791

ETFDH mutations as a major cause of riboflavin-responsive multiple acyl-CoA dehydrogenation deficiency.

PubMed

Olsen, Rikke K J; Olpin, Simon E; Andresen, Brage S; Miedzybrodzka, Zofia H; Pourfarzam, Morteza; Merinero, Begoña; Frerman, Frank E; Beresford, Michael W; Dean, John C S; Cornelius, Nanna; Andersen, Oluf; Oldfors, Anders; Holme, Elisabeth; Gregersen, Niels; Turnbull, Douglass M; Morris, Andrew A M

2007-08-01

Multiple acyl-CoA dehydrogenation deficiency (MADD) is a disorder of fatty acid, amino acid and choline metabolism that can result from defects in two flavoproteins, electron transfer flavoprotein (ETF) or ETF: ubiquinone oxidoreductase (ETF:QO). Some patients respond to pharmacological doses of riboflavin. It is unknown whether these patients have defects in the flavoproteins themselves or defects in the formation of the cofactor, FAD, from riboflavin. We report 15 patients from 11 pedigrees. All the index cases presented with encephalopathy or muscle weakness or a combination of these symptoms; several had previously suffered cyclical vomiting. Urine organic acid and plasma acyl-carnitine profiles indicated MADD. Clinical and biochemical parameters were either totally or partly corrected after riboflavin treatment. All patients had mutations in the gene for ETF:QO. In one patient, we show that the ETF:QO mutations are associated with a riboflavin-sensitive impairment of ETF:QO activity. This patient also had partial deficiencies of flavin-dependent acyl-CoA dehydrogenases and respiratory chain complexes, most of which were restored to control levels after riboflavin treatment. Low activities of mitochondrial flavoproteins or respiratory chain complexes have been reported previously in two of our patients with ETF:QO mutations. We postulate that riboflavin-responsive MADD may result from defects of ETF:QO combined with general mitochondrial dysfunction. This is the largest collection of riboflavin-responsive MADD patients ever reported, and the first demonstration of the molecular genetic basis for the disorder.

Crystal Structures of Xanthomonas campestris OleA Reveal Features That Promote Head-to-Head Condensation of Two Long-Chain Fatty Acids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goblirsch, Brandon R.; Frias, Janice A.; Wackett, Lawrence P.

2012-10-25

OleA is a thiolase superfamily enzyme that has been shown to catalyze the condensation of two long-chain fatty acyl-coenzyme A (CoA) substrates. The enzyme is part of a larger gene cluster responsible for generating long-chain olefin products, a potential biofuel precursor. In thiolase superfamily enzymes, catalysis is achieved via a ping-pong mechanism. The first substrate forms a covalent intermediate with an active site cysteine that is followed by reaction with the second substrate. For OleA, this conjugation proceeds by a nondecarboxylative Claisen condensation. The OleA from Xanthomonas campestris has been crystallized and its structure determined, along with inhibitor-bound and xenon-derivatizedmore » structures, to improve our understanding of substrate positioning in the context of enzyme turnover. OleA is the first characterized thiolase superfamily member that has two long-chain alkyl substrates that need to be bound simultaneously and therefore uniquely requires an additional alkyl binding channel. The location of the fatty acid biosynthesis inhibitor, cerulenin, that possesses an alkyl chain length in the range of known OleA substrates, in conjunction with a single xenon binding site, leads to the putative assignment of this novel alkyl binding channel. Structural overlays between the OleA homologues, 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase and the fatty acid biosynthesis enzyme FabH, allow assignment of the two remaining channels: one for the thioester-containing pantetheinate arm and the second for the alkyl group of one substrate. A short {beta}-hairpin region is ordered in only one of the crystal forms, and that may suggest open and closed states relevant for substrate binding. Cys143 is the conserved catalytic cysteine within the superfamily, and the site of alkylation by cerulenin. The alkylated structure suggests that a glutamic acid residue (Glu117{beta}) likely promotes Claisen condensation by acting as the catalytic base

Strategies for Correcting Very Long Chain Acyl-CoA Dehydrogenase Deficiency*

PubMed Central

Tenopoulou, Margarita; Chen, Jie; Bastin, Jean; Bennett, Michael J.; Ischiropoulos, Harry; Doulias, Paschalis-Thomas

2015-01-01

Very long acyl-CoA dehydrogenase (VLCAD) deficiency is a genetic pediatric disorder presenting with a spectrum of phenotypes that remains for the most part untreatable. Here, we present a novel strategy for the correction of VLCAD deficiency by increasing mutant VLCAD enzymatic activity. Treatment of VLCAD-deficient fibroblasts, which express distinct mutant VLCAD protein and exhibit deficient fatty acid β-oxidation, with S-nitroso-N-acetylcysteine induced site-specific S-nitrosylation of VLCAD mutants at cysteine residue 237. Cysteine 237 S-nitrosylation was associated with an 8–17-fold increase in VLCAD-specific activity and concomitant correction of acylcarnitine profile and β-oxidation capacity, two hallmarks of the disorder. Overall, this study provides biochemical evidence for a potential therapeutic modality to correct β-oxidation deficiencies. PMID:25737446

Tissue-specific regulation of medium-chain acyl-CoA dehydrogenase gene by thyroid hormones in the developing rat.

PubMed

Djouadi, F; Riveau, B; Merlet-Benichou, C; Bastin, J

1997-05-15

During development, gene expression of medium-chain acyl-CoA dehydrogenase (MCAD), a nuclear-encoded mitochondrial enzyme that catalyses the first step of medium-chain fatty acid beta-oxidation, is highly regulated in tissues in accordance with fatty acid utilization, but the factors involved in this regulation are largely unknown. To investigate a possible role of thyroid hormones, rat pups were made hypothyroid by the administration of propylthiouracyl to the mother from day 12 of gestation, and their kidneys, heart and liver were removed on postnatal day 16 to determine MCAD mRNA abundance, protein level and enzyme activity. Similar experiments were run in 3,3',5-tri-iodothyronine (T3)-replaced hypothyroid (1 microg of T3/100 g body weight from postnatal day 5 to 15) and euthyroid pups. Hypothyroidism led to an increase in MCAD mRNA abundance in kidney and a decrease in abundance in heart, but had no effect in liver. The protein levels and enzyme activity were lowered in hypothyroid heart and kidney, suggesting that hypothyroidism affects post-transcriptional steps of gene expression in the kidney. All the effects of hypothyroidism were completely reversed in both heart and kidney by T3 replacement. Injection of a single T3 dose into 16-day-old euthyroid rats also led to tissue-specific changes in mRNA abundance. Nuclear run-on assays performed from hypothyroid and hypothyroid plus T3 rats showed that T3 stimulates MCAD gene transcription in heart and represses it in the kidney. These results indicate that the postnatal rise in circulating T3 is essential to the developmental regulation of the MCAD gene in vivo.

Physicochemical Parameters Affecting the Electrospray Ionization Efficiency of Amino Acids after Acylation

PubMed Central

2017-01-01

Electrospray ionization (ESI) is widely used in liquid chromatography coupled to mass spectrometry (LC–MS) for the analysis of biomolecules. However, the ESI process is still not completely understood, and it is often a matter of trial and error to enhance ESI efficiency and, hence, the response of a given set of compounds. In this work we performed a systematic study of the ESI response of 14 amino acids that were acylated with organic acid anhydrides of increasing chain length and with poly(ethylene glycol) (PEG) changing certain physicochemical properties in a predictable manner. By comparing the ESI response of 70 derivatives, we found that there was a strong correlation between the calculated molecular volume and the ESI response, while correlation with hydrophobicity (log P values), pKa, and the inverse calculated surface tension was significantly lower although still present, especially for individual derivatized amino acids with increasing acyl chain lengths. Acylation with PEG containing five ethylene glycol units led to the largest gain in ESI response. This response was maximal independent of the calculated physicochemical properties or the type of amino acid. Since no actual physicochemical data is available for most derivatized compounds, the responses were also used as input for a quantitative structure–property relationship (QSPR) model to find the best physicochemical descriptors relating to the ESI response from molecular structures using the amino acids and their derivatives as a reference set. A topological descriptor related to molecular size (SPAN) was isolated next to a descriptor related to the atomic composition and structural groups (BIC0). The validity of the model was checked with a test set of 43 additional compounds that were unrelated to amino acids. While prediction was generally good (R2 > 0.9), compounds containing halogen atoms or nitro groups gave a lower predicted ESI response. PMID:28737384

Enhanced Cellular Uptake of Short Polyarginine Peptides through Fatty Acylation and Cyclization

PubMed Central

2015-01-01

Many of the reported arginine-rich cell-penetrating peptides (CPPs) for the enhanced delivery of drugs are linear peptides composed of more than seven arginine residues to retain the cell penetration properties. Herein, we synthesized a class of nine polyarginine peptides containing 5 and 6 arginines, namely, R5 and R6. We further explored the effect of acylation with long chain fatty acids (i.e., octanoic acid, dodecanoic acid, and hexadecanoic acid) and cyclization on the cell penetrating properties of the peptides. The fluorescence-labeled acylated cyclic peptide dodecanoyl-[R5] and linear peptide dodecanoyl-(R5) showed approximately 13.7- and 10.2-fold higher cellular uptake than that of control 5,6-carboxyfluorescein, respectively. The mechanism of the peptide internalization into cells was found to be energy-dependent endocytosis. Dodecanoyl-[R5] and dodecanoyl-[R6] enhanced the intracellular uptake of a fluorescence-labeled cell-impermeable negatively charged phosphopeptide (F′-GpYEEI) in human ovarian cancer cells (SK-OV-3) by 3.4-fold and 5.5-fold, respectively, as shown by flow cytometry. The cellular uptake of F′-GpYEEI in the presence of hexadecanoyl-[R5] was 9.3- and 6.0-fold higher than that in the presence of octanoyl-[R5] and dodecanoyl-[R5], respectively. Dodecanoyl-[R5] enhanced the cellular uptake of the phosphopeptide by 1.4–2.5-fold higher than the corresponding linear peptide dodecanoyl-(R5) and those of representative CPPs, such as hepta-arginine (CR7) and TAT peptide. These results showed that a combination of acylation by long chain fatty acids and cyclization on short arginine-containing peptides can improve their cell-penetrating property, possibly through efficient interaction of rigid positively charged R and hydrophobic dodecanoyl moiety with the corresponding residues in the cell membrane phospholipids. PMID:24978295

A high-performance liquid chromatography-based radiometric assay for acyl-CoA:alcohol transacylase from jojoba.

PubMed

Garver, W S; Kemp, J D; Kuehn, G D

1992-12-01

Acyl-CoA:alcohol transacylase catalyzes the final step in the biosynthesis of storage liquid wax esters from acyl-CoA fatty acids and fatty alcohols in a limited number of microbes, algae, and Simmondsia chinensis Link (jojoba). An improved and automated method of enzyme assay for this catalyst from cotyledons of jojoba is described. The assay method uses reversed-phase C18 high performance liquid chromatography (HPLC) to separate the labeled C30:1 liquid wax product, [14C]-dodecanyl-octadecenoate, from the unreacted substrate, [14C]octadecenoyl-CoA (oleyl-CoA), and other components produced from enzymes present in the crude homogenate of jojoba cotyledons, including [14C]-octadecenoic acid (oleic acid) and [14C]octadecenol (oleyol). Methods are also described for microscale chemical synthesis in one vessel of 14C-radiolabeled substrates and products for the transacylase. These labeled reagents are required to confirm the HPLC separations of reaction products. The radioactive components are quantitated using an on-line flow-through scintillation detector enabling sensitive and precise analysis of the reaction products.

Design and synthesis of 2-nitroimidazoles with variable alkylating and acylating functionality.

PubMed

Winters, Thomas; Sercel, Anthony; Suto, Carla; Elliott, William; Leopold, Wilbur; Leopold, Judith; Showalter, Hollis

2014-01-01

The synthesis of a small series of 2-nitroimidazoles in which the β-amino alcohol side chain was amidated with a range of alkylating/acylating functionality is described. Synthetic methodologies were developed that generally provided for selective N-acyl versus N,O-bisacyl products. In vitro, target analogs showed minimal radiosensitization activity, with only a few exhibiting a sensitizer enhancement ratio (SER) >2.0 and C(1.6) values comparable to reference agents RB-6145 and RSU-1069. In an assay to determine potential to alkylate biomolecules, representative analogs showed <1% of the alkylating activity of RSU-1069. In vivo, one analog showed an enhancement ratio of 1.6 relative to vehicle control when tested in B6C3F1 mice with an implanted KHT sarcoma. The data reinforce prior findings that there is a correlation between alkylation potential and in vivo activity.

Characterization of Site-Specific Mutations in a Short-Chain-Length/Medium-Chain-Length Polyhydroxyalkanoate Synthase: In Vivo and In Vitro Studies of Enzymatic Activity and Substrate Specificity

PubMed Central

Chuah, Jo-Ann; Tomizawa, Satoshi; Yamada, Miwa; Tsuge, Takeharu; Doi, Yoshiharu

2013-01-01

Saturation point mutagenesis was carried out at position 479 in the polyhydroxyalkanoate (PHA) synthase from Chromobacterium sp. strain USM2 (PhaCCs) with specificities for short-chain-length (SCL) [(R)-3-hydroxybutyrate (3HB) and (R)-3-hydroxyvalerate (3HV)] and medium-chain-length (MCL) [(R)-3-hydroxyhexanoate (3HHx)] monomers in an effort to enhance the specificity of the enzyme for 3HHx. A maximum 4-fold increase in 3HHx incorporation and a 1.6-fold increase in PHA biosynthesis, more than the wild-type synthase, was achieved using selected mutant synthases. These increases were subsequently correlated with improved synthase activity and increased preference of PhaCCs for 3HHx monomers. We found that substitutions with uncharged residues were beneficial, as they resulted in enhanced PHA production and/or 3HHx incorporation. Further analysis led to postulations that the size and geometry of the substrate-binding pocket are determinants of PHA accumulation, 3HHx fraction, and chain length specificity. In vitro activities for polymerization of 3HV and 3HHx monomers were consistent with in vivo substrate specificities. Ultimately, the preference shown by wild-type and mutant synthases for either SCL (C4 and C5) or MCL (C6) substrates substantiates the fundamental classification of PHA synthases. PMID:23584780

Model-based Confirmation of Alternative Substrates of Mitochondrial Electron Transport Chain

PubMed Central

Kleessen, Sabrina; Araújo, Wagner L.; Fernie, Alisdair R.; Nikoloski, Zoran

2012-01-01

Discrimination of metabolic models based on high throughput metabolomics data, reflecting various internal and external perturbations, is essential for identifying the components that contribute to the emerging behavior of metabolic processes. Here, we investigate 12 different models of the mitochondrial electron transport chain (ETC) in Arabidopsis thaliana during dark-induced senescence in order to elucidate the alternative substrates to this metabolic pathway. Our findings demonstrate that the coupling of the proposed computational approach, based on dynamic flux balance analysis, with time-resolved metabolomics data results in model-based confirmations of the hypotheses that, during dark-induced senescence in Arabidopsis, (i) under conditions where the main substrate for the ETC are not fully available, isovaleryl-CoA dehydrogenase and 2-hydroxyglutarate dehydrogenase are able to donate electrons to the ETC, (ii) phytanoyl-CoA does not act even as an indirect substrate of the electron transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase complex, and (iii) the mitochondrial γ-aminobutyric acid transporter has functional significance in maintaining mitochondrial metabolism. Our study provides a basic framework for future in silico studies of alternative pathways in mitochondrial metabolism under extended darkness whereby the role of its components can be computationally discriminated based on available molecular profile data. PMID:22334689

Model-based confirmation of alternative substrates of mitochondrial electron transport chain.

PubMed

Kleessen, Sabrina; Araújo, Wagner L; Fernie, Alisdair R; Nikoloski, Zoran

2012-03-30

Discrimination of metabolic models based on high throughput metabolomics data, reflecting various internal and external perturbations, is essential for identifying the components that contribute to the emerging behavior of metabolic processes. Here, we investigate 12 different models of the mitochondrial electron transport chain (ETC) in Arabidopsis thaliana during dark-induced senescence in order to elucidate the alternative substrates to this metabolic pathway. Our findings demonstrate that the coupling of the proposed computational approach, based on dynamic flux balance analysis, with time-resolved metabolomics data results in model-based confirmations of the hypotheses that, during dark-induced senescence in Arabidopsis, (i) under conditions where the main substrate for the ETC are not fully available, isovaleryl-CoA dehydrogenase and 2-hydroxyglutarate dehydrogenase are able to donate electrons to the ETC, (ii) phytanoyl-CoA does not act even as an indirect substrate of the electron transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase complex, and (iii) the mitochondrial γ-aminobutyric acid transporter has functional significance in maintaining mitochondrial metabolism. Our study provides a basic framework for future in silico studies of alternative pathways in mitochondrial metabolism under extended darkness whereby the role of its components can be computationally discriminated based on available molecular profile data.

Novel approach in LC-MS/MS using MRM to generate a full profile of acyl-CoAs: discovery of acyl-dephospho-CoAs.

PubMed

Li, Qingling; Zhang, Shenghui; Berthiaume, Jessica M; Simons, Brigitte; Zhang, Guo-Fang

2014-03-01

A metabolomic approach to selectively profile all acyl-CoAs was developed using a programmed multiple reaction monitoring (MRM) method in LC-MS/MS and was employed in the analysis of various rat organs. The programmed MRM method possessed 300 mass ion transitions with the mass difference of 507 between precursor ion (Q1) and product ion (Q3), and the precursor ion started from m/z 768 and progressively increased one mass unit at each step. Acyl-dephospho-CoAs resulting from the dephosphorylation of acyl-CoAs were identified by accurate MS and fragmentation. Acyl-dephospho-CoAs were also quantitatively scanned by the MRM method with the mass difference of 427 between Q1 and Q3 mass ions. Acyl-CoAs and dephospho-CoAs were assayed with limits of detection ranging from 2 to 133 nM. The accuracy of the method was demonstrated by assaying a range of concentrations of spiked acyl-CoAs with the results of 80-114%. The distribution of acyl-CoAs reflects the metabolic status of each organ. The physiological role of dephosphorylation of acyl-CoAs remains to be further characterized. The methodology described herein provides a novel strategy in metabolomic studies to quantitatively and qualitatively profile all potential acyl-CoAs and acyl-dephospho-CoAs.

Structural insights into GDP-mediated regulation of a bacterial acyl-CoA thioesterase.

PubMed

Khandokar, Yogesh B; Srivastava, Parul; Cowieson, Nathan; Sarker, Subir; Aragao, David; Das, Shubagata; Smith, Kate M; Raidal, Shane R; Forwood, Jade K

2017-12-15

Thioesterases catalyze the cleavage of thioester bonds within many activated fatty acids and acyl-CoA substrates. They are expressed ubiquitously in both prokaryotes and eukaryotes and are subdivided into 25 thioesterase families according to their catalytic active site, protein oligomerization, and substrate specificity. Although many of these enzyme families are well-characterized in terms of function and substrate specificity, regulation across most thioesterase families is poorly understood. Here, we characterized a TE6 thioesterase from the bacterium Neisseria meningitidis Structural analysis with X-ray crystallographic diffraction data to 2.0-Å revealed that each protein subunit harbors a hot dog-fold and that the TE6 enzyme forms a hexamer with D3 symmetry. An assessment of thioesterase activity against a range of acyl-CoA substrates revealed the greatest activity against acetyl-CoA, and structure-guided mutagenesis of putative active site residues identified Asn 24 and Asp 39 as being essential for activity. Our structural analysis revealed that six GDP nucleotides bound the enzyme in close proximity to an intersubunit disulfide bond interactions that covalently link thioesterase domains in a double hot dog dimer. Structure-guided mutagenesis of residues within the GDP-binding pocket identified Arg 93 as playing a key role in the nucleotide interaction and revealed that GDP is required for activity. All mutations were confirmed to be specific and not to have resulted from structural perturbations by X-ray crystallography. This is the first report of a bacterial GDP-regulated thioesterase and of covalent linkage of thioesterase domains through a disulfide bond, revealing structural similarities with ADP regulation in the human ACOT12 thioesterase. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Unique plasma metabolomic signatures of individuals with inherited disorders of long-chain fatty acid oxidation

USDA-ARS?s Scientific Manuscript database

Blood and urine acylcarnitine profiles are commonly used to diagnose long-chain fatty acid oxidation disorders (FAOD: i.e., long-chain hydroxy-acyl-CoA dehydrogenase [LCHAD] and carnitine palmitoyltransferase 2 [CPT2] deficiency), but the global metabolic impact of long-chain FAOD has not been repor...

A microarray of ubiquitylated proteins for profiling deubiquitylase activity reveals the critical roles of both chain and substrate.

PubMed

Loch, Christian M; Strickler, James E

2012-11-01

Substrate ubiquitylation is a reversible process critical to cellular homeostasis that is often dysregulated in many human pathologies including cancer and neurodegeneration. Elucidating the mechanistic details of this pathway could unlock a large store of information useful to the design of diagnostic and therapeutic interventions. Proteomic approaches to the questions at hand have generally utilized mass spectrometry (MS), which has been successful in identifying both ubiquitylation substrates and profiling pan-cellular chain linkages, but is generally unable to connect the two. Interacting partners of the deubiquitylating enzymes (DUBs) have also been reported by MS, although substrates of catalytically competent DUBs generally cannot be. Where they have been used towards the study of ubiquitylation, protein microarrays have usually functioned as platforms for the identification of substrates for specific E3 ubiquitin ligases. Here, we report on the first use of protein microarrays to identify substrates of DUBs, and in so doing demonstrate the first example of microarray proteomics involving multiple (i.e., distinct, sequential and opposing) enzymatic activities. This technique demonstrates the selectivity of DUBs for both substrate and type (mono- versus poly-) of ubiquitylation. This work shows that the vast majority of DUBs are monoubiquitylated in vitro, and are incapable of removing this modification from themselves. This work also underscores the critical role of utilizing both ubiquitin chains and substrates when attempting to characterize DUBs. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics. Copyright © 2012 Elsevier B.V. All rights reserved.

An Acrobatic Substrate Metamorphosis Reveals a Requirement for Substrate Conformational Dynamics in Trypsin Proteolysis*

PubMed Central

Kayode, Olumide; Wang, Ruiying; Pendlebury, Devon F.; Cohen, Itay; Henin, Rachel D.; Hockla, Alexandra; Soares, Alexei S.; Papo, Niv; Caulfield, Thomas R.; Radisky, Evette S.

2016-01-01

The molecular basis of enzyme catalytic power and specificity derives from dynamic interactions between enzyme and substrate during catalysis. Although considerable effort has been devoted to understanding how conformational dynamics within enzymes affect catalysis, the role of conformational dynamics within protein substrates has not been addressed. Here, we examine the importance of substrate dynamics in the cleavage of Kunitz-bovine pancreatic trypsin inhibitor protease inhibitors by mesotrypsin, finding that the varied conformational dynamics of structurally similar substrates can profoundly impact the rate of catalysis. A 1.4-Å crystal structure of a mesotrypsin-product complex formed with a rapidly cleaved substrate reveals a dramatic conformational change in the substrate upon proteolysis. By using long all-atom molecular dynamics simulations of acyl-enzyme intermediates with proteolysis rates spanning 3 orders of magnitude, we identify global and local dynamic features of substrates on the nanosecond-microsecond time scale that correlate with enzymatic rates and explain differential susceptibility to proteolysis. By integrating multiple enhanced sampling methods for molecular dynamics, we model a viable conformational pathway between substrate-like and product-like states, linking substrate dynamics on the nanosecond-microsecond time scale with large collective substrate motions on the much slower time scale of catalysis. Our findings implicate substrate flexibility as a critical determinant of catalysis. PMID:27810896

Synergistic N-Heterocyclic Carbene/Palladium-Catalyzed Reactions of Aldehyde Acyl Anions with either Diarylmethyl or Allylic Carbonates.

PubMed

Yasuda, Shigeo; Ishii, Takuya; Takemoto, Shunsuke; Haruki, Hiroki; Ohmiya, Hirohisa

2018-03-05

Benzylation and allylation of aldehyde acyl anions were enabled by the merger of a thiazolium N-heterocyclic carbene (NHC) catalyst and a palladium/bisphosphine catalyst in a synergistic manner. Owing to the mildness of the reaction conditions, various functional groups were tolerated in the substrates. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Modulation of FadR Binding Capacity for Acyl-CoA Fatty Acids Through Structure-Guided Mutagenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bacik, John-Paul; Yeager, Chris M.; Twary, Scott N.

FadR is a versatile global regulator in Escherichia coli that controls fatty acid metabolism and thereby modulates the ability of this bacterium to grow using fatty acids or acetate as the sole carbon source. FadR regulates fatty acid metabolism in response to intra-cellular concentrations of acyl-CoA lipids. The ability of FadR to bind acyl-CoA fatty acids is hence of significant interest for the engineering of biosynthetic pathways for the production of lipid-based biofuels and commodity chemicals. Based on the available crystal structure of E. coli bound to myristoyl- CoA, we predicted amino acid positions within the effector binding pocket thatmore » would alter the ability of FadR to bind acyl-CoA fatty acids without affecting DNA binding. We utilized fluorescence polarization to characterize the in-vitro binding properties of wild type and mutant FadR. We found that a Leu102Ala mutant enhanced binding of the effector, likely by increasing the size of the binding pocket for the acyl moiety of the molecule. Conversely, the elimination of the guanidine side chain (Arg213Ala and Arg213Met mutants) of the CoA moiety binding site severely diminished the ability of FadR to bind the acyl-CoA effector. These results demonstrate the ability to fine tune FadR binding capacity. The validation of an efficient method to fully characterize all the binding events involved in the specific activity (effector and DNA operator binding) of FadR has allowed us to increase our understanding of the role of specific amino acids in the binding and recognition of acyl-CoA fatty acids and will greatly facilitate efforts aimed at engineering tunable FadR regulators for synthetic biology.« less

Modulation of FadR Binding Capacity for Acyl-CoA Fatty Acids Through Structure-Guided Mutagenesis

DOE PAGES

Bacik, John-Paul; Yeager, Chris M.; Twary, Scott N.; ...

2015-09-18

FadR is a versatile global regulator in Escherichia coli that controls fatty acid metabolism and thereby modulates the ability of this bacterium to grow using fatty acids or acetate as the sole carbon source. FadR regulates fatty acid metabolism in response to intra-cellular concentrations of acyl-CoA lipids. The ability of FadR to bind acyl-CoA fatty acids is hence of significant interest for the engineering of biosynthetic pathways for the production of lipid-based biofuels and commodity chemicals. Based on the available crystal structure of E. coli bound to myristoyl- CoA, we predicted amino acid positions within the effector binding pocket thatmore » would alter the ability of FadR to bind acyl-CoA fatty acids without affecting DNA binding. We utilized fluorescence polarization to characterize the in-vitro binding properties of wild type and mutant FadR. We found that a Leu102Ala mutant enhanced binding of the effector, likely by increasing the size of the binding pocket for the acyl moiety of the molecule. Conversely, the elimination of the guanidine side chain (Arg213Ala and Arg213Met mutants) of the CoA moiety binding site severely diminished the ability of FadR to bind the acyl-CoA effector. These results demonstrate the ability to fine tune FadR binding capacity. The validation of an efficient method to fully characterize all the binding events involved in the specific activity (effector and DNA operator binding) of FadR has allowed us to increase our understanding of the role of specific amino acids in the binding and recognition of acyl-CoA fatty acids and will greatly facilitate efforts aimed at engineering tunable FadR regulators for synthetic biology.« less

Exploring Lactobacillus reuteri DSM20016 as a biocatalyst for transformation of longer chain 1,2-diols: Limits with microcompartment.

PubMed

Chen, Lu; Hatti-Kaul, Rajni

2017-01-01

Lactobacillus reuteri metabolises glycerol efficiently to form 3-hydroxypropionic acid (3-HP) and 1,3-propanediol (1,3PDO) by the same mechanism as that for 1,2-propanediol (1,2PDO) conversion to propionic acid and propanol via its propanediol utilization (pdu) pathway. Pdu enzymes are encoded by the pdu-operon, which also contain genes encoding the shell proteins of the microcompartment housing the metabolic pathway. In this work the selectivity and kinetics of the reactions catalysed by L. reuteri DSM20016 Pdu enzymes glycerol dehydratase (GDH), 1,3-propanediol oxidoreductase (PduQ) and coenzyme-A acylating propionaldehyde dehydrogenase (PduP), produced recombinantly, was investigated against corresponding substrates of different chain lengths. Glycerol dehydratase exhibited activity against C2-C4 polyols, with the highest activity against glycerol and 1,2-propanediol (1,2-PDO). A double mutant of the pduC gene of GDH (PduC-S302A/Q337A) was constructed that displayed lowered activity against glycerol and 1,2PDO but extended the substrate range upto C6-diol. The best substrate for both PduQ and PduP was 3-hydroxypropanal (3HPA), although PduP exhibited nearly 10-fold higher specific activity. The enzymes also showed some activity against C3-C10 aliphatic aldehydes, with PduP having higher relative activity. Subsequently, transformation of polyols using whole cells of L. reuteri containing the wild type- and mutated GDH, respectively, confirmed the reduced activity of the mutant against glycerol and 1,2PDO, but its activity against longer substrates was negligible. In contrast, recombinant Escherichia coli BL21(DE3) cells harboring the GDH variant converted diols with up to C6 carbon chain length to their respective aldehydes, suggesting that the protein shell of the microcompartment in L. reuteri posed a barrier to the passage of longer chain substrate.

Defining the extreme substrate specificity of Euonymus alatus diacylglycerol acetyltransferase, an unusual membrane-bound O-acyltransferase

DOE PAGES

Bansal, Sunil; Durrett, Timothy P.

2016-11-08

Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) synthesizes the unusually structured 3-acetyl-1,2-diacylglycerols (acetyl-TAG) found in the seeds of a few plant species. A member of the membrane-bound O-acyltransferase (MBOAT) family, EaDAcT transfers the acetyl group from acetyl-CoA to sn-1,2-diacylglycerol (DAG) to produce acetyl-TAG. In vitro assays demonstrated that the enzyme is also able to utilize butyryl-CoA and hexanoyl-CoA as acyl donors, though with much less efficiency compared with acetyl-CoA. Acyl-CoAs longer than eight carbons were not used by EaDAcT. This extreme substrate specificity of EaDAcT distinguishes it from all other MBOATs which typically catalyze the transfer of much longer acyl groups. Inmore » vitro selectivity experiments revealed that EaDAcT preferentially acetylated DAG molecules containing more double bonds over those with less. However, the enzyme was also able to acetylate saturated DAG containing medium chain fatty acids, albeit with less efficiency. Interestingly, EaDAcT could only acetylate the free hydroxyl group of sn-1,2-DAG but not the available hydroxyl groups in sn-1,3-DAG or in monoacylglycerols (MAG). Consistent with its similarity to the jojoba wax synthase, EaDAcT could acetylate fatty alcohols in vitro to produce alkyl acetates. Likewise, when coexpressed in yeast with a fatty acyl-CoA reductase capable of producing fatty alcohols, EaDAcT synthesized alkyl acetates although the efficiency of production was low. As a result, this improved understanding of EaDAcT specificity confirms that the enzyme preferentially utilizes acetyl-CoA to acetylate sn-1,2-DAGs and will be helpful in engineering the production of acetyl-TAG with improved functionality in transgenic plants.« less

Defining the extreme substrate specificity of Euonymus alatus diacylglycerol acetyltransferase, an unusual membrane-bound O-acyltransferase

PubMed Central

Bansal, Sunil; Durrett, Timothy P.

2016-01-01

Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) synthesizes the unusually structured 3-acetyl-1,2-diacylglycerols (acetyl-TAG) found in the seeds of a few plant species. A member of the membrane-bound O-acyltransferase (MBOAT) family, EaDAcT transfers the acetyl group from acetyl-CoA to sn-1,2-diacylglycerol (DAG) to produce acetyl-TAG. In vitro assays demonstrated that the enzyme is also able to utilize butyryl-CoA and hexanoyl-CoA as acyl donors, though with much less efficiency compared with acetyl-CoA. Acyl-CoAs longer than eight carbons were not used by EaDAcT. This extreme substrate specificity of EaDAcT distinguishes it from all other MBOATs which typically catalyze the transfer of much longer acyl groups. In vitro selectivity experiments revealed that EaDAcT preferentially acetylated DAG molecules containing more double bonds over those with less. However, the enzyme was also able to acetylate saturated DAG containing medium chain fatty acids, albeit with less efficiency. Interestingly, EaDAcT could only acetylate the free hydroxyl group of sn-1,2-DAG but not the available hydroxyl groups in sn-1,3-DAG or in monoacylglycerols (MAG). Consistent with its similarity to the jojoba wax synthase, EaDAcT could acetylate fatty alcohols in vitro to produce alkyl acetates. Likewise, when coexpressed in yeast with a fatty acyl-CoA reductase capable of producing fatty alcohols, EaDAcT synthesized alkyl acetates although the efficiency of production was low. This improved understanding of EaDAcT specificity confirms that the enzyme preferentially utilizes acetyl-CoA to acetylate sn-1,2-DAGs and will be helpful in engineering the production of acetyl-TAG with improved functionality in transgenic plants. PMID:27688773

Defining the extreme substrate specificity of Euonymus alatus diacylglycerol acetyltransferase, an unusual membrane-bound O-acyltransferase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bansal, Sunil; Durrett, Timothy P.

Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) synthesizes the unusually structured 3-acetyl-1,2-diacylglycerols (acetyl-TAG) found in the seeds of a few plant species. A member of the membrane-bound O-acyltransferase (MBOAT) family, EaDAcT transfers the acetyl group from acetyl-CoA to sn-1,2-diacylglycerol (DAG) to produce acetyl-TAG. In vitro assays demonstrated that the enzyme is also able to utilize butyryl-CoA and hexanoyl-CoA as acyl donors, though with much less efficiency compared with acetyl-CoA. Acyl-CoAs longer than eight carbons were not used by EaDAcT. This extreme substrate specificity of EaDAcT distinguishes it from all other MBOATs which typically catalyze the transfer of much longer acyl groups. Inmore » vitro selectivity experiments revealed that EaDAcT preferentially acetylated DAG molecules containing more double bonds over those with less. However, the enzyme was also able to acetylate saturated DAG containing medium chain fatty acids, albeit with less efficiency. Interestingly, EaDAcT could only acetylate the free hydroxyl group of sn-1,2-DAG but not the available hydroxyl groups in sn-1,3-DAG or in monoacylglycerols (MAG). Consistent with its similarity to the jojoba wax synthase, EaDAcT could acetylate fatty alcohols in vitro to produce alkyl acetates. Likewise, when coexpressed in yeast with a fatty acyl-CoA reductase capable of producing fatty alcohols, EaDAcT synthesized alkyl acetates although the efficiency of production was low. As a result, this improved understanding of EaDAcT specificity confirms that the enzyme preferentially utilizes acetyl-CoA to acetylate sn-1,2-DAGs and will be helpful in engineering the production of acetyl-TAG with improved functionality in transgenic plants.« less

An Acrobatic Substrate Metamorphosis Reveals a Requirement for Substrate Conformational Dynamics in Trypsin Proteolysis

DOE PAGES

Kayode, Olumide; Wang, Ruiying; Pendlebury, Devon F.; ...

2016-11-03

The molecular basis of enzyme catalytic power and specificity derives from dynamic interactions between enzyme and substrate during catalysis. While considerable effort has been devoted to understanding how conformational dynamics within enzymes affect catalysis, the role of conformational dynamics within protein substrates has not been addressed. Here in this paper, we examine the importance of substrate dynamics in the cleavage of Kunitz-BPTI protease inhibitors by mesotrypsin, finding that the varied conformational dynamics of structurally similar substrates can profoundly impact the rate of catalysis. A 1.4 Å crystal structure of a mesotrypsin-product complex formed with a rapidly cleaved substrate reveals amore » dramatic conformational change in the substrate upon proteolysis. Using long all-atom molecular dynamics simulations of acyl-enzyme intermediates with proteolysis rates spanning three orders of magnitude, we identify global and local dynamic features of substrates on the ns-μs timescale that correlate with enzymatic rates and explain differential susceptibility to proteolysis. By integrating multiple enhanced sampling methods for molecular dynamics, we model a viable conformational pathway between substratelike and product-like states, linking substrate dynamics on the ns-μs timescale with large collective substrate motions on the much slower timescale of catalysis. Our findings implicate substrate flexibility as a critical determinant of catalysis.« less

Age-dependent decline in acyl-ghrelin concentrations and reduced association of acyl-ghrelin and growth hormone in healthy older adults.

PubMed

Nass, Ralf; Farhy, Leon S; Liu, Jianhua; Pezzoli, Suzan S; Johnson, Michael L; Gaylinn, Bruce D; Thorner, Michael O

2014-02-01

Acyl-ghrelin is thought to have both orexigenic effects and to stimulate GH release. A possible cause of the anorexia of aging is an age-dependent decrease in circulating acyl-ghrelin levels. The purpose of the study was to compare acyl-ghrelin and GH concentrations between healthy old and young adults and to examine the relationship of acyl-ghrelin and GH secretion in both age groups. Six healthy older adults (age 62-74 y, body mass index range 20.9-29 kg/m(2)) and eight healthy young men (aged 18-28 y, body mass index range 20.6-26.2 kg/m(2)) had frequent blood samples drawn for hormone measurements every 10 minutes for 24 hours. Ghrelin was measured in an in-house, two-site sandwich ELISA specific for full-length acyl-ghrelin. GH was measured in a sensitive assay (Immulite 2000), and GH peaks were determined by deconvolution analysis. The acyl-ghrelin/GH association was estimated from correlations between amplitudes of individual GH secretory events and the average acyl-ghrelin concentration in the 60-minute interval preceding each GH burst. Twenty-four-hour mean (±SEM) GH (0.48 ± 0.14 vs 2.2 ± 0.3 μg/L, P < .005) and acyl-ghrelin (14.7 ± 2.3 vs 27.8 ± 3.9 pg/mL, P < .05) levels were significantly lower in older adults compared with young adults. Twenty-four-hour cortisol concentrations were higher in the old than the young adults (15.1 ± 1.0 vs 10.6 ± 0.9 μg/dL, respectively, P < .01). The ghrelin/GH association was more than 3-fold lower in the older group compared with the young adults (0.16 ± 0.12 vs 0.69 ± 0.04, P < .001). These results provide further evidence of an age-dependent decline in circulating acyl-ghrelin levels, which might play a role both in the decline of GH and in the anorexia of aging. Our data also suggest that with normal aging, endogenous acyl-ghrelin levels are less tightly linked to GH regulation.

Branched-chain amino acid restriction in Zucker-fatty rats improves muscle insulin sensitivity by enhancing efficiency of fatty acid oxidation and acyl-glycine export.

PubMed

White, Phillip J; Lapworth, Amanda L; An, Jie; Wang, Liping; McGarrah, Robert W; Stevens, Robert D; Ilkayeva, Olga; George, Tabitha; Muehlbauer, Michael J; Bain, James R; Trimmer, Jeff K; Brosnan, M Julia; Rolph, Timothy P; Newgard, Christopher B

2016-07-01

A branched-chain amino acid (BCAA)-related metabolic signature is strongly associated with insulin resistance and predictive of incident diabetes and intervention outcomes. To better understand the role that this metabolite cluster plays in obesity-related metabolic dysfunction, we studied the impact of BCAA restriction in a rodent model of obesity in which BCAA metabolism is perturbed in ways that mirror the human condition. Zucker-lean rats (ZLR) and Zucker-fatty rats (ZFR) were fed either a custom control, low fat (LF) diet, or an isonitrogenous, isocaloric LF diet in which all three BCAA (Leu, Ile, Val) were reduced by 45% (LF-RES). We performed comprehensive metabolic and physiologic profiling to characterize the effects of BCAA restriction on energy balance, insulin sensitivity, and glucose, lipid and amino acid metabolism. LF-fed ZFR had higher levels of circulating BCAA and lower levels of glycine compared to LF-fed ZLR. Feeding ZFR with the LF-RES diet lowered circulating BCAA to levels found in LF-fed ZLR. Activity of the rate limiting enzyme in the BCAA catabolic pathway, branched chain keto acid dehydrogenase (BCKDH), was lower in liver but higher in skeletal muscle of ZFR compared to ZLR and was not responsive to diet in either tissue. BCAA restriction had very little impact on metabolites studied in liver of ZFR where BCAA content was low, and BCKDH activity was suppressed. However, in skeletal muscle of LF-fed ZFR compared to LF-fed ZLR, where BCAA content and BCKDH activity were increased, accumulation of fatty acyl CoAs was completely normalized by dietary BCAA restriction. BCAA restriction also normalized skeletal muscle glycine content and increased urinary acetyl glycine excretion in ZFR. These effects were accompanied by lower RER and improved skeletal muscle insulin sensitivity in LF-RES fed ZFR as measured by hyperinsulinemic-isoglycemic clamp. Our data are consistent with a model wherein elevated circulating BCAA contribute to development of

Production of a Brassica napus low-molecular mass acyl-coenzyme A-binding protein in Arabidopsis alters the acyl-coenzyme A pool and acyl composition of oil in seeds

USDA-ARS?s Scientific Manuscript database

Low-molecular mass (10 kD) cytosolic acyl-coenzyme A-binding protein (ACBP) has a substantial influence over fatty acid (FA) composition in oilseeds, possibly via an effect on the partitioning of acyl groups between elongation and desaturation pathways. Previously, we demonstrated that the expressio...

Physical Characteristics of Tetrahydroxy and Acylated Derivatives of Jojoba Liquid Wax in Lubricant Applications

PubMed Central

Biresaw, Girma; Gordon, Sherald

2018-01-01

Jojoba liquid wax is a mixture of esters of long-chain fatty acids and fatty alcohols mainly C38:2–C46:2. The oil exhibits excellent emolliency on the skin and, therefore, is a component in many personal care cosmetic formulations. The virgin oil is a component of the seed of the jojoba (Simmondsia chinensis) plant which occurs naturally in the Sonora Desert in the United States and northwestern Mexico as well as in the northeastern Sahara desert. The seed contains 50–60% oil by dry weight. The plant has been introduced into Australia, Argentina, and Israel for commercial production of the jojoba oil. As a natural lubricant, we are seeking to explore its potential as a renewable industrial lubricant additive. Thus, we have chemically modified the carbon-carbon double bonds in the oil structure in order to improve its already good resistance to air oxidation so as to enhance its utility as well as its shelf life in nonpersonal care applications. To achieve this goal, we have hydroxylated its –C=C– bonds. Acylation of the resulting hydroxyl moieties has generated short-chain vicinal acyl substituents on the oil which keep the wax liquid, improving its cold flow properties and also protecting it from auto-oxidation and rancidity. PMID:29484216

Physical Characteristics of Tetrahydroxy and Acylated Derivatives of Jojoba Liquid Wax in Lubricant Applications.

PubMed

Harry-O'kuru, Rogers E; Biresaw, Girma; Gordon, Sherald; Xu, Jingyuan

2018-01-01

Jojoba liquid wax is a mixture of esters of long-chain fatty acids and fatty alcohols mainly C38:2-C46:2. The oil exhibits excellent emolliency on the skin and, therefore, is a component in many personal care cosmetic formulations. The virgin oil is a component of the seed of the jojoba ( Simmondsia chinensis ) plant which occurs naturally in the Sonora Desert in the United States and northwestern Mexico as well as in the northeastern Sahara desert. The seed contains 50-60% oil by dry weight. The plant has been introduced into Australia, Argentina, and Israel for commercial production of the jojoba oil. As a natural lubricant, we are seeking to explore its potential as a renewable industrial lubricant additive. Thus, we have chemically modified the carbon-carbon double bonds in the oil structure in order to improve its already good resistance to air oxidation so as to enhance its utility as well as its shelf life in nonpersonal care applications. To achieve this goal, we have hydroxylated its -C=C- bonds. Acylation of the resulting hydroxyl moieties has generated short-chain vicinal acyl substituents on the oil which keep the wax liquid, improving its cold flow properties and also protecting it from auto-oxidation and rancidity.

delta 6 Hexadecenoic acid is synthesized by the activity of a soluble delta 6 palmitoyl-acyl carrier protein desaturase in Thunbergia alata endosperm.

PubMed

Cahoon, E B; Cranmer, A M; Shanklin, J; Ohlrogge, J B

1994-11-04

delta 6 Hexadecenoic acid (16:1 delta 6) composes more than 80% of the seed oil of Thunbergia alata. Studies were conducted to determine the biosynthetic origin of the double bond of this unusual fatty acid. Assays of fractions of developing T. alata seed endosperm with [1-14C]palmitoyl (16:0)-acyl carrier protein (ACP) revealed the presence of a soluble delta 6 desaturase activity. This activity was greatest when 16:0-ACP was provided as a substrate, whereas no desaturation of the coenzyme A ester of this fatty acid was detected. In addition, delta 6 16:0-ACP desaturase activity in T. alata endosperm extracts was dependent on the presence of ferredoxin and molecular oxygen and was stimulated by catalase. To further characterize this enzyme, a cDNA encoding a diverged acyl-ACP desaturase was isolated from a T. alata endosperm cDNA library using polymerase chain reaction with degenerate oligonucleotides corresponding to conserved amino acid sequences in delta 9 stearoyl (18:0)- and delta 4 16:0-ACP desaturases. The primary structure of the mature peptide encoded by this cDNA shares 66% identity with the mature castor delta 9 18:0-ACP desaturase and 57% identity with the mature coriander delta 4 16:0-ACP desaturase. Extracts of Escherichia coli that express the T. alata cDNA catalyzed the delta 6 desaturation of 16:0-ACP. These results demonstrate that 16:1 delta 6 in T. alata endosperm is formed by the activity of a soluble delta 6 16:0-ACP desaturase that is structurally related to the delta 9 18:0- and delta 4 16:0-ACP desaturases. Implications of this work to an understanding of active site structures of acyl-ACP desaturases are discussed.

Purification of a Jojoba Embryo Fatty Acyl-Coenzyme A Reductase and Expression of Its cDNA in High Erucic Acid Rapeseed

PubMed Central

Metz, James G.; Pollard, Michael R.; Anderson, Lana; Hayes, Thomas R.; Lassner, Michael W.

2000-01-01

The jojoba (Simmondsia chinensis) plant produces esters of long-chain alcohols and fatty acids (waxes) as a seed lipid energy reserve. This is in contrast to the triglycerides found in seeds of other plants. We purified an alcohol-forming fatty acyl-coenzyme A reductase (FAR) from developing embryos and cloned the cDNA encoding the enzyme. Expression of a cDNA in Escherichia coli confers FAR activity upon those cells and results in the accumulation of fatty alcohols. The FAR sequence shows significant homology to an Arabidopsis protein of unknown function that is essential for pollen development. When the jojoba FAR cDNA is expressed in embryos of Brassica napus, long-chain alcohols can be detected in transmethylated seed oils. Resynthesis of the gene to reduce its A plus T content resulted in increased levels of alcohol production. In addition to free alcohols, novel wax esters were detected in the transgenic seed oils. In vitro assays revealed that B. napus embryos have an endogenous fatty acyl-coenzyme A: fatty alcohol acyl-transferase activity that could account for this wax synthesis. Thus, introduction of a single cDNA into B. napus results in a redirection of a portion of seed oil synthesis from triglycerides to waxes. PMID:10712526

Purification of a jojoba embryo fatty acyl-coenzyme A reductase and expression of its cDNA in high erucic acid rapeseed.

PubMed

Metz, J G; Pollard, M R; Anderson, L; Hayes, T R; Lassner, M W

2000-03-01

The jojoba (Simmondsia chinensis) plant produces esters of long-chain alcohols and fatty acids (waxes) as a seed lipid energy reserve. This is in contrast to the triglycerides found in seeds of other plants. We purified an alcohol-forming fatty acyl-coenzyme A reductase (FAR) from developing embryos and cloned the cDNA encoding the enzyme. Expression of a cDNA in Escherichia coli confers FAR activity upon those cells and results in the accumulation of fatty alcohols. The FAR sequence shows significant homology to an Arabidopsis protein of unknown function that is essential for pollen development. When the jojoba FAR cDNA is expressed in embryos of Brassica napus, long-chain alcohols can be detected in transmethylated seed oils. Resynthesis of the gene to reduce its A plus T content resulted in increased levels of alcohol production. In addition to free alcohols, novel wax esters were detected in the transgenic seed oils. In vitro assays revealed that B. napus embryos have an endogenous fatty acyl-coenzyme A: fatty alcohol acyl-transferase activity that could account for this wax synthesis. Thus, introduction of a single cDNA into B. napus results in a redirection of a portion of seed oil synthesis from triglycerides to waxes.

Molecular Characterization of the Elaeis guineensis Medium-Chain Fatty Acid Diacylglycerol Acyltransferase DGAT1-1 by Heterologous Expression in Yarrowia lipolytica.

PubMed

Aymé, Laure; Jolivet, Pascale; Nicaud, Jean-Marc; Chardot, Thierry

2015-01-01

Diacylglycerol acyltransferases (DGAT) are involved in the acylation of sn-1,2-diacylglycerol. Palm kernel oil, extracted from Elaeis guineensis (oil palm) seeds, has a high content of medium-chain fatty acids mainly lauric acid (C12:0). A putative E. guineensis diacylglycerol acyltransferase gene (EgDGAT1-1) is expressed at the onset of lauric acid accumulation in the seed endosperm suggesting that it is a determinant of medium-chain triacylglycerol storage. To test this hypothesis, we thoroughly characterized EgDGAT1-1 activity through functional complementation of a Yarrowia lipolytica mutant strain devoid of neutral lipids. EgDGAT1-1 expression is sufficient to restore triacylglycerol accumulation in neosynthesized lipid droplets. A comparative functional study with Arabidopsis thaliana DGAT1 highlighted contrasting substrate specificities when the recombinant yeast was cultured in lauric acid supplemented medium. The EgDGAT1-1 expressing strain preferentially accumulated medium-chain triacylglycerols whereas AtDGAT1 expression induced long-chain triacylglycerol storage in Y. lipolytica. EgDGAT1-1 localized to the endoplasmic reticulum where TAG biosynthesis takes place. Reestablishing neutral lipid accumulation in the Y. lipolytica mutant strain did not induce major reorganization of the yeast microsomal proteome. Overall, our findings demonstrate that EgDGAT1-1 is an endoplasmic reticulum DGAT with preference for medium-chain fatty acid substrates, in line with its physiological role in palm kernel. The characterized EgDGAT1-1 could be used to promote medium-chain triacylglycerol accumulation in microbial-produced oil for industrial chemicals and cosmetics.

Molecular Characterization of the Elaeis guineensis Medium-Chain Fatty Acid Diacylglycerol Acyltransferase DGAT1-1 by Heterologous Expression in Yarrowia lipolytica

PubMed Central

Aymé, Laure; Jolivet, Pascale; Nicaud, Jean-Marc; Chardot, Thierry

2015-01-01

Diacylglycerol acyltransferases (DGAT) are involved in the acylation of sn-1,2-diacylglycerol. Palm kernel oil, extracted from Elaeis guineensis (oil palm) seeds, has a high content of medium-chain fatty acids mainly lauric acid (C12:0). A putative E. guineensis diacylglycerol acyltransferase gene (EgDGAT1-1) is expressed at the onset of lauric acid accumulation in the seed endosperm suggesting that it is a determinant of medium-chain triacylglycerol storage. To test this hypothesis, we thoroughly characterized EgDGAT1-1 activity through functional complementation of a Yarrowia lipolytica mutant strain devoid of neutral lipids. EgDGAT1-1 expression is sufficient to restore triacylglycerol accumulation in neosynthesized lipid droplets. A comparative functional study with Arabidopsis thaliana DGAT1 highlighted contrasting substrate specificities when the recombinant yeast was cultured in lauric acid supplemented medium. The EgDGAT1-1 expressing strain preferentially accumulated medium-chain triacylglycerols whereas AtDGAT1 expression induced long-chain triacylglycerol storage in Y. lipolytica. EgDGAT1-1 localized to the endoplasmic reticulum where TAG biosynthesis takes place. Reestablishing neutral lipid accumulation in the Y. lipolytica mutant strain did not induce major reorganization of the yeast microsomal proteome. Overall, our findings demonstrate that EgDGAT1-1 is an endoplasmic reticulum DGAT with preference for medium-chain fatty acid substrates, in line with its physiological role in palm kernel. The characterized EgDGAT1-1 could be used to promote medium-chain triacylglycerol accumulation in microbial-produced oil for industrial chemicals and cosmetics. PMID:26581109

Identification of N-acyl homoserine lactones produced by Gluconacetobacter diazotrophicus PAL5 cultured in complex and synthetic media.

PubMed

Nieto-Peñalver, Carlos G; Bertini, Elisa V; de Figueroa, Lucía I C

2012-07-01

The endophytic diazotrophic Gluconacetobacter diazotrophicus PAL5 was originally isolated from sugarcane (Saccharum officinarum). The biological nitrogen fixation, phytohormones secretion, solubilization of mineral nutrients and phytopathogen antagonism allow its classification as a plant growth-promoting bacterium. The recent genomic sequence of PAL5 unveiled the presence of a quorum sensing (QS) system. QS are regulatory mechanisms that, through the production of signal molecules or autoinducers, permit a microbial population the regulation of the physiology in a coordinated manner. The most studied autoinducers in gram-negative bacteria are the N-acyl homoserine lactones (AHLs). The usage of biosensor strains evidenced the presence of AHL-like molecules in cultures of G. diazotrophicus PAL5 grown in complex and synthetic media. Analysis of AHLs performed by LC-APCI-MS permitted the identification of eight different signal molecules, including C6-, C8-, C10-, C12- and C14-HSL. Mass spectra confirmed that this diazotrophic strain also synthesizes autoinducers with carbonyl substitutions in the acyl chain. No differences in the profile of AHLs could be determined under both culture conditions. However, although the level of short-chain AHLs was not affected, a decrease of 30% in the production of long-chain AHLs could be measured in synthetic medium.

Production of a Brassica napus Low-Molecular Mass Acyl-Coenzyme A-Binding Protein in Arabidopsis Alters the Acyl-Coenzyme A Pool and Acyl Composition of Oil in Seeds.

PubMed

Yurchenko, Olga; Singer, Stacy D; Nykiforuk, Cory L; Gidda, Satinder; Mullen, Robert T; Moloney, Maurice M; Weselake, Randall J

2014-06-01

Low-molecular mass (10 kD) cytosolic acyl-coenzyme A-binding protein (ACBP) has a substantial influence over fatty acid (FA) composition in oilseeds, possibly via an effect on the partitioning of acyl groups between elongation and desaturation pathways. Previously, we demonstrated that the expression of a Brassica napus ACBP (BnACBP) complementary DNA in the developing seeds of Arabidopsis (Arabidopsis thaliana) resulted in increased levels of polyunsaturated FAs at the expense of eicosenoic acid (20:1 cisΔ11 ) and saturated FAs in seed oil. In this study, we investigated whether alterations in the FA composition of seed oil at maturity were correlated with changes in the acyl-coenzyme A (CoA) pool in developing seeds of transgenic Arabidopsis expressing BnACBP. Our results indicated that both the acyl-CoA pool and seed oil of transgenic Arabidopsis lines expressing cytosolic BnACBP exhibited relative increases in linoleic acid (18:2 cisΔ9,12 ; 17.9%-44.4% and 7%-13.2%, respectively) and decreases in 20:1 cisΔ11 (38.7%-60.7% and 13.8%-16.3%, respectively). However, alterations in the FA composition of the acyl-CoA pool did not always correlate with those seen in the seed oil. In addition, we found that targeting of BnACBP to the endoplasmic reticulum resulted in FA compositional changes that were similar to those seen in lines expressing cytosolic BnACBP, with the most prominent exception being a relative reduction in α-linolenic acid (18:3 cisΔ9,12,15 ) in both the acyl-CoA pool and seed oil of the former (48.4%-48.9% and 5.3%-10.4%, respectively). Overall, these data support the role of ACBP in acyl trafficking in developing seeds and validate its use as a biotechnological tool for modifying the FA composition of seed oil. © 2014 American Society of Plant Biologists. All Rights Reserved.

Extensive domain motion and electron transfer in the human electron transferring flavoprotein.medium chain Acyl-CoA dehydrogenase complex.

PubMed

Toogood, Helen S; van Thiel, Adam; Basran, Jaswir; Sutcliffe, Mike J; Scrutton, Nigel S; Leys, David

2004-07-30

The crystal structure of the human electron transferring flavoprotein (ETF).medium chain acyl-CoA dehydrogenase (MCAD) complex reveals a dual mode of protein-protein interaction, imparting both specificity and promiscuity in the interaction of ETF with a range of structurally distinct primary dehydrogenases. ETF partitions the functions of partner binding and electron transfer between (i) the recognition loop, which acts as a static anchor at the ETF.MCAD interface, and (ii) the highly mobile redox active FAD domain. Together, these enable the FAD domain of ETF to sample a range of conformations, some compatible with fast interprotein electron transfer. Disorders in amino acid or fatty acid catabolism can be attributed to mutations at the protein-protein interface. Crucially, complex formation triggers mobility of the FAD domain, an induced disorder that contrasts with general models of protein-protein interaction by induced fit mechanisms. The subsequent interfacial motion in the MCAD.ETF complex is the basis for the interaction of ETF with structurally diverse protein partners. Solution studies using ETF and MCAD with mutations at the protein-protein interface support this dynamic model and indicate ionic interactions between MCAD Glu(212) and ETF Arg alpha(249) are likely to transiently stabilize productive conformations of the FAD domain leading to enhanced electron transfer rates between both partners.

α-Amidoalkylating agents from N-acyl-α-amino acids: 1-(N-acylamino)alkyltriphenylphosphonium salts.

PubMed

Mazurkiewicz, Roman; Adamek, Jakub; Październiok-Holewa, Agnieszka; Zielińska, Katarzyna; Simka, Wojciech; Gajos, Anna; Szymura, Karol

2012-02-17

N-Acyl-α-amino acids were efficiently transformed in a two-step procedure into 1-N-(acylamino)alkyltriphenylphosphonium salts, new powerful α-amidoalkylating agents. The effect of the α-amino acid structure, the base used [MeONa or a silica gel-supported piperidine (SiO(2)-Pip)], and the main electrolysis parameters (current density, charge consumption) on the yield and selectivity of the electrochemical decarboxylative α-methoxylation of N-acyl-α-amino acids (Hofer-Moest reaction) was investigated. For most proteinogenic and all studied unproteinogenic α-amino acids, very good results were obtained using a substoichiometric amount of SiO(2)-Pip as the base. Only in the cases of N-acylated cysteine, methionine, and tryptophan, attempts to carry out the Hofer-Moest reaction in the applied conditions failed, probably because of the susceptibility of these α-amino acids to an electrochemical oxidation on the side chain. The methoxy group of N-(1-methoxyalkyl)amides was effectively displaced with the triphenylphosphonium group by dissolving an equimolar amount of N-(1-methoxyalkyl)amide and triphenylphosphonium tetrafluoroborate in CH(2)Cl(2) at room temperature for 30 min, followed by the precipitation of 1-N-(acylamino)alkyltriphenylphosphonium salt with Et(2)O.

Elevated Medium-Chain Acylcarnitines Are Associated With Gestational Diabetes Mellitus and Early Progression to Type 2 Diabetes and Induce Pancreatic β-Cell Dysfunction.

PubMed

Batchuluun, Battsetseg; Al Rijjal, Dana; Prentice, Kacey J; Eversley, Judith A; Burdett, Elena; Mohan, Haneesha; Bhattacharjee, Alpana; Gunderson, Erica P; Liu, Ying; Wheeler, Michael B

2018-05-01

Specific circulating metabolites have emerged as important risk factors for the development of diabetes. The acylcarnitines (acylCs) are a family of metabolites known to be elevated in type 2 diabetes (T2D) and linked to peripheral insulin resistance. However, the effect of acylCs on pancreatic β-cell function is not well understood. Here, we profiled circulating acylCs in two diabetes cohorts: 1 ) women with gestational diabetes mellitus (GDM) and 2 ) women with recent GDM who later developed impaired glucose tolerance (IGT), new-onset T2D, or returned to normoglycemia within a 2-year follow-up period. We observed a specific elevation in serum medium-chain (M)-acylCs, particularly hexanoyl- and octanoylcarnitine, among women with GDM and individuals with T2D without alteration in long-chain acylCs. Mice treated with M-acylCs exhibited glucose intolerance, attributed to impaired insulin secretion. Murine and human islets exposed to elevated levels of M-acylCs developed defects in glucose-stimulated insulin secretion and this was directly linked to reduced mitochondrial respiratory capacity and subsequent ability to couple glucose metabolism to insulin secretion. In conclusion, our study reveals that an elevation in circulating M-acylCs is associated with GDM and early stages of T2D onset and that this elevation directly impairs β-cell function. © 2018 by the American Diabetes Association.

Structure of Mycobacterium tuberculosis mtFabD, a malonyl-CoA:acyl carrier protein transacylase (MCAT).

PubMed

Ghadbane, Hemza; Brown, Alistair K; Kremer, Laurent; Besra, Gurdyal S; Fütterer, Klaus

2007-10-01

Mycobacteria display a unique and unusual cell-wall architecture, central to which is the membrane-proximal mycolyl-arabinogalactan-peptidoglycan core (mAGP). The biosynthesis of mycolic acids, which form the outermost layer of the mAGP core, involves malonyl-CoA:acyl carrier protein transacylase (MCAT). This essential enzyme catalyses the transfer of malonyl from coenzyme A to acyl carrier protein AcpM, thus feeding these two-carbon units into the chain-elongation cycle of the type II fatty-acid synthase. The crystal structure of M. tuberculosis mtFabD, the mycobacterial MCAT, has been determined to 3.0 A resolution by multi-wavelength anomalous dispersion. Phasing was facilitated by Ni2+ ions bound to the 20-residue N-terminal affinity tag, which packed between the two independent copies of mtFabD.

An Acrobatic Substrate Metamorphosis Reveals a Requirement for Substrate Conformational Dynamics in Trypsin Proteolysis.

PubMed

Kayode, Olumide; Wang, Ruiying; Pendlebury, Devon F; Cohen, Itay; Henin, Rachel D; Hockla, Alexandra; Soares, Alexei S; Papo, Niv; Caulfield, Thomas R; Radisky, Evette S

2016-12-16

The molecular basis of enzyme catalytic power and specificity derives from dynamic interactions between enzyme and substrate during catalysis. Although considerable effort has been devoted to understanding how conformational dynamics within enzymes affect catalysis, the role of conformational dynamics within protein substrates has not been addressed. Here, we examine the importance of substrate dynamics in the cleavage of Kunitz-bovine pancreatic trypsin inhibitor protease inhibitors by mesotrypsin, finding that the varied conformational dynamics of structurally similar substrates can profoundly impact the rate of catalysis. A 1.4-Å crystal structure of a mesotrypsin-product complex formed with a rapidly cleaved substrate reveals a dramatic conformational change in the substrate upon proteolysis. By using long all-atom molecular dynamics simulations of acyl-enzyme intermediates with proteolysis rates spanning 3 orders of magnitude, we identify global and local dynamic features of substrates on the nanosecond-microsecond time scale that correlate with enzymatic rates and explain differential susceptibility to proteolysis. By integrating multiple enhanced sampling methods for molecular dynamics, we model a viable conformational pathway between substrate-like and product-like states, linking substrate dynamics on the nanosecond-microsecond time scale with large collective substrate motions on the much slower time scale of catalysis. Our findings implicate substrate flexibility as a critical determinant of catalysis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Riboflavin-Responsive and -Non-responsive Mutations in FAD Synthase Cause Multiple Acyl-CoA Dehydrogenase and Combined Respiratory-Chain Deficiency.

PubMed

Olsen, Rikke K J; Koňaříková, Eliška; Giancaspero, Teresa A; Mosegaard, Signe; Boczonadi, Veronika; Mataković, Lavinija; Veauville-Merllié, Alice; Terrile, Caterina; Schwarzmayr, Thomas; Haack, Tobias B; Auranen, Mari; Leone, Piero; Galluccio, Michele; Imbard, Apolline; Gutierrez-Rios, Purificacion; Palmfeldt, Johan; Graf, Elisabeth; Vianey-Saban, Christine; Oppenheim, Marcus; Schiff, Manuel; Pichard, Samia; Rigal, Odile; Pyle, Angela; Chinnery, Patrick F; Konstantopoulou, Vassiliki; Möslinger, Dorothea; Feichtinger, René G; Talim, Beril; Topaloglu, Haluk; Coskun, Turgay; Gucer, Safak; Botta, Annalisa; Pegoraro, Elena; Malena, Adriana; Vergani, Lodovica; Mazzà, Daniela; Zollino, Marcella; Ghezzi, Daniele; Acquaviva, Cecile; Tyni, Tiina; Boneh, Avihu; Meitinger, Thomas; Strom, Tim M; Gregersen, Niels; Mayr, Johannes A; Horvath, Rita; Barile, Maria; Prokisch, Holger

2016-06-02

Multiple acyl-CoA dehydrogenase deficiencies (MADDs) are a heterogeneous group of metabolic disorders with combined respiratory-chain deficiency and a neuromuscular phenotype. Despite recent advances in understanding the genetic basis of MADD, a number of cases remain unexplained. Here, we report clinically relevant variants in FLAD1, which encodes FAD synthase (FADS), as the cause of MADD and respiratory-chain dysfunction in nine individuals recruited from metabolic centers in six countries. In most individuals, we identified biallelic frameshift variants in the molybdopterin binding (MPTb) domain, located upstream of the FADS domain. Inasmuch as FADS is essential for cellular supply of FAD cofactors, the finding of biallelic frameshift variants was unexpected. Using RNA sequencing analysis combined with protein mass spectrometry, we discovered FLAD1 isoforms, which only encode the FADS domain. The existence of these isoforms might explain why affected individuals with biallelic FLAD1 frameshift variants still harbor substantial FADS activity. Another group of individuals with a milder phenotype responsive to riboflavin were shown to have single amino acid changes in the FADS domain. When produced in E. coli, these mutant FADS proteins resulted in impaired but detectable FADS activity; for one of the variant proteins, the addition of FAD significantly improved protein stability, arguing for a chaperone-like action similar to what has been reported in other riboflavin-responsive inborn errors of metabolism. In conclusion, our studies identify FLAD1 variants as a cause of potentially treatable inborn errors of metabolism manifesting with MADD and shed light on the mechanisms by which FADS ensures cellular FAD homeostasis. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

The Lipopolysaccharide Lipid A Long-Chain Fatty Acid Is Important for Rhizobium leguminosarum Growth and Stress Adaptation in Free-Living and Nodule Environments.

PubMed

Bourassa, Dianna V; Kannenberg, Elmar L; Sherrier, D Janine; Buhr, R Jeffrey; Carlson, Russell W

2017-02-01

Rhizobium bacteria live in soil and plant environments, are capable of inducing symbiotic nodules on legumes, invade these nodules, and develop into bacteroids that fix atmospheric nitrogen into ammonia. Rhizobial lipopolysaccharide (LPS) is anchored in the bacterial outer membrane through a specialized lipid A containing a very long-chain fatty acid (VLCFA). VLCFA function for rhizobial growth in soil and plant environments is not well understood. Two genes, acpXL and lpxXL, encoding acyl carrier protein and acyltransferase, are among the six genes required for biosynthesis and transfer of VLCFA to lipid A. Rhizobium leguminosarum mutant strains acpXL, acpXL - /lpxXL - , and lpxXL - were examined for LPS structure, viability, and symbiosis. Mutations in acpXL and lpxXL abolished VLCFA attachment to lipid A. The acpXL mutant transferred a shorter acyl chain instead of VLCFA. Strains without lpxXL neither added VLCFA nor a shorter acyl chain. In all strains isolated from nodule bacteria, lipid A had longer acyl chains compared with laboratory-cultured bacteria, whereas mutant strains displayed altered membrane properties, modified cationic peptide sensitivity, and diminished levels of cyclic β-glucans. In pea nodules, mutant bacteroids were atypically formed and nitrogen fixation and senescence were affected. The role of VLCFA for rhizobial environmental fitness is discussed.

Studies to further investigate the inhibition of human liver microsomal CYP2C8 by the acyl-β-glucuronide of gemfibrozil.

PubMed

Jenkins, S M; Zvyaga, T; Johnson, S R; Hurley, J; Wagner, A; Burrell, R; Turley, W; Leet, J E; Philip, T; Rodrigues, A D

2011-12-01

In previous studies, gemfibrozil acyl-β-glucuronide, but not gemfibrozil, was found to be a mechanism-based inhibitor of cytochrome P450 2C8. To better understand whether this inhibition is specific for gemfibrozil acyl-β-glucuronide or whether other glucuronide conjugates are potential substrates for inhibition of this enzyme, we evaluated several pharmaceutical compounds (as their acyl glucuronides) as direct-acting and metabolism-dependent inhibitors of CYP2C8 in human liver microsomes. Of 11 compounds that were evaluated as their acyl glucuronide conjugates, only gemfibrozil acyl-β-glucuronide exhibited mechanism-based inhibition, indicating that CYP2C8 mechanism-based inhibition is very specific to certain glucuronide conjugates. Structural analogs of gemfibrozil were synthesized, and their glucuronide conjugates were prepared to further examine the mechanism of inhibition. When the aromatic methyl groups on the gemfibrozil moiety were substituted with trifluoromethyls, the resulting glucuronide conjugate was a weaker inhibitor of CYP2C8 and mechanism-based inhibition was abolished. However, the glucuronide conjugates of monomethyl gemfibrozil analogs were mechanism-based inhibitors of CYP2C8, although not as potent as gemfibrozil acyl-β-glucuronide itself. The ortho-monomethyl analog was a more potent inhibitor than the meta-monomethyl analog, indicating that CYP2C8 favors the ortho position for oxidation and potential inhibition. Molecular modeling of gemfibrozil acyl-β-glucuronide in the CYP2C8 active site is consistent with the ortho-methyl position being the favored site of covalent attachment to the heme. Moreover, hydrogen bonding to four residues (Ser100, Ser103, Gln214, and Asn217) is implicated.

Xanthomonas campestris RpfB is a Fatty Acyl-CoA Ligase Required to Counteract the Thioesterase Activity of the RpfF Diffusible Signal Factor (DSF) Synthase

PubMed Central

Bi, Hongkai; Yu, Yonghong; Dong, Huijuan; Wang, Haihong; Cronan, John E.

2014-01-01

SUMMARY In Xanthomonas campestris pv. campestris (Xcc), the proteins encoded by the rpf (regulator of pathogenicity factor) gene cluster produce and sense a fatty acid signal molecule called diffusible signaling factor (DSF, 2(Z)-11-methyldodecenoic acid). RpfB was reported to be involved in DSF processing and was predicted to encode an acyl-CoA ligase. We report that RpfB activates a wide range of fatty acids to their CoA esters in vitro. Moreover, RpfB can functionally replace the paradigm bacterial acyl-CoA ligase, Escherichia coli FadD, in the E. coli β-oxidation pathway and deletion of RpfB from the Xcc genome results in a strain unable to utilize fatty acids as carbon sources. An essential RpfB function in the pathogenicity factor pathway was demonstrated by the properties of a strain deleted for both the rpfB and rpfC genes. The ΔrpfB ΔrpfC strain grew poorly and lysed upon entering stationary phase. Deletion of rpfF, the gene encoding the DSF synthetic enzyme, restored normal growth to this strain. RpfF is a dual function enzyme that synthesizes DSF by dehydration of a 3-hydroxyacyl-acyl carrier protein (ACP) fatty acid synthetic intermediate and also cleaves the thioester bond linking DSF to ACP. However, the RpfF thioesterase activity is of broad specificity and upon elimination of its RpfC inhibitor RpfF attains maximal activity and its thioesterase activity proceeds to block membrane lipid synthesis by cleavage of acyl-ACP intermediates. This resulted in release of the nascent acyl chains to the medium as free fatty acids. This lack of acyl chains for phospholipid synthesis results in cell lysis unless RpfB is present to counteract the RpfF thioesterase activity by catalyzing uptake and activation of the free fatty acids to give acyl-CoAs that can be utilized to restore membrane lipid synthesis. Heterologous expression of a different fatty acid activating enzyme, the Vibrio harveyi acyl-ACP synthetase, replaced RpfB in counteracting the effects of

A Canonical Biotin Synthesis Enzyme, 8-Amino-7-Oxononanoate Synthase (BioF), Utilizes Different Acyl Chain Donors in Bacillus subtilis and Escherichia coli.

PubMed

Manandhar, Miglena; Cronan, John E

2018-01-01

BioF (8-amino-7-oxononanoate synthase) is a strictly conserved enzyme that catalyzes the first step in assembly of the fused heterocyclic rings of biotin. The BioF acyl chain donor has long been thought to be pimeloyl-CoA. Indeed, in vitro the Escherichia coli and Bacillus sphaericus enzymes have been shown to condense pimeloyl-CoA with l-alanine in a pyridoxal 5'-phosphate-dependent reaction with concomitant CoA release and decarboxylation of l-alanine. However, recent in vivo studies of E. coli and Bacillus subtilis suggested that the BioF proteins of the two bacteria could have different specificities for pimelate thioesters in that E. coli BioF may utilize either pimeloyl coenzyme A (CoA) or the pimelate thioester of the acyl carrier protein (ACP) of fatty acid synthesis. In contrast, B. subtilis BioF seemed likely to be specific for pimeloyl-CoA and unable to utilize pimeloyl-ACP. We now report genetic and in vitro data demonstrating that B. subtilis BioF specifically utilizes pimeloyl-CoA. IMPORTANCE Biotin is an essential vitamin required by mammals and birds because, unlike bacteria, plants, and some fungi, these organisms cannot make biotin. Currently, the biotin included in vitamin tablets and animal feeds is made by chemical synthesis. This is partly because the biosynthetic pathways in bacteria are incompletely understood. This paper defines an enzyme of the Bacillus subtilis pathway and shows that it differs from that of Escherichia coli in the ability to utilize specific precursors. These bacteria have been used in biotin production and these data may aid in making biotin produced by biotechnology commercially competitive with that produced by chemical synthesis. Copyright © 2017 American Society for Microbiology.

Water and solute permeability of rat lung caveolae: high permeabilities explained by acyl chain unsaturation.

PubMed

Hill, Warren G; Almasri, Eyad; Ruiz, W Giovanni; Apodaca, Gerard; Zeidel, Mark L

2005-07-01

Caveolae are invaginated membrane structures with high levels of cholesterol, sphingomyelin, and caveolin protein that are predicted to exist as liquid-ordered domains with low water permeability. We isolated a caveolae-enriched membrane fraction without detergents from rat lung and characterized its permeability properties to nonelectrolytes and protons. Membrane permeability to water was 2.85 +/- 0.41 x 10(-3) cm/s, a value 5-10 times higher than expected based on comparisons with other cholesterol and sphingolipid-enriched membranes. Permeabilities to urea, ammonia, and protons were measured and found to be moderately high for urea and ammonia at 8.85 +/- 2.40 x 10(-7)and 6.84 +/- 1.03 x 10(-2) respectively and high for protons at 8.84 +/- 3.06 x 10(-2) cm/s. To examine whether caveolin or other integral membrane proteins were responsible for high permeabilities, liposomes designed to mimic the lipids of the inner and outer leaflets of the caveolar membrane were made. Osmotic water permeability to both liposome compositions were determined and a combined inner/outer leaflet water permeability was calculated and found to be close to that of native caveolae at 1.58 +/- 1.1 x 10(-3) cm/s. In caveolae, activation energy for water flux was high (19.4 kcal/mol) and water permeability was not inhibited by HgCl2; however, aquaporin 1 was detectable by immunoblotting. Immunostaining of rat lung with AQP1 and caveolin antisera revealed very low levels of colocalization. We conclude that aquaporin water channels do not contribute significantly to the observed water flux and that caveolae have relatively high water and solute permeabilities due to the high degree of unsaturation in their fatty acyl chains.

Safety Assessment of Acyl Glucuronides-A Simplified Paradigm.

PubMed

Smith, Dennis A; Hammond, Timothy; Baillie, Thomas A

2018-06-01

While simple O - (ether-linked) and N -glucuronide drug conjugates generally are unreactive and considered benign from a safety perspective, the acyl glucuronides that derive from metabolism of carboxylic acid-containing xenobiotics can exhibit a degree of chemical reactivity that is dependent upon their molecular structure. As a result, concerns have arisen over the safety of acyl glucuronides as a class, several members of which have been implicated in the toxicity of their respective parent drugs. However, direct evidence in support of these claims remains sparse, and due to frequently encountered species differences in the systemic exposure to acyl glucuronides (both of the parent drug and oxidized derivatives thereof), coupled with their instability in aqueous media and potential to undergo chemical rearrangement (acyl migration), qualification of these conjugates by traditional safety assessment methods can be very challenging. In this Commentary, we discuss alternative (non-acyl glucuronide) mechanisms by which carboxylic acids may cause serious adverse reactions, and propose a novel, practical approach to compare systemic exposure to acyl glucuronide metabolites in humans to that in animal species used in preclinical safety assessment based on relative estimates of the total body burden of these circulating conjugates. Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.

Conformational Flexibility of Metazoan Fatty Acid Synthase Enables Catalysis

PubMed Central

Brignole, Edward J.; Smith, Stuart; Asturias, Francisco J.

2008-01-01

The metazoan cytosolic fatty acid synthase (FAS) contains all of the enzymes required for de novo fatty acid biosynthesis covalently linked around two reaction chambers. While the 3D architecture of FAS has been mostly defined, it is unclear how reaction intermediates can transfer between distant catalytic domains. Using single-particle electron microscopy we have identified a near continuum of conformations consistent with remarkable flexibility of FAS. The distribution of conformations was influenced by the presence of substrates and altered by different catalytic mutations suggesting a direct correlation between conformation and specific enzymatic activities. 3D reconstructions were interpreted by docking high-resolution structures of individual domains and illustrate that the substrate loading and condensation domains dramatically swing and swivel to access substrates within either reaction chamber. Concomitant rearrangement of the β-carbon processing domains synchronizes acyl-chain reduction in one chamber with acyl-chain elongation in the other. PMID:19151726

Metabolic regulation of ghrelin O-acyl transferase (GOAT) expression in the mouse hypothalamus, pituitary, and stomach.

PubMed

Gahete, Manuel D; Córdoba-Chacón, Jose; Salvatori, Roberto; Castaño, Justo P; Kineman, Rhonda D; Luque, Raul M

2010-04-12

Ghrelin acts as an endocrine link connecting physiological processes regulating food intake, body composition, growth, and energy balance. Ghrelin is the only peptide known to undergo octanoylation. The enzyme mediating this process, ghrelin O-acyltransferase (GOAT), is expressed in the gastrointestinal tract (GI; primary source of circulating ghrelin) as well as other tissues. The present study demonstrates that stomach GOAT mRNA levels correlate with circulating acylated-ghrelin levels in fasted and diet-induced obese mice. In addition, GOAT was found to be expressed in both the pituitary and hypothalamus (two target tissues of ghrelin's actions), and regulated in response to metabolic status. Using primary pituitary cell cultures as a model system to study the regulation of GOAT expression, we found that acylated-ghrelin, but not desacyl-ghrelin, increased GOAT expression. In addition, growth-hormone-releasing hormone (GHRH) and leptin increased, while somatostatin (SST) decreased GOAT expression. The physiologic relevance of these later results is supported by the observation that pituitary GOAT expression in mice lacking GHRH, SST and leptin showed opposite changes to those observed after in vitro treatment with the corresponding peptides. Therefore, it seems plausible that these hormones directly contribute to the regulation of pituitary GOAT. Interestingly, in all the models studied, pituitary GOAT expression paralleled changes in the expression of a dominant spliced-variant of ghrelin (In2-ghrelin) and therefore this transcript may be a primary substrate for pituitary GOAT. Collectively, these observations support the notion that the GI tract is not the only source of acylated-ghrelin, but in fact locally produced des-acylated-ghrelin could be converted to acylated-ghrelin within target tissues by locally active GOAT, to mediate its tissue-specific effects.

Plasma levels of acylated ghrelin in patients with functional dyspepsia

PubMed Central

Kim, Yeon Soo; Lee, Joon Seong; Lee, Tae Hee; Cho, Joo Young; Kim, Jin Oh; Kim, Wan Jung; Kim, Hyun Gun; Jeon, Seong Ran; Jeong, Hoe Su

2012-01-01

AIM: To investigate the relationship between plasma acylated ghrelin levels and the pathophysiology of functional dyspepsia. METHODS: Twenty-two female patients with functional dyspepsia and twelve healthy volunteers were recruited for the study. The functional dyspepsia patients were each diagnosed based on the Rome III criteria. Eligible patients completed a questionnaire concerning the severity of 10 symptoms. Plasma acylated ghrelin levels before and after a meal were determined in the study participants using a commercial human acylated enzyme immunoassay kit; electrogastrograms were performed for 50 min before and after a standardized 10-min meal containing 265 kcal. RESULTS: There were no significant differences in plasma acylated ghrelin levels between healthy volunteers and patients with functional dyspepsia. However, in patients with functional dyspepsia, there was a negative correlation between fasting plasma acylated ghrelin levels and the sum score of epigastric pain (r = -0.427, P = 0.047) and a positive correlation between the postprandial/fasting plasma acylated ghrelin ratio and the sum score of early satiety (r = 0.428, P =0.047). Additionally, there was a negative correlation between fasting acylated ghrelin plasma levels and fasting normogastria (%) (r = -0.522, P = 0.013). Interestingly, two functional dyspepsia patients showed paradoxically elevated plasma acylated ghrelin levels after the meal. CONCLUSION: Abnormal plasma acylated ghrelin levels before or after a meal may be related to several of the dyspeptic symptoms seen in patients with functional dyspepsia. PMID:22611317

Characterization of CG6178 gene product with high sequence similarity to firefly luciferase in Drosophila melanogaster.

PubMed

Oba, Yuichi; Ojika, Makoto; Inouye, Satoshi

2004-03-31

This is the first identification of a long-chain fatty acyl-CoA synthetase in Drosophila by enzymatic characterization. The gene product of CG6178 (CG6178) in Drosophila melanogaster genome, which has a high sequence similarity to firefly luciferase, has been expressed and characterized. CG6178 showed long-chain fatty acyl-CoA synthetic activity in the presence of ATP, CoA and Mg(2+), suggesting a fatty acyl adenylate is an intermediate. Recently, it was revealed that firefly luciferase has two catalytic functions, monooxygenase (luciferase) and AMP-mediated CoA ligase (fatty acyl-CoA synthetase). However, unlike firefly luciferase, CG6178 did not show luminescence activity in the presence of firefly luciferin, ATP, CoA and Mg(2+). The enzymatic properties of CG6178 including substrate specificity, pH dependency and optimal temperature were close to those of firefly luciferase and rat fatty acyl-CoA synthetase. Further, phylogenic analyses strongly suggest that the firefly luciferase gene may have evolved from a fatty acyl-CoA synthetase gene as a common ancestral gene.

Involvement of PlsX and the acyl-phosphate dependent sn-glycerol-3-phosphate acyltransferase PlsY in the initial stage of glycerolipid synthesis in Bacillus subtilis.

PubMed

Hara, Yoshinori; Seki, Masahide; Matsuoka, Satoshi; Hara, Hiroshi; Yamashita, Atsushi; Matsumoto, Kouji

2008-12-01

The gene responsible for the first acylation of sn-glycerol-3-phosphate (G3P) in Bacillus subtilis has not yet been determined with certainty. The product of this first acylation, lysophosphatidic acid (LPA), is subsequently acylated again to form phosphatidic acid (PA), the primary precursor to membrane glycerolipids. A novel G3P acyltransferase (GPAT), the gene product of plsY, which uses acyl-phosphate formed by the plsX gene product, has recently been found to synthesize LPA in Streptococcus pneumoniae. We found that in B. subtilis growth arrests after repression of either a plsY homologue or a plsX homologue were overcome by expression of E. coli plsB, which encodes an acyl-acylcarrier protein (acyl-ACP)-dependent GPAT, although in the case of plsX repression a high level of plsB expression was required. B. subtilis has, therefore, a capability to use the acyl-ACP dependent GPAT of PlsB. Simultaneous expression of plsY and plsX suppressed the glycerol requirement of a strict glycerol auxotrophic derivative of the E. coli plsB26 mutant, although either one alone did not. Membrane fractions from B. subtilis cells catalyzed palmitoylphosphate-dependent acylation of [14C]-labeled G3P to synthesize [14C]-labeled LPA, whereas those from DeltaplsY cells did not. The results indicate unequivocally that PlsY is an acyl-phosphate dependent GPAT. Expression of plsX corrected the glycerol auxotrophy of a DeltaygiH (the deleted allele of an E. coli homologue of plsY) derivative of BB26-36 (plsB26 plsX50), suggesting an essential role of plsX other than substrate supply for acyl-phosphate dependent LPA synthesis. Two-hybrid examinations suggested that PlsY is associated with PlsX and that each may exist in multimeric form.

Condensing enzymes from Cuphea wrightii associated with medium chain fatty acid biosynthesis.

PubMed

Slabaugh, M B; Leonard, J M; Knapp, S J

1998-03-01

Seed oils of most Cuphea species contain > 90% medium chain (C8-C14) fatty acids. Thioesterases with specificity for these substrates are important determinants of the medium chain phenotype. The role of condensing enzymes, however, has not been investigated. cDNA clones encoding beta-ketoacyl-acyl carrier protein (ACP) synthase (KAS) were isolated from C. wrightii, a C10/C12-producing species. Deduced amino acid sequences of four unique clones were approximately 60% identical to plant KAS I sequences and approximately 75% identical to a distinct class of KAS sequences recently identified in castor and barley. A 46 kDa protein that was observed only in developing and mature seed was detected using antiserum directed against recombinant Cuphea KAS protein. The 46 kDa protein was abundant in developing seeds of six medium chain-producing Cuphea species but barely detected in one long chain-producing species. A 48 kDa protein identified immunologically as KAS I was expressed in both medium and long chain-producing Cuphea species and was detected in all tissues tested. In in vitro assays, extracts from C. wrightii and C. viscosissima developing embryos were unable to extend fatty acid chains beyond C10 following treatment with 10 microns cerulenin, a potent inhibitor of KAS I. However, a C. viscosissima mutant, cpr-1, whose seed oils are deficient in caprate relative to wild type, was impaired in extension of C8 to C10 in this assay and Western analysis revealed a specific deficiency in 46 kDa KAS in cpr-1 embryos. These results implicate cerulenin-resistant condensing activity in production of medium chain fatty acids in Cuphea.

Sex-related difference in the inductions by perfluoro-octanoic acid of peroxisomal beta-oxidation, microsomal 1-acylglycerophosphocholine acyltransferase and cytosolic long-chain acyl-CoA hydrolase in rat liver.

PubMed Central

Kawashima, Y; Uy-Yu, N; Kozuka, H

1989-01-01

Inductions by perfluoro-octanoic acid (PFOA) of hepatomegaly, peroxisomal beta-oxidation, microsomal 1-acylglycerophosphocholine acyltransferase and cytosolic long-chain acyl-CoA hydrolase were compared in liver between male and female rats. Marked inductions of these four parameters were seen concurrently in liver of male rats, whereas the inductions in liver of female rats were far less pronounced. The sex-related difference in the response of rat liver to PFOA was much more marked than that seen with p-chlorophenoxyisobutyric acid (clofibric acid) or 2,2'-(decamethylenedithio)diethanol (tiadenol). Hormonal manipulations revealed that this sex-related difference in the inductions is strongly dependent on sex hormones, namely that testosterone is necessary for the inductions, whereas oestradiol prevented the inductions by PFOA. PMID:2570571

Endoglucanase Peripheral Loops Facilitate Complexation of Glucan Chains on Cellulose via Adaptive Coupling to the Emergent Substrate Structures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, Yuchun; Beckham, Gregg T.; Himmel, Michael E.

We examine how the catalytic domain of a glycoside hydrolase family 7 endoglucanase catalytic domain (Cel7B CD) facilitates complexation of cellulose chains from a crystal surface. With direct relevance to the science of biofuel production, this problem also represents a model system of biopolymer processing by proteins in Nature. Interactions of Cel7B CD with a cellulose microfibril along different paths of complexation are characterized by mapping the atomistic fluctuations recorded in free-energy simulations onto the parameters of a coarse-grain model. The resulting patterns of protein-biopolymer couplings also uncover the sequence signatures of the enzyme in peeling off glucan chains frommore » the microfibril substrate. We show that the semiopen active site of Cel7B CD exhibits similar barriers and free energies of complexation over two distinct routes; namely, scooping of a chain into the active-site cleft and threading from the chain end into the channel. On the other hand, the complexation energetics strongly depends on the surface packing of the targeted chain and the resulting interaction sites with the enzyme. A revealed principle is that Cel7B CD facilitates cellulose deconstruction via adaptive coupling to the emergent substrate. The flexible, peripheral segments of the protein outside of the active-site cleft are able to accommodate the varying features of cellulose along the simulated paths of complexation. The general strategy of linking physics-based molecular interactions to protein sequence could also be helpful in elucidating how other protein machines process biopolymers.« less

Oxonitriles: A Grignard Addition-Acylation Route to Enamides

PubMed Central

Wei, Guoqing; Zhang, Zhiyu; Steward, Omar W.

2008-01-01

Sequential addition of three different Grignard reagents and pivaloyl chloride to 3-oxo-1-cyclohexene-1-carbonitrile installs four new bonds to generate a diverse array of cyclic enamides. Remarkably, formation of the C-magnesiated nitrile intermediate is followed by preferential acylation by pivaloyl chloride rather than consumption by in situ Grignard reagent. Rapid N-acylation of the C-magnesiated nitrile generates an acyl ketenimine that reacts readily with Grignard reagents, or a trialkyl zincate, effectively assembling highly substituted, cyclic enamides. PMID:17020332

Cloning, tissue distribution, functional characterization and nutritional regulation of a fatty acyl Elovl5 elongase in chu's croaker Nibea coibor.

PubMed

Lin, Zhideng; Huang, Yisheng; Zou, Weiguang; Rong, Hua; Hao, Meiling; Wen, Xiaobo

2018-06-15

Enzymes that lengthen the carbon chain of polyunsaturated fatty acids (PUFA) are key to the biosynthesis of the long-chain polyunsaturated fatty acids (LC-PUFA). Here we report on the molecular cloning, tissue distribution, functional characterization and nutritional regulation of a elovl5 gene from Nibea coibor. The full-length cDNA was 1315 bp, including a 5-untranslated region (UTR) of 134 bp, a 3-UTR of 296 bp and an open reading frame of 885 bp, which specified a peptide of 294 amino acids. Bioinformatics analysis showed that the deduced peptide sequence possessed all the characteristic features of microsomal fatty acyl elongases, including the so-called histidine box (HXXHH), the canonical C-terminal endoplasmic reticulum retention signal, several predicted transmembrane regions and other highly conserved motifs. Expression of elovl5 was strongly observed in stomach, and more weakly in kidney, spleen, intestine, brain, eye, liver, gill, muscle and heart. Functional characterization revealed that the chu's croaker Elovl5 was able to elongate both C18 and C20 PUFA substrates. Nutritional study indicated that the hepatic expression of elovl5 could be up-regulated by low dietary n-3 LC-PUFA. These results may contribute to better understanding the LC-PUFA biosynthetic pathway and regulation mechanism in chu's croaker. Copyright © 2018. Published by Elsevier B.V.

Effects of supercritical carbon dioxide on immobile bound polymer chains on solid substrates

NASA Astrophysics Data System (ADS)

Sen, Mani; Asada, Mitsunori; Jiang, Naisheng; Endoh, Maya K.; Akgun, Bulent; Satija, Sushil; Koga, Tadanori

2013-03-01

Adsorbed polymer layers formed on flat solid substrates have recently been the subject of extensive studies because it is postulated to control the dynamics of technologically relevant polymer thin films, for example, in lithography. Such adsorbed layers have been reported to hinder the mobility of polymer chains in thin films even at a large length scale. Consequently, this bound layer remains immobile regardless of processing techniques (i.e. thermal annealing, solvent dissolution, etc). Here, we investigate the use of supercritical carbon dioxide (scCO2) as a novel plasticizer for bound polystyrene layers formed on silicon substrates. In-situ swelling and interdiffusion experiments using neutron reflectivity were performed. As a result, we found the anomalous plasticization effects of scCO2 on the bound polymer layers near the critical point where the anomalous adsorption of CO2 molecules in polymer thin films has been reported previously. Acknowledgement: We acknowledge the financial support from NSF Grant No. CMMI-084626.

Diverse profiles of N-acyl-homoserine lactones in biofilm forming strains of Cronobacter sakazakii.

PubMed

Singh, Niharika; Patil, Amrita; Prabhune, Asmita A; Raghav, Mamta; Goel, Gunjan

2017-04-03

The present study investigates the role of quorum sensing (QS) molecules expressed by C. sakazakii in biofilm formation and extracellular polysaccharide expression. The QS signaling was detected using Chromobacterium violaceum 026 and Agrobacterium tumefaciens NTL4(pZLR4) based bioassay. Long chain N-acyl-homoserine lactones (AHLs) with C6- C18 chain length were identified using High Performance Liquid Chromatography and Liquid Chromatography-High Resolution Mass Spectrometry. A higher Specific Biofilm Formation (SBF) index (p < 0.05) with the presence of genes associated with cellulose biosynthesis (bcsA, bcsC and bcsG) was observed in the strains. AHLs and their mechanisms can serve as novel targets for developing technologies to eradicate and prevent biofilm formation by C. sakazakii.

Synthesis and biological activities of turkesterone 11α-acyl derivatives

PubMed Central

Dinan, Laurence; Bourne, Pauline; Whiting, Pensri; Tsitsekli, Ada; Saatov, Ziyadilla; Dhadialla, Tarlochan S.; Hormann, Robert E.; Lafont, René; Coll, Josep

2003-01-01

Turkesterone is a phytoecdysteroid possessing an 11α-hydroxyl group. It is an analogue of the insect steroid hormone 20-hydroxyecdysone. Previous ecdysteroid QSAR and molecular modelling studies predicted that the cavity of the ligand binding domain of the ecdysteroid receptor would possess space in the vicinity of C-11/C-12 of the ecdysteroid. We report the regioselective synthesis of a series of turkesterone 11α-acyl derivatives in order to explore this possibility. The structures of the analogues have been unambiguously determined by spectroscopic means (NMR and low-resolution mass spectrometry). Purity was verified by HPLC. Biological activities have been determined in Drosophila melanogaster BII cell-based bioassay for ecdysteroid agonists and in an in vitro radioligand-displacement assay using bacterially-expressed D. melanogaster EcR/USP receptor proteins. The 11α-acyl derivatives do retain a significant amount of biological activity relative to the parent ecdysteroid. Further, although activity initially drops with the extension of the acyl chain length (C2 to C4), it then increases (C6 to C10), before decreasing again (C14 and C20). The implications of these findings for the interaction of ecdysteroids with the ecdysteroid receptor and potential applications in the generation of affinity-labelled and fluorescently-tagged ecdysteroids are discussed. Abbreviation: CoMFA comparative molecular field analysis DCM dichloromethane DMF dimethylformamide DMP 2,2-dimethoxypropane 4D-QSAR 4-dimensional quantitative structure-activity relationship EcR ecdysteroid receptor EcRE ecdysteroid response element HPLC high-performance liquid chromatography LBD ligand-binding domain NMR nuclear magnetic resonance ponA ponasterone A QSAR quantitative structure-activity relationship RXR retinoid X receptor SAR structure-activity relationship SPE solid-phase extraction THF tetrahydrofuran TLC thin-layer chromatography p-TsOH para-toluenesulphonic acid USP ultraspiracle UV

Oxonitriles: a grignard addition-acylation route to enamides.

PubMed

Fleming, Fraser F; Wei, Guoqing; Zhang, Zhiyu; Steward, Omar W

2006-10-12

[reaction: see text] Sequential addition of three different Grignard reagents and pivaloyl chloride to 3-oxo-1-cyclohexene-1-carbonitrile installs four new bonds to generate a diverse array of cyclic enamides. Remarkably, formation of the C-magnesiated nitrile intermediate is followed by preferential acylation by pivaloyl chloride rather than consumption by an in situ Grignard reagent. Rapid N-acylation of the C-magnesiated nitrile generates an acyl ketenimine that reacts readily with Grignard reagents or a trialkylzincate, effectively assembling highly substituted, cyclic enamides.

Disparate effects of oxidation on plasma acyltransferase activities: inhibition of cholesterol esterification but stimulation of transesterification of oxidized phospholipids.

PubMed

Subbaiah, P V; Liu, M

1996-05-31

Oxidation of lipoproteins results in the formation of several polar phospholipids with pro-inflammatory and pro-atherogenic properties. To examine the possible role of lecithin/cholesterol acyltransferase (LCAT) in the metabolism of these oxidized phospholipids, we oxidized whole plasma with either Cu(2+) or a free-radical generator, and determined the various activities of LCAT. Oxidation caused a reduction in plasma phosphatidylcholine (PC), an increase in a short-chain polar PC (SCP-PC), and an inhibition of the transfer of long-chain acyl groups to cholesterol (LCAT activity) or to lyso PC (lysolecithin acyltransferase (LAT) I activity). However, the transfer of short-chain acyl groups from SCP-PC to lyso PCLAT II activity) was stimulated several fold, in direct correlation with the degree of oxidation. LAT II activity was not stimulated by oxidation in LCAT-deficient plasma, showing that it is carried out by LCAT. Oxidized normal plasma exhibited low LCAT activity even in the presence of exogenous proteoliposome substrate, indicating that the depletion of substrate PC was not responsible for the loss of activity. Oxidation of isolated LDL or HDL abolished their ability to support LCAT and LAT I activities of exogenous enzyme, but promoted the LAT II activity. Purified LCAT lost its LCAT and LAT I functions, but not its LAT II function, when oxidized in vitro. These results show that while oxidation of plasma causes a loss of LCAT's ability to transfer long-chain acyl groups, its ability to transfer short-chain acyl groups, from SCP-PC is retained, and even stimulated, suggesting that LCAT may have a physiological role in the metabolism of oxidized PC in plasma.

In silico prediction of acyl glucuronide reactivity

NASA Astrophysics Data System (ADS)

Potter, Tim; Lewis, Richard; Luker, Tim; Bonnert, Roger; Bernstein, Michael A.; Birkinshaw, Timothy N.; Thom, Stephen; Wenlock, Mark; Paine, Stuart

2011-11-01

Drugs and drug candidates containing a carboxylic acid moiety, including many widely used non-steroidal anti-inflammatory drugs (NSAIDs) are often metabolized to form acyl glucuronides (AGs). NSAIDs such as Ibuprofen are amongst the most widely used drugs on the market, whereas similar carboxylic acid drugs such as Suprofen have been withdrawn due to adverse events. Although the link between these AG metabolites and toxicity is not proven, there is circumstantial literature evidence to suggest that more reactive acyl glucuronides may, in some cases, present a greater risk of exhibiting toxic effects. We wished therefore to rank the reactivity of potential new carboxylate-containing drug candidates, and performed kinetic studies on synthetic acyl glucuronides to benchmark our key compounds. Driven by the desire to quickly rank the reactivity of compounds without the need for lengthy synthesis of the acyl glucuronide, a correlation was established between the degradation half-life of the acyl glucuronide and the half life for the hydrolysis of the more readily available methyl ester derivative. This finding enabled a considerable broadening of chemical property space to be investigated. The need for kinetic measurements was subsequently eliminated altogether by correlating the methyl ester hydrolysis half-life with the predicted 13C NMR chemical shift of the carbonyl carbon together with readily available steric descriptors in a PLS model. This completely in silico prediction of acyl glucuronide reactivity is applicable within the earliest stages of drug design with low cost and acceptable accuracy to guide intelligent molecular design. This reactivity data will be useful alongside the more complex additional pharmacokinetic exposure and distribution data that is generated later in the drug discovery process for assessing the overall toxicological risk of acidic drugs.

The Bacillus subtilis Acyl Lipid Desaturase Is a Δ5 Desaturase

PubMed Central

Altabe, Silvia G.; Aguilar, Pablo; Caballero, Gerardo M.; de Mendoza, Diego

2003-01-01

Bacillus subtilis was recently reported to synthesize unsaturated fatty acids (UFAs) with a double bond at positions Δ5, Δ7, and Δ9 (M. H. Weber, W. Klein, L. Muller, U. M. Niess, and M. A. Marahiel, Mol. Microbiol. 39:1321-1329, 2001). Since this finding would have considerable importance in the double-bond positional specificity displayed by the B. subtilis acyl lipid desaturase, we have attempted to confirm this observation. We report that the double bond of UFAs synthesized by B. subtilis is located exclusively at the Δ5 position, regardless of the growth temperature and the length chain of the fatty acids. PMID:12730185

Terminal Olefin Profiles and Phylogenetic Analyses of Olefin Synthases of Diverse Cyanobacterial Species.

PubMed

Zhu, Tao; Scalvenzi, Thibault; Sassoon, Nathalie; Lu, Xuefeng; Gugger, Muriel

2018-07-01

Cyanobacteria can synthesize alkanes and alkenes, which are considered to be infrastructure-compatible biofuels. In terms of physiological function, cyanobacterial hydrocarbons are thought to be essential for membrane flexibility for cell division, size, and growth. The genetic basis for the biosynthesis of terminal olefins (1-alkenes) is a modular type I polyketide synthase (PKS) termed olefin synthase (Ols). The modular architectures of Ols and structural characteristics of alkenes have been investigated only in a few species of the small percentage (approximately 10%) of cyanobacteria that harbor putative Ols pathways. In this study, investigations of the domains, modular architectures, and phylogenies of Ols in 28 cyanobacterial strains suggested distinctive pathway evolution. Structural feature analyses revealed 1-alkenes with three carbon chain lengths (C 15 , C 17 , and C 19 ). In addition, the total cellular fatty acid profile revealed the diversity of the carbon chain lengths, while the fatty acid feeding assay indicated substrate carbon chain length specificity of cyanobacterial Ols enzymes. Finally, in silico analyses suggested that the N terminus of the modular Ols enzyme exhibited characteristics typical of a fatty acyl-adenylate ligase (FAAL), suggesting a mechanism of fatty acid activation via the formation of acyl-adenylates. Our results shed new light on the diversity of cyanobacterial terminal olefins and a mechanism for substrate activation in the biosynthesis of these olefins. IMPORTANCE Cyanobacterial terminal olefins are hydrocarbons with promising applications as advanced biofuels. Despite the basic understanding of the genetic basis of olefin biosynthesis, the structural diversity and phylogeny of the key modular olefin synthase (Ols) have been poorly explored. An overview of the chemical structural traits of terminal olefins in cyanobacteria is provided in this study. In addition, we demonstrated by in vivo fatty acid feeding assays that

Precise Nanoelectronics with Adatom Chains

NASA Technical Reports Server (NTRS)

Yamada, Toshishige

1999-01-01

Adatom chains on an atomically regulated substrate will be building components in future precise nanoelectronics. Adatoms need to be secured with chemical bonding, but then electronic isolation between the adatom and substrate systems is not guaranteed. A one-dimensional model shows that good isolation with existence of surface states is expected on an s-p crossing substrate such as Si, Ge, or GaAs, reflecting the bulk nature of the substrate. Isolation is better if adatoms are electronically similar to the substrate atoms, and can be manipulated by hydrogenation. Chain structures with group IV adatoms with two chemical bonds, or group III adatoms with one chemical bond, are semiconducting, reflecting the surface nature of the substrate. These structures are unintentionally doped due to the charge transfer across the chemical bonds. Physical properties of adatom chains have to be determined for the unified adatom-substrate system.

Protein-Protein Interactions, Not Substrate Recognition, Dominate the Turnover of Chimeric Assembly Line Polyketide Synthases*

PubMed Central

Klaus, Maja; Ostrowski, Matthew P.; Austerjost, Jonas; Robbins, Thomas; Lowry, Brian; Cane, David E.; Khosla, Chaitan

2016-01-01

The potential for recombining intact polyketide synthase (PKS) modules has been extensively explored. Both enzyme-substrate and protein-protein interactions influence chimeric PKS activity, but their relative contributions are unclear. We now address this issue by studying a library of 11 bimodular and 8 trimodular chimeric PKSs harboring modules from the erythromycin, rifamycin, and rapamycin synthases. Although many chimeras yielded detectable products, nearly all had specific activities below 10% of the reference natural PKSs. Analysis of selected bimodular chimeras, each with the same upstream module, revealed that turnover correlated with the efficiency of intermodular chain translocation. Mutation of the acyl carrier protein (ACP) domain of the upstream module in one chimera at a residue predicted to influence ketosynthase-ACP recognition led to improved turnover. In contrast, replacement of the ketoreductase domain of the upstream module by a paralog that produced the enantiomeric ACP-bound diketide caused no changes in processing rates for each of six heterologous downstream modules compared with those of the native diketide. Taken together, these results demonstrate that protein-protein interactions play a larger role than enzyme-substrate recognition in the evolution or design of catalytically efficient chimeric PKSs. PMID:27246853

Sialomucins are characteristically O-acylated in poorly differentiated and colloid prostatic adenocarcinomas.

PubMed

Sáez, C; Japón, M A; Conde, A F; Poveda, M A; Luna-Moré, S; Segura, D I

1998-12-01

Mucinous glycoproteins are secreted by prostatic adenocarcinomas and might play important roles in tumor invasion and metastasis. Their histochemical properties on routine biopsy specimens have not been fully characterized. We present a histochemical study of mucin in 21 prostatic adenocarcinomas, with particular focus on the demonstration of different types of sialomucins. We applied the following histochemical techniques to routinely processed, formalin-fixed, paraffin-embedded tissue sections: Alcian blue (pH 2.5) and periodic acid-Schiff to reveal both acidic and neutral mucins; high iron diamine and Alcian blue (pH 2.5) to show sulfated and acidic nonsulfated mucosubstances simultaneously; periodic acid borohydride, potassium hydroxide, and periodic acid-Schiff to demonstrate O-acylated sialic acids; periodic acid thionine-Schiff, potassium hydroxide, and periodic acid-Schiff to differentiate pre-existing glycols from those revealed after saponification procedures; and periodic acid borohydride and periodic acid-Schiff to show C9-O-acylated sialic acid. These techniques are useful tools for demonstrating neutral and acidic (sialo- and sulfo-) mucins and di(C8,C9- or C7,C9-)-O-acylated, tri(C7,C8,C9-)-O-acylated and mono(C9)-O-acylated sialomucins. Most prostatic adenocarcinomas showed acidic mucins, with sialomucins predominating over sulfomucins. Well-differentiated and moderately differentiated noncolloid tumors had non-O-acylated sialomucins. Poorly differentiated tumors contained mono-O-acylated (C9) sialomucins, and colloid-type tumors secreted mono-, di-, and tri-O-acylated sialoglycoproteins. Acidic mucins, mainly sialomucins, constitute the major secretory component in prostatic adenocarcinomas, and our results show that the O-acylation of these sialoglycoproteins inversely correlates with tumor differentiation. Well-differentiated and moderately differentiated tumors are not O-acylated, whereas the poorly differentiated ones characteristically have O-acylated

Long-Chain Fatty Acyl Coenzyme A Ligase FadD2 Mediates Intrinsic Pyrazinamide Resistance in Mycobacterium tuberculosis

PubMed Central

Rosen, Brandon C.; Dillon, Nicholas A.; Peterson, Nicholas D.; Minato, Yusuke

2016-01-01

ABSTRACT Pyrazinamide (PZA) is a first-line tuberculosis (TB) drug that has been in clinical use for 60 years yet still has an unresolved mechanism of action. Based upon the observation that the minimum concentration of PZA required to inhibit the growth of Mycobacterium tuberculosis is approximately 1,000-fold higher than that of other first-line drugs, we hypothesized that M. tuberculosis expresses factors that mediate intrinsic resistance to PZA. To identify genes associated with intrinsic PZA resistance, a library of transposon-mutagenized Mycobacterium bovis BCG strains was screened for strains showing hypersusceptibility to the active form of PZA, pyrazinoic acid (POA). Disruption of the long-chain fatty acyl coenzyme A (CoA) ligase FadD2 enhanced POA susceptibility by 16-fold on agar medium, and the wild-type level of susceptibility was restored upon expression of fadD2 from an integrating mycobacterial vector. Consistent with the recent observation that POA perturbs mycobacterial CoA metabolism, the fadD2 mutant strain was more vulnerable to POA-mediated CoA depletion than the wild-type strain. Ectopic expression of the M. tuberculosis pyrazinamidase PncA, necessary for conversion of PZA to POA, in the fadD2 transposon insertion mutant conferred at least a 16-fold increase in PZA susceptibility under active growth conditions in liquid culture at neutral pH. Importantly, deletion of fadD2 in M. tuberculosis strain H37Rv also resulted in enhanced susceptibility to POA. These results indicate that FadD2 is associated with intrinsic PZA and POA resistance and provide a proof of concept for the target-based potentiation of PZA activity in M. tuberculosis. PMID:27855077

Acyl donors for native chemical ligation.

PubMed

Yan, Bingjia; Shi, Weiwei; Ye, Linzhi; Liu, Lei

2018-04-11

Native chemical ligation (NCL) has become one of the most important methods in chemical syntheses of proteins. Recently, in order to expand its scope, considerable effort has been devoted to tuning the C-terminal acyl donor thioesters used in NCL. This article reviews the recent advances in the design of C-terminal acyl donors, their precursors and surrogates, and highlights some noteworthy progress that may lead the future direction of protein chemical synthesis. Copyright © 2018 Elsevier Ltd. All rights reserved.

Tunable Oleo-Furan Surfactants by Acylation of Renewable Furans

DOE PAGES

Park, Dae Sung; Joseph, Kristeen E.; Koehle, Maura; ...

2016-10-19

One important advance in fluid surface control was the amphiphilic surfactant composed of coupled molecular structures (i.e., hydrophilic and hydrophobic) to reduce surface tension between two distinct fluid phases. However, implementation of simple surfactants has been hindered by the broad range of applications in water containing alkaline earth metals (i.e., hard water). This disrupts surfactant function and requires extensive use of undesirable and expensive chelating additives. We show that sugar-derived furans can be linked with triglyceride-derived fatty acid chains via Friedel–Crafts acylation within single layer (SPP) zeolite catalysts. Finally, these alkylfuran surfactants independently suppress the effects of hard water whilemore » simultaneously permitting broad tunability of size, structure, and function, which can be optimized for superior capability for forming micelles and solubilizing in water.« less

Tunable Oleo-Furan Surfactants by Acylation of Renewable Furans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Dae Sung; Joseph, Kristeen E.; Koehle, Maura

2016-11-23

An important advance in fluid surface control was the amphiphilic surfactant comprised of coupled molecular structures (i.e. hydrophilic and hydrophobic) to reduce surface tension between two distinct fluid phases. However, implementation of simple surfactants has been hindered by the broad range of applications in water containing alkaline earth metals (i.e. hard water), which disrupt surfactant function and require extensive use of undesirable and expensive chelating additives. Here we show that sugar-derived furans can be linked with triglyceride-derived fatty acid chains via Friedel-Crafts acylation within single layer (SPP) zeolite catalysts. These alkylfuran surfactants independently suppress the effects of hard water whilemore » simultaneously permitting broad tunability of size, structure, and function, which can be optimized for superior capability for forming micelles and solubilizing in water.« less

Label-free measurement of the yeast short chain TAG lipase activity by ESI-MS after one-step esterification.

PubMed

Ham, Hye Jin; Seo, Jongcheol; Yoon, Hye-Joo; Shin, Seung Koo

2017-03-01

Triacylglycerol (TAG) lipases hydrolyze ester bonds in TAG and release diacylglycerol (DAG), monoacylglycerol (MAG), and FA. We present a one-step chemical derivatization method for label-free quantification of a mixture of TAG, DAG, and MAG following lipase assay by ESI-MS. Because the ionization efficiencies of TAG, DAG, and MAG are not identical, lipase reaction products, DAG and MAG, are derivatized to TAG species by esterifying their hydroxyl groups using acyl chloride, whose acyl chain contains one less (or one more) -CH 2 group than that of substrate TAG. This resulted in three TAG species that were separated by 14 Da from one another and exhibited similar ion responses representing their molar amounts in the mass spectra. A good linear correlation was observed between peak intensity ratios and molar ratios in calibration curve. This method enables simultaneous quantification of TAG, DAG, and MAG in lipase assay and, in turn, allows stoichiometric determination of the concentrations of FAs released from TAG and DAG separately. By applying this strategy to measure both TAG and DAG lipolytic activities of the yeast Tgl2 lipase, we demonstrated its usefulness in studying enzymatic catalysis, as lipase enzymes often show dissimilar activities toward these lipids. Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.

Detecting and characterizing N-acyl-homoserine lactone signal molecules by thin-layer chromatography

PubMed Central

Shaw, Paul D.; Ping, Gao; Daly, Sean L.; Cha, Chung; Cronan, John E.; Rinehart, Kenneth L.; Farrand, Stephen K.

1997-01-01

Many Gram-negative bacteria regulate gene expression in response to their population size by sensing the level of acyl-homoserine lactone signal molecules which they produce and liberate to the environment. We have developed an assay for these signals that couples separation by thin-layer chromatography with detection using Agrobacterium tumefaciens harboring lacZ fused to a gene that is regulated by autoinduction. With the exception of N-butanoyl-l-homoserine lactone, the reporter detected acyl-homoserine lactones with 3-oxo-, 3-hydroxy-, and 3-unsubstituted side chains of all lengths tested. The intensity of the response was proportional to the amount of the signal molecule chromatographed. Each of the 3-oxo- and the 3-unsubstituted derivatives migrated with a unique mobility. Using the assay, we showed that some bacteria produce as many as five detectable signal molecules. Structures could be assigned tentatively on the basis of mobility and spot shape. The dominant species produced by Pseudomonas syringae pv. tabaci chromatographed with the properties of N-(3-oxohexanoyl)-l-homoserine lactone, a structure that was confirmed by mass spectrometry. An isolate of Pseudomonas fluorescens produced five detectable species, three of which had novel chromatographic properties. These were identified as the 3-hydroxy- forms of N-hexanoyl-, N-octanoyl-, and N-decanoyl-l-homoserine lactone. The assay can be used to screen cultures of bacteria for acyl-homoserine lactones, for quantifying the amounts of these molecules produced, and as an analytical and preparative aid in determining the structures of these signal molecules. PMID:9177164

Acyl-CoA:Lysophosphatidylethanolamine Acyltransferase Activity Regulates Growth of Arabidopsis1

PubMed Central

Jasieniecka-Gazarkiewicz, Katarzyna; Lager, Ida; Carlsson, Anders S.; Gutbrod, Katharina; Peisker, Helga; Dörmann, Peter; Stymne, Sten; Banaś, Antoni

2017-01-01

Arabidopsis (Arabidopsis thaliana) contains two enzymes (encoded by the At1g80950 and At2g45670 genes) preferentially acylating lysophosphatidylethanolamine (LPE) with acyl-coenzyme A (CoA), designated LYSOPHOSPHATIDYLETHANOLAMINE ACYLTRANSFERASE1 (LPEAT1) and LPEAT2. The transfer DNA insertion mutant lpeat2 and the double mutant lpeat1 lpeat2 showed impaired growth, smaller leaves, shorter roots, less seed setting, and reduced lipid content per fresh weight in roots and seeds and large increases in LPE and lysophosphatidylcholine (LPC) contents in leaves. Microsomal preparations from leaves of these mutants showed around 70% decrease in acylation activity of LPE with 16:0-CoA compared with wild-type membranes, whereas the acylation with 18:1-CoA was much less affected, demonstrating that other lysophospholipid acyltransferases than the two LPEATs could acylate LPE. The above-mentioned effects were less pronounced in the single lpeat1 mutant. Overexpression of either LPEAT1 or LPEAT2 under the control of the 35S promotor led to morphological changes opposite to what was seen in the transfer DNA mutants. Acyl specificity studies showed that LPEAT1 utilized 16:0-CoA at the highest rate of 11 tested acyl-CoAs, whereas LPEAT2 utilized 20:0-CoA as the best acyl donor. Both LPEATs could acylate either sn position of ether analogs of LPC. The data show that the activities of LPEAT1 and LPEAT2 are, in a complementary way, involved in growth regulation in Arabidopsis. It is shown that LPEAT activity (especially LPEAT2) is essential for maintaining adequate levels of phosphatidylethanolamine, LPE, and LPC in the cells. PMID:28408542

Binding of the human "electron transferring flavoprotein" (ETF) to the medium chain acyl-CoA dehydrogenase (MCAD) involves an arginine and histidine residue.

PubMed

Parker, Antony R

2003-10-01

The interaction between the "electron transferring flavoprotein" (ETF) and medium chain acyl-CoA dehydrogenase (MCAD) enables successful flavin to flavin electron transfer, crucial for the beta-oxidation of fatty acids. The exact biochemical determinants for ETF binding to MCAD are unknown. Here we show that binding of human ETF, to MCAD, was inhibited by 2,3-butanedione and diethylpyrocarbonate (DEPC) and reversed by incubation with free arginine and hydroxylamine respectively. Spectral analyses of native ETF vs modified ETF suggested that flavin binding was not affected and that the loss of ETF activity with MCAD involved modification of one ETF arginine residue and one ETF histidine residue respectively. MCAD and octanoyl-CoA protected ETF against inactivation by both 2,3-butanedione and DEPC indicating that the arginine and histidine residues are present in or around the MCAD binding site. Comparison of exposed arginine and histidine residues among different ETF species, however, indicates that arginine residues are highly conserved but that histidine residues are not. These results lead us to conclude that this single arginine residue is essential for the binding of ETF to MCAD, but that the single histidine residue, although involved, is not.

Mechanism-based inhibitors of MenE, an acyl-CoA synthetase involved in bacterial menaquinone biosynthesis†

PubMed Central

Lu, Xuequan; Zhang, Huaning; Tonge, Peter J.; Tan, Derek S.

2008-01-01

Menaquinone (vitamin K2) is an essential component of the electron transfer chain in many pathogens, including Mycobacterium tuberculosis and Staphylococcus aureus, and menaquinone biosynthesis is a potential target for antibiotic drug discovery. We report herein a series of mechanism-based inhibitors of MenE, an acyl-CoA synthetase that catalyzes adenylation and thioesterification of o-succinylbenzoic acid (OSB) during menaquinone biosynthesis. The most potent compound inhibits MenE with an IC50 value of 5.7 μM. PMID:18762421

Regioselective Acylation of Diols and Triols: The Cyanide Effect.

PubMed

Peng, Peng; Linseis, Michael; Winter, Rainer F; Schmidt, Richard R

2016-05-11

Central topics of carbohydrate chemistry embrace structural modifications of carbohydrates and oligosaccharide synthesis. Both require regioselectively protected building blocks that are mainly available via indirect multistep procedures. Hence, direct protection methods targeting a specific hydroxy group are demanded. Dual hydrogen bonding will eventually differentiate between differently positioned hydroxy groups. As cyanide is capable of various kinds of hydrogen bonding and as it is a quite strong sterically nondemanding base, regioselective O-acylations should be possible at low temperatures even at sterically congested positions, thus permitting formation and also isolation of the kinetic product. Indeed, 1,2-cis-diols, having an equatorial and an axial hydroxy group, benzoyl cyanide or acetyl cyanide as an acylating agent, and DMAP as a catalyst yield at -78 °C the thermodynamically unfavorable axial O-acylation product; acyl migration is not observed under these conditions. This phenomenon was substantiated with 3,4-O-unproteced galacto- and fucopyranosides and 2,3-O-unprotected mannopyranosides. Even for 3,4,6-O-unprotected galactopyranosides as triols, axial 4-O-acylation is appreciably faster than O-acylation of the primary 6-hydroxy group. The importance of hydrogen bonding for this unusual regioselectivity could be confirmed by NMR studies and DFT calculations, which indicate favorable hydrogen bonding of cyanide to the most acidic axial hydroxy group supported by hydrogen bonding of the equatorial hydroxy group to the axial oxygen. Thus, the "cyanide effect" is due to dual hydrogen bonding of the axial hydroxy group which enhances the nucleophilicity of the respective oxygen atom, permitting an even faster reaction for diols than for mono-ols. In contrast, fluoride as a counterion favors dual hydrogen bonding to both hydroxy groups leading to equatorial O-acylation.

The role of beta-ketoacyl-acyl carrier protein synthase III in the condensation steps of fatty acid biosynthesis in sunflower.

PubMed

González-Mellado, Damián; von Wettstein-Knowles, Penny; Garcés, Rafael; Martínez-Force, Enrique

2010-05-01

The beta-ketoacyl-acyl carrier protein synthase III (KAS III; EC 2.3.1.180) is a condensing enzyme catalyzing the initial step of fatty acid biosynthesis using acetyl-CoA as primer. To determine the mechanisms involved in the biosynthesis of fatty acids in sunflower (Helianthus annuus L.) developing seeds, a cDNA coding for HaKAS III (EF514400) was isolated, cloned and sequenced. Its protein sequence is as much as 72% identical to other KAS III-like ones such as those from Perilla frutescens, Jatropha curcas, Ricinus communis or Cuphea hookeriana. Phylogenetic study of the HaKAS III homologous proteins infers its origin from cyanobacterial ancestors. A genomic DNA gel blot analysis revealed that HaKAS III is a single copy gene. Expression levels of this gene, examined by Q-PCR, revealed higher levels in developing seeds storing oil than in leaves, stems, roots or seedling cotyledons. Heterologous expression of HaKAS III in Escherichia coli altered their fatty acid content and composition implying an interaction of HaKAS III with the bacterial FAS complex. Testing purified HaKAS III recombinant protein by adding to a reconstituted E. coli FAS system lacking condensation activity revealed a novel substrate specificity. In contrast to all hitherto characterized plant KAS IIIs, the activities of which are limited to the first cycles of intraplastidial fatty acid biosynthesis yielding C6 chains, HaKAS III participates in at least four cycles resulting in C10 chains.

Unexpected Hydrolytic Instability of N-Acylated Amino Acid Amides and Peptides

PubMed Central

2015-01-01

Remote amide bonds in simple N-acyl amino acid amide or peptide derivatives 1 can be surprisingly unstable hydrolytically, affording, in solution, variable amounts of 3 under mild acidic conditions, such as trifluoroacetic acid/water mixtures at room temperature. This observation has important implications for the synthesis of this class of compounds, which includes N-terminal-acylated peptides. We describe the factors contributing to this instability and how to predict and control it. The instability is a function of the remote acyl group, R2CO, four bonds away from the site of hydrolysis. Electron-rich acyl R2 groups accelerate this reaction. In the case of acyl groups derived from substituted aromatic carboxylic acids, the acceleration is predictable from the substituent’s Hammett σ value. N-Acyl dipeptides are also hydrolyzed under typical cleavage conditions. This suggests that unwanted peptide truncation may occur during synthesis or prolonged standing in solution when dipeptides or longer peptides are acylated on the N-terminus with electron-rich aromatic groups. When amide hydrolysis is an undesired secondary reaction, as can be the case in the trifluoroacetic acid-catalyzed cleavage of amino acid amide or peptide derivatives 1 from solid-phase resins, conditions are provided to minimize that hydrolysis. PMID:24617596

Enzymatic hydrolysis of short-chain lecithin/long-chain phospholipid unilamellar vesicles: sensitivity of phospholipases to matrix phase state.

PubMed

Gabriel, N E; Agman, N V; Roberts, M F

1987-11-17

Short-chain lecithin/long-chain phospholipid unilamellar vesicles (SLUVs), unlike pure long-chain lecithin vesicles, are excellent substrates for water-soluble phospholipases. Hemolysis assays show that greater than 99.5% of the short-chain lecithin is partitioned in the bilayer. In these binary component vesicles, the short-chain species is the preferred substrate, while the long-chain phospholipid can be treated as an inhibitor (phospholipase C) or poor substrate (phospholipase A2). For phospholipase C Bacillus cereus, apparent Km and Vmax values show that bilayer-solubilized diheptanoylphosphatidylcholine (diheptanoyl-PC) is nearly as good a substrate as pure micellar diheptanoyl-PC, although the extent of short-chain lecithin hydrolysis depends on the phase state of the long-chain lipid. For phospholipase A2 Naja naja naja, both Km and Vmax values show a greater range: in a gel-state matrix, diheptanoyl-PC is hydrolyzed with micellelike kinetic parameters; in a liquid-crystalline matrix, the short-chain lecithin becomes comparable to the long-chain component. Both enzymes also show an anomalous increase in specific activity toward diheptanoyl-PC around the phase transition temperature of the long-chain phospholipid. Since the short-chain lecithin does not exhibit a phase transition, this must reflect fluctuations in head-group area or vertical motions of the short-chain lecithin caused by surrounding long-chain lecithin molecules. These results are discussed in terms of a specific model for SLUV hydrolysis and a general explanation for the "interfacial activation" observed with water-soluble phospholipases.

New acylated clionasterol glycosides from Valeriana officinalis.

PubMed

Pullela, Srinivas V; Choi, Young Whan; Khan, Shabana I; Khan, Ikhlas A

2005-10-01

The chloroform extract of Valeriana officinalis led to the isolation of clionasterol-3-O-beta-D-glucopyranoside and a mixture of 6'-O-acyl-beta-D-glucosyl-clionasterols. The acyl moieties were identified as hexadecanoyl, 8 E,11 E-octadecadienoyl and 14-methylpentadecanoyl by alkaline hydrolysis followed by GC-MS analysis. The isolated compounds did not exhibit any anti-inflammatory, anticancer or cytotoxic activity when tested in a variety of in vitro cell-based assays.

Atomic Chain Electronics

NASA Technical Reports Server (NTRS)

Yamada, Toshishige; Saini, Subhash (Technical Monitor)

1998-01-01

Adatom chains, precise structures artificially created on an atomically regulated surface, are the smallest possible candidates for future nanoelectronics. Since all the devices are created by combining adatom chains precisely prepared with atomic precision, device characteristics are predictable, and free from deviations due to accidental structural defects. In this atomic dimension, however, an analogy to the current semiconductor devices may not work. For example, Si structures are not always semiconducting. Adatom states do not always localize at the substrate surface when adatoms form chemical bonds to the substrate atoms. Transport properties are often determined for the entire system of the chain and electrodes, and not for chains only. These fundamental issues are discussed, which will be useful for future device considerations.

In Vitro Enzymatic Synthesis of New Penicillins Containing Keto Acids as Side Chains

PubMed Central

Ferrero, Miguel A.; Reglero, Angel; Martínez-Blanco, Honorina; Fernández-Valverde, Martiniano; Luengo, Jose M.

1991-01-01

Seven different penicillins containing α-ketobutyric, β-ketobutyric, γ-ketovaleric, α-ketohexanoic, δ-ketohexanoic, ε-ketoheptanoic, and α-ketooctanoic acids as side chains have been synthesized in vitro by incubating the enzymes phenylacetyl coenzyme A (CoA) ligase from Pseudomonas putida and acyl-CoA:6-aminopenicillanic acid acyltransferase from Penicillium chrysogenum with CoA, ATP, Mg2+, dithiothreitol, 6-aminopenicillanic acid, and the corresponding side chain precursor. PMID:1952871

Activity of 3-Ketosteroid 9α-Hydroxylase (KshAB) Indicates Cholesterol Side Chain and Ring Degradation Occur Simultaneously in Mycobacterium tuberculosis*

PubMed Central

Capyk, Jenna K.; Casabon, Israël; Gruninger, Robert; Strynadka, Natalie C.; Eltis, Lindsay D.

2011-01-01

Mycobacterium tuberculosis (Mtb), a significant global pathogen, contains a cholesterol catabolic pathway. Although the precise role of cholesterol catabolism in Mtb remains unclear, the Rieske monooxygenase in this pathway, 3-ketosteroid 9α-hydroxylase (KshAB), has been identified as a virulence factor. To investigate the physiological substrate of KshAB, a rhodococcal acyl-CoA synthetase was used to produce the coenzyme A thioesters of two cholesterol derivatives: 3-oxo-23,24-bisnorchol-4-en-22-oic acid (forming 4-BNC-CoA) and 3-oxo-23,24-bisnorchola-1,4-dien-22-oic acid (forming 1,4-BNC-CoA). The apparent specificity constant (kcat/Km) of KshAB for the CoA thioester substrates was 20–30 times that for the corresponding 17-keto compounds previously proposed as physiological substrates. The apparent KmO2 was 90 ± 10 μm in the presence of 1,4-BNC-CoA, consistent with the value for two other cholesterol catabolic oxygenases. The Δ1 ketosteroid dehydrogenase KstD acted with KshAB to cleave steroid ring B with a specific activity eight times greater for a CoA thioester than the corresponding ketone. Finally, modeling 1,4-BNC-CoA into the KshA crystal structure suggested that the CoA moiety binds in a pocket at the mouth of the active site channel and could contribute to substrate specificity. These results indicate that the physiological substrates of KshAB are CoA thioester intermediates of cholesterol side chain degradation and that side chain and ring degradation occur concurrently in Mtb. This finding has implications for steroid metabolites potentially released by the pathogen during infection and for the design of inhibitors for cholesterol-degrading enzymes. The methodologies and rhodococcal enzymes used to generate thioesters will facilitate the further study of cholesterol catabolism. PMID:21987574

Activity of 3-ketosteroid 9α-hydroxylase (KshAB) indicates cholesterol side chain and ring degradation occur simultaneously in Mycobacterium tuberculosis.

PubMed

Capyk, Jenna K; Casabon, Israël; Gruninger, Robert; Strynadka, Natalie C; Eltis, Lindsay D

2011-11-25

Mycobacterium tuberculosis (Mtb), a significant global pathogen, contains a cholesterol catabolic pathway. Although the precise role of cholesterol catabolism in Mtb remains unclear, the Rieske monooxygenase in this pathway, 3-ketosteroid 9α-hydroxylase (KshAB), has been identified as a virulence factor. To investigate the physiological substrate of KshAB, a rhodococcal acyl-CoA synthetase was used to produce the coenzyme A thioesters of two cholesterol derivatives: 3-oxo-23,24-bisnorchol-4-en-22-oic acid (forming 4-BNC-CoA) and 3-oxo-23,24-bisnorchola-1,4-dien-22-oic acid (forming 1,4-BNC-CoA). The apparent specificity constant (k(cat)/K(m)) of KshAB for the CoA thioester substrates was 20-30 times that for the corresponding 17-keto compounds previously proposed as physiological substrates. The apparent K(m)(O(2)) was 90 ± 10 μM in the presence of 1,4-BNC-CoA, consistent with the value for two other cholesterol catabolic oxygenases. The Δ(1) ketosteroid dehydrogenase KstD acted with KshAB to cleave steroid ring B with a specific activity eight times greater for a CoA thioester than the corresponding ketone. Finally, modeling 1,4-BNC-CoA into the KshA crystal structure suggested that the CoA moiety binds in a pocket at the mouth of the active site channel and could contribute to substrate specificity. These results indicate that the physiological substrates of KshAB are CoA thioester intermediates of cholesterol side chain degradation and that side chain and ring degradation occur concurrently in Mtb. This finding has implications for steroid metabolites potentially released by the pathogen during infection and for the design of inhibitors for cholesterol-degrading enzymes. The methodologies and rhodococcal enzymes used to generate thioesters will facilitate the further study of cholesterol catabolism.

Comparative study on digestive lipase activities on the self emulsifying excipient Labrasol, medium chain glycerides and PEG esters.

PubMed

Fernandez, Sylvie; Jannin, Vincent; Rodier, Jean-David; Ritter, Nicolas; Mahler, Bruno; Carrière, Frédéric

2007-05-01

Labrasol is a lipid-based self-emulsifying excipient used in the preparation of lipophilic drugs intended for oral delivery. It is mainly composed of PEG esters and glycerides with medium acyl chains, which are potential substrates for digestive lipases. The hydrolysis of Labrasol by porcine pancreatic extracts, human pancreatic juice and several purified digestive lipases was investigated in the present study. Classical human pancreatic lipase (HPL) and porcine pancreatic lipase, which are the main lipases involved in the digestion of dietary triglycerides, showed very low levels of activity on the entire Labrasol excipient as well as on separated fractions of glycerides and PEG esters. On the other hand, gastric lipase, pancreatic lipase-related protein 2 (PLRP2) and carboxyl ester hydrolase (CEH) showed high specific activities on Labrasol. These lipases were found to hydrolyze the main components of Labrasol (PEG esters and monoglycerides) used as individual substrates, whereas these esters were found to be poor substrates for HPL. The lipolytic activity of pancreatic extracts and human pancreatic juice on Labrasol(R) is therefore mainly due to the combined action of CEH and PLRP2. These two pancreatic enzymes, together with gastric lipase, are probably the main enzymes involved in the in vivo lipolysis of Labrasol taken orally.

Domain analysis of 3 Keto Acyl-CoA synthase for structural variations in Vitis vinifera and Oryza brachyantha using comparative modelling.

PubMed

Sagar, Mamta; Pandey, Neetesh; Qamar, Naseha; Singh, Brijendra; Shukla, Akanksha

2015-03-01

The long chain fatty acids incorporated into plant lipids are derived from the iterative addition of C2 units which is provided by malonyl-CoA to an acyl-CoA after interactions with 3-ketoacyl-CoA synthase (KCS), found in several plants. This study provides functional characterization of three 3 ketoacyl CoA synthase like proteins in Vitis vinifera (one) and Oryza brachyantha (two proteins). Sequence analysis reveals that protein of Oryza brachyantha shows 96% similarity to a hypothetical protein in Sorghum bicolor; total 11 homologs were predicted in Sorghum bicolor. Conserved domain prediction confirm the presence of FAE1/Type III polyketide synthase-like protein, Thiolase-like, subgroup; Thiolase-like and 3-Oxoacyl-ACP synthase III, C-terminal and chalcone synthase like domain but very long chain 3-keto acyl CoA domain is absent. All three proteins were found to have Chalcone and stilbene synthases C terminal domain which is similar to domain of thiolase and β keto acyl synthase. Its N terminal domain is absent in J3M9Z7 protein of Oryza brachyantha and F6HH63 protein of Vitis vinifera. Differences in N-terminal domain is responsible for distinguish activity. The J3MF16 protein of Oryza brachyantha contains N terminal domain and C terminal domain and characterized using annotation of these domains. Domains Gcs (streptomyces coelicolor) and Chalcone-stilbene synthases (KAS) in 2-pyrone synthase (Gerbera hybrid) and chalcone synthase 2 (Medicago sativa) were found to be present in three proteins. This similarity points toward anthocyanin biosynthetic process. Similarity to chalcone synthase 2 reveals its possible role in Naringenine and Chalcone synthase like activity. In 3 keto acyl CoA synthase of Oryza brachyantha. Active site residues C-240, H-407, N-447 are present in J3MF16 protein that are common in these three protein at different positions. Structural variations among dimer interface, product binding site, malonyl-CoA binding sites, were predicted in

Crystal structure of delta9 stearoyl-acyl carrier protein desaturase from castor seed and its relationship to other di-iron proteins.

PubMed Central

Lindqvist, Y; Huang, W; Schneider, G; Shanklin, J

1996-01-01

The three-dimensional structure of recombinant homodimeric delta9 stearoyl-acyl carrier protein desaturase, the archetype of the soluble plant fatty acid desaturases that convert saturated to unsaturated fatty acids, has been determined by protein crystallographic methods to a resolution of 2.4 angstroms. The structure was solved by a combination of single isomorphous replacement, anomalous contribution from the iron atoms to the native diffraction data and 6-fold non-crystallographic symmetry averaging. The 363 amino acid monomer consists of a single domain of 11 alpha-helices. Nine of these form an antiparallel helix bundle. The enzyme subunit contains a di-iron centre, with ligands from four of the alpha-helices in the helix bundle. The iron ions are bound in a highly symmetric environment, with one of the irons forming interactions with the side chains of E196 and H232 and the second iron with the side chains of E105 and H146. Two additional glutamic acid side chains, from E143 and E229, are within coordination distance to both iron ions. A water molecule is found within the second coordination sphere from the iron atoms. The lack of electron density corresponding to a mu-oxo bridge, and the long (4.2 angstroms) distance between the iron ions suggests that this probably represents the diferrous form of the enzyme. A deep channel which probably binds the fatty acid extends from the surface into the interior of the enzyme. Modelling of the substrate, stearic acid, into this channel places the delta9 carbon atom in the vicinity of one of the iron ions. Images PMID:8861937

Crystal structure of delta9 stearoyl-acyl carrier protein desaturase from castor seed and its relationship to other di-iron proteins.

PubMed

Lindqvist, Y; Huang, W; Schneider, G; Shanklin, J

1996-08-15

The three-dimensional structure of recombinant homodimeric delta9 stearoyl-acyl carrier protein desaturase, the archetype of the soluble plant fatty acid desaturases that convert saturated to unsaturated fatty acids, has been determined by protein crystallographic methods to a resolution of 2.4 angstroms. The structure was solved by a combination of single isomorphous replacement, anomalous contribution from the iron atoms to the native diffraction data and 6-fold non-crystallographic symmetry averaging. The 363 amino acid monomer consists of a single domain of 11 alpha-helices. Nine of these form an antiparallel helix bundle. The enzyme subunit contains a di-iron centre, with ligands from four of the alpha-helices in the helix bundle. The iron ions are bound in a highly symmetric environment, with one of the irons forming interactions with the side chains of E196 and H232 and the second iron with the side chains of E105 and H146. Two additional glutamic acid side chains, from E143 and E229, are within coordination distance to both iron ions. A water molecule is found within the second coordination sphere from the iron atoms. The lack of electron density corresponding to a mu-oxo bridge, and the long (4.2 angstroms) distance between the iron ions suggests that this probably represents the diferrous form of the enzyme. A deep channel which probably binds the fatty acid extends from the surface into the interior of the enzyme. Modelling of the substrate, stearic acid, into this channel places the delta9 carbon atom in the vicinity of one of the iron ions.

Half-of-the-Sites Reactivity of the Castor Δ9-18:0-Acyl Carrier Protein Desaturase.

PubMed

Liu, Qin; Chai, Jin; Moche, Martin; Guy, Jodie; Lindqvist, Ylva; Shanklin, John

2015-09-01

Fatty acid desaturases regulate the unsaturation status of cellular lipids. They comprise two distinct evolutionary lineages, a soluble class found in the plastids of higher plants and an integral membrane class found in plants, yeast (Saccharomyces cerevisiae), animals, and bacteria. Both classes exhibit a dimeric quaternary structure. Here, we test the functional significance of dimeric organization of the soluble castor Δ9-18:0-acyl carrier protein desaturase, specifically, the hypothesis that the enzyme uses an alternating subunit half-of-the-sites reactivity mechanism whereby substrate binding to one subunit is coordinated with product release from the other subunit. Using a fluorescence resonance energy transfer assay, we demonstrated that dimers stably associate at concentrations typical of desaturase assays. An active site mutant T104K/S202E, designed to occlude the substrate binding cavity, was expressed, purified, and its properties validated by x-ray crystallography, size exclusion chromatography, and activity assay. Heterodimers comprising distinctly tagged wild-type and inactive mutant subunits were purified at 1:1 stoichiometry. Despite having only one-half the number of active sites, purified heterodimers exhibit equivalent activity to wild-type homodimers, consistent with half-of-the-sites reactivity. However, because multiple rounds of turnover were observed, we conclude that substrate binding to one subunit is not required to facilitate product release from the second subunit. The observed half-of-the-sites reactivity could potentially buffer desaturase activity from oxidative inactivation. That soluble desaturases require only one active subunit per dimer for full activity represents a mechanistic difference from the membrane class of desaturases such as the Δ9-acyl-CoA, Ole1p, from yeast, which requires two catalytically competent subunits for activity. © 2015 American Society of Plant Biologists. All Rights Reserved.

Acyl-protein thioesterase 2 catalyzes the deacylation of peripheral membrane-associated GAP-43.

PubMed

Tomatis, Vanesa M; Trenchi, Alejandra; Gomez, Guillermo A; Daniotti, Jose L

2010-11-30

An acylation/deacylation cycle is necessary to maintain the steady-state subcellular distribution and biological activity of S-acylated peripheral proteins. Despite the progress that has been made in identifying and characterizing palmitoyltransferases (PATs), much less is known about the thioesterases involved in protein deacylation. In this work, we investigated the deacylation of growth-associated protein-43 (GAP-43), a dually acylated protein at cysteine residues 3 and 4. Using fluorescent fusion constructs, we measured in vivo the rate of deacylation of GAP-43 and its single acylated mutants in Chinese hamster ovary (CHO)-K1 and human HeLa cells. Biochemical and live cell imaging experiments demonstrated that single acylated mutants were completely deacylated with similar kinetic in both cell types. By RT-PCR we observed that acyl-protein thioesterase 1 (APT-1), the only bona fide thioesterase shown to mediate deacylation in vivo, is expressed in HeLa cells, but not in CHO-K1 cells. However, APT-1 overexpression neither increased the deacylation rate of single acylated GAP-43 nor affected the steady-state subcellular distribution of dually acylated GAP-43 both in CHO-K1 and HeLa cells, indicating that GAP-43 deacylation is not mediated by APT-1. Accordingly, we performed a bioinformatic search to identify putative candidates with acyl-protein thioesterase activity. Among several candidates, we found that APT-2 is expressed both in CHO-K1 and HeLa cells and its overexpression increased the deacylation rate of single acylated GAP-43 and affected the steady-state localization of diacylated GAP-43 and H-Ras. Thus, the results demonstrate that APT-2 is the protein thioesterase involved in the acylation/deacylation cycle operating in GAP-43 subcellular distribution.

KAS IV: a 3-ketoacyl-ACP synthase from Cuphea sp. is a medium chain specific condensing enzyme.

PubMed

Dehesh, K; Edwards, P; Fillatti, J; Slabaugh, M; Byrne, J

1998-08-01

cDNA clones encoding a novel 3-ketoacyl-ACP synthase (KAS) have been isolated from Cuphea. The amino acid sequence of this enzyme is different from the previously characterized classes of KASs, designated KAS I and III, and similar to those designated as KAS II. To define the acyl chain specificity of this enzyme, we generated transgenic Brassica plants over-expressing the cDNA encoded protein in a seed specific manner. Expression of this enzyme in transgenic Brassica seeds which normally do not produce medium chain fatty acids does not result in any detectable modification of the fatty acid profile. However, co-expression of the Cuphea KAS with medium chain specific thioesterases, capable of production of either 12:0 or 8:0/10:0 fatty acids in seed oil, strongly enhances the levels of these medium chain fatty acids as compared with seed oil of plants expressing the thioesterases alone. By contrast, co-expression of the Cuphea KAS along with an 18:0/18.1-ACP thioesterase does not result in any detectable modification of the fatty acids. These data indicate that the Cuphea KAS reported here has a different acyl-chain specificity to the previously characterized KAS I, II and III. Therefore, we designate this enzyme KAS IV, a medium chain specific condensing enzyme.

Dissecting the total transition state stabilization provided by amino acid side chains at orotidine 5'-monophosphate decarboxylase: a two-part substrate approach.

PubMed

Barnett, Shonoi A; Amyes, Tina L; Wood, Bryant M; Gerlt, John A; Richard, John P

2008-07-29

Kinetic analysis of decarboxylation catalyzed by S154A, Q215A, and S154A/Q215A mutant yeast orotidine 5'-monophosphate decarboxylases with orotidine 5'-monophosphate (OMP) and with a truncated nucleoside substrate (EO) activated by phosphite dianion shows (1) the side chain of Ser-154 stabilizes the transition state through interactions with the pyrimidine rings of OMP or EO, (2) the side chain of Gln-215 interacts with the phosphodianion group of OMP or with phosphite dianion, and (3) the interloop hydrogen bond between the side chains of Ser-154 and Gln-215 orients the amide side chain of Gln-215 to interact with the phosphodianion group of OMP or with phosphite dianion.

The modules of trans-acyltransferase assembly lines redefined with a central acyl carrier protein.

PubMed

Vander Wood, Drew A; Keatinge-Clay, Adrian T

2018-06-01

Here, the term "module" is redefined for trans-acyltransferase (trans-AT) assembly lines to agree with how its domains cooperate and evolutionarily co-migrate. The key domain in both the polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) modules of assembly lines is the acyl carrier protein (ACP). ACPs not only relay growing acyl chains through the assembly line but also collaborate with enzymes in modules, both in cis and in trans, to add a specific chemical moiety. A ketosynthase (KS) downstream of ACP often plays the role of gatekeeper, ensuring that only a single intermediate generated by the enzymes of a module is passed downstream. Bioinformatic analysis of 526 ACPs from 33 characterized trans-AT assembly lines reveals ACPs from the same module type generally clade together, reflective of the co-evolution of these domains with their cognate enzymes. While KSs downstream of ACPs from the same module type generally also clade together, KSs upstream of ACPs do not-in disagreement with the traditional definition of a module. Beyond nomenclature, the presented analysis impacts our understanding of module function, the evolution of assembly lines, pathway prediction, and assembly line engineering. © 2018 Wiley Periodicals, Inc.

Acyl Meldrum's acid derivatives: application in organic synthesis

NASA Astrophysics Data System (ADS)

Janikowska, K.; Rachoń, J.; Makowiec, S.

2014-07-01

This review is focused on an important class of Meldrum's acid derivatives commonly known as acyl Meldrum's acids. The preparation methods of these compounds are considered including the recently proposed and rather rarely used ones. The chemical properties of acyl Meldrum's acids are described in detail, including thermal stability and reactions with various nucleophiles. The possible mechanisms of these transformations are analyzed. The bibliography includes 134 references.

Cloning and characterization of unusual fatty acid desaturases from Anemone leveillei: identification of an acyl-coenzyme A C20 Delta5-desaturase responsible for the synthesis of sciadonic acid.

PubMed

Sayanova, Olga; Haslam, Richard; Venegas Caleron, Monica; Napier, Johnathan A

2007-05-01

The seed oil of Anemone leveillei contains significant amounts of sciadonic acid (20:3Delta(5,11,14); SA), an unusual non-methylene-interrupted fatty acid with pharmaceutical potential similar to arachidonic acid. Two candidate cDNAs (AL10 and AL21) for the C(20) Delta(5cis)-desaturase from developing seeds of A. leveillei were functionally characterized in transgenic Arabidopsis (Arabidopsis thaliana) plants. The open reading frames of both Delta(5)-desaturases showed some similarity to presumptive acyl-coenzyme A (CoA) desaturases found in animals and plants. When expressed in transgenic Arabidopsis, AL21 showed a broad range of substrate specificity, utilizing both saturated (16:0 and 18:0) and unsaturated (18:2, n-6 and 18:3, n-3) substrates. In contrast, AL10 did not show any activity in wild-type Arabidopsis. Coexpression of AL10 or AL21 with a C(18) Delta(9)-elongase in transgenic Arabidopsis plants resulted in the production of SA and juniperonic fatty acid (20:4Delta(5,11,14,17)). Thus, AL10 acted only on C(20) polyunsaturated fatty acids in a manner analogous to "front-end" desaturases. However, neither AL10 nor AL21 contain the cytochrome b(5) domain normally present in this class of enzymes. Acyl-CoA profiling of transgenic Arabidopsis plants and developing A. leveillei seeds revealed significant accumulation of Delta(5)-unsaturated fatty acids as acyl-CoAs compared to the accumulation of these fatty acids in total lipids. Positional analysis of triacylglycerols of A. leveillei seeds showed that Delta(5)-desaturated fatty acids were present in both sn-2 and sn-1 + sn-3 positions, although the majority of 16:1Delta(5), 18:1Delta(5), and SA was present at the sn-2 position. Our data provide biochemical evidence for the A. leveillei Delta(5)-desaturases using acyl-CoA substrates.

Organometallic macromolecules with piano stool coordination repeating units: chain configuration and stimulated solution behaviour.

PubMed

Cao, Kai; Ward, Jonathan; Amos, Ryan C; Jeong, Moon Gon; Kim, Kyoung Taek; Gauthier, Mario; Foucher, Daniel; Wang, Xiaosong

2014-09-11

Theoretical calculations illustrate that organometallic macromolecules with piano stool coordination repeating units (Fe-acyl complex) adopt linear chain configuration with a P-Fe-C backbone surrounded by aromatic groups. The macromolecules show molecular weight-dependent and temperature stimulated solution behaviour in DMSO.

Regioselective and stereospecific acylation across oxirane- and silyloxy systems as a novel strategy to the synthesis of enantiomerically pure mono-, di- and triglycerides.

PubMed

Stamatov, Stephan D; Stawinski, Jacek

2007-12-07

A trifluoroacetate-catalyzed opening of the oxirane ring of glycidyl derivatives bearing allylic acyl or alkyl functionalities with trifluoroacetic anhydride (TFAA), provides an efficient entry to configurationally homogeneous 1(3)-acyl- or 1(3)-O-alkyl-sn-glycerols. Selective introduction of tert-butyldimethylsilyl- (TBDMS), or triisopropylsilyl- (TIPS) transient protections at the terminal sites within these key intermediates secures 1(3)-acyl- or 1(3)-O-alkyl-3(1)-O-TBDMS (or TIPS)-sn-glycerols as general bifunctional precursors to 1,2(2,3)-diacyl-, 1(3)-O-alkyl-2-acyl- and 1,3-diacyl-sn-glycerols and hence triester isosters. Incorporation of a requisite acyl residue at the central carbon of the silylated synthons with a subsequent Et(3)N.3HF-promoted, direct trichloroacetylation across the siloxy system by trichloroacetic anhydride (TCAA), followed by cleavage of the trichloroacetyl group, affords the respective 1,2(2,3)-diacyl- or 1(3)-O-alkyl-2-acyl-sn-glycerols. Alternatively, a reaction sequence involving: (i) attachment of a trichloroacetyl fragment at the stereogenic C2-centre of the monosilylated glycerides; (ii) replacement of the silyl moiety by a short- or long-chain carboxylic acid residue by means of the acylating agent: tetra-n-butylammonium bromide (TBABr)-carboxylic acid anhydride (CAA)-trimethylsilyl bromide (TMSBr); and (iii) removal of the trichloroacetyl replacement, provides pure 1,3-diacyl-sn-glycerols. The TBABr-CAA-TMSBr reagent system allows also a one-step conversion of 1,2-diacylglycerol silyl ethers into homochiral triglycerides with predefined asymmetry and degree of unsaturation. These compounds can also be accessed via a two-step one-pot approach where the trichloroacetyl derivatives of 1,2(2,3)- or 1,3-diacyl-sn-glycerols serve as triester building blocks for establishing the third ester bond at preselected C3(1)- or C2-positions within the glycerol skeleton at the very last synthetic stage. In all instances, the target compounds

Treatment of cardiomyopathy and rhabdomyolysis in long-chain fat oxidation disorders using an anaplerotic odd-chain triglyceride

PubMed Central

Roe, Charles R.; Sweetman, Lawrence; Roe, Diane S.; David, France; Brunengraber, Henri

2002-01-01

The current dietary treatment of long-chain fatty acid oxidation defects (high carbohydrate with medium-even-chain triglycerides and reduced amounts of long-chain fats) fails, in many cases, to prevent cardiomyopathy, rhabdomyolysis, and muscle weakness. We hypothesized that the apparent defect in energy production results from a depletion of the catalytic intermediates of the citric acid cycle via leakage through cell membranes (cataplerosis). We further hypothesized that replacing dietary medium-even-chain fatty acids (precursors of acetyl-CoA) by medium-odd-chain fatty acids (precursors of acetyl-CoA and anaplerotic propionyl-CoA) would restore energy production and improve cardiac and skeletal muscle function. We fed subjects with long-chain defects a controlled diet in which the fat component was switched from medium-even-chain triglycerides to triheptanoin. In three patients with very-long-chain acyl-CoA dehydrogenase deficiency, this treatment led rapidly to clinical improvement that included the permanent disappearance of chronic cardiomyopathy, rhabdomyolysis, and muscle weakness (for more than 2 years in one child), and of rhabdomyolysis and weakness in the others. There was no evidence of propionyl overload in these patients. The treatment has been well tolerated for up to 26 months and opens new avenues for the management of patients with mitochondrial fat oxidation disorders. PMID:12122118

Rapid Acyl-Homoserine Lactone Quorum Signal Biodegradation in Diverse Soils†

PubMed Central

Wang, Ya-Juan; Leadbetter, Jared Renton

2005-01-01

Signal degradation impacts all communications. Although acyl-homoserine lactone (acyl-HSL) quorum-sensing signals are known to be degraded by defined laboratory cultures, little is known about their stability in nature. Here, we show that acyl-HSLs are biodegraded in soils sampled from diverse U.S. sites and by termite hindgut contents. When amended to samples at physiologically relevant concentrations, 14C-labeled acyl-HSLs were mineralized to 14CO2 rapidly and, at most sites examined, without lag. A lag-free turf soil activity was characterized in further detail. Heating or irradiation of the soil prior to the addition of radiolabel abolished mineralization, whereas protein synthesis inhibitors did not. Mineralization exhibited an apparent Km of 1.5 μM acyl-HSL, ca. 1,000-fold lower than that reported for a purified acyl-HSL lactonase. Under optimal conditions, acyl-HSL degradation proceeded at a rate of 13.4 nmol · h−1 · g of fresh weight soil−1. Bioassays established that the final extent of signal inactivation was greater than for its full conversion to CO2 but that the two processes were well coupled kinetically. A most probable number of 4.6 × 105 cells · g of turf soil−1 degraded physiologically relevant amounts of hexanoyl-[1-14C]HSL to 14CO2. It would take chemical lactonolysis months to match the level of signal decay achieved in days by the observed biological activity. Rapid decay might serve either to quiet signal cross talk that might otherwise occur between spatially separated microbial aggregates or as a full system reset. Depending on the context, biological signal decay might either promote or complicate cellular communications and the accuracy of population density-based controls on gene expression in species-rich ecosystems. PMID:15746331

An annotated database of Arabidopsis mutants of acyl lipid metabolism

DOE PAGES

McGlew, Kathleen; Shaw, Vincent; Zhang, Meng; ...

2014-12-10

Mutants have played a fundamental role in gene discovery and in understanding the function of genes involved in plant acyl lipid metabolism. The first mutant in Arabidopsis lipid metabolism ( fad4) was described in 1985. Since that time, characterization of mutants in more than 280 genes associated with acyl lipid metabolism has been reported. This review provides a brief background and history on identification of mutants in acyl lipid metabolism, an analysis of the distribution of mutants in different areas of acyl lipid metabolism and presents an annotated database (ARALIPmutantDB) of these mutants. The database provides information on the phenotypesmore » of mutants, pathways and enzymes/proteins associated with the mutants, and allows rapid access via hyperlinks to summaries of information about each mutant and to literature that provides information on the lipid composition of the mutants. Mutants for at least 30 % of the genes in the database have multiple names, which have been compiled here to reduce ambiguities in searches for information. Furthermore, the database should also provide a tool for exploring the relationships between mutants in acyl lipid-related genes and their lipid phenotypes and point to opportunities for further research.« less

Structure of Mycobacterium tuberculosis mtFabD, a malonyl-CoA:acyl carrier protein transacylase (MCAT)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ghadbane, Hemza; Brown, Alistair K.; Kremer, Laurent

2007-10-01

Binding of Ni{sup 2+} ions to the uncleaved affinity tag facilitated de novo phasing of the crystal structure of M. tuberculosis mtFabD to 3.0 Å resolution. Mycobacteria display a unique and unusual cell-wall architecture, central to which is the membrane-proximal mycolyl-arabinogalactan-peptidoglycan core (mAGP). The biosynthesis of mycolic acids, which form the outermost layer of the mAGP core, involves malonyl-CoA:acyl carrier protein transacylase (MCAT). This essential enzyme catalyses the transfer of malonyl from coenzyme A to acyl carrier protein AcpM, thus feeding these two-carbon units into the chain-elongation cycle of the type II fatty-acid synthase. The crystal structure of M. tuberculosismore » mtFabD, the mycobacterial MCAT, has been determined to 3.0 Å resolution by multi-wavelength anomalous dispersion. Phasing was facilitated by Ni{sup 2+} ions bound to the 20-residue N-terminal affinity tag, which packed between the two independent copies of mtFabD.« less

Characterization of the "Escherichia Coli" Acyl Carrier Protein Phosphodiesterase

ERIC Educational Resources Information Center

Thomas, Jacob

2009-01-01

Acyl carrier protein (ACP) is a small essential protein that functions as a carrier of the acyl intermediates of fatty acid synthesis. ACP requires the posttranslational attachment of a 4'phosphopantetheine functional group, derived from CoA, in order to perform its metabolic function. A Mn[superscript 2+] dependent enzymatic activity that removes…

Mechanism of MenE Inhibition by Acyl-Adenylate Analogues and Discovery of Novel Antibacterial Agents

PubMed Central

Sharma, Indrajeet; Lavaud, Lubens J.; Ngo, Stephen C.; Shek, Roger; Rajashankar, Kanagalaghatta R.; French, Jarrod B.; Tan, Derek S.; Tonge, Peter J.

2015-01-01

MenE is an o-succinylbenzoyl-CoA (OSB-CoA) synthetase in the bacterial menaquinone biosynthesis pathway and is a promising target for the development of novel antibacterial agents. The enzyme catalyzes CoA ligation via an acyl-adenylate intermediate, and we have previously reported tight-binding inhibitors of MenE based on stable acyl-sulfonyladenosine analogues of this intermediate, including OSB-AMS (1) which has an IC50 value of ≤ 25 nM for the Escherichia coli MenE. Herein, we show that OSB-AMS reduces menaquinone levels in S. aureus, consistent with its proposed mechanism of action, despite the observation that the antibacterial activity of OSB-AMS is ~1000-fold lower than the IC50 for enzyme inhibition. To inform the synthesis of MenE inhibitors with improved antibacterial activity, we have undertaken a structure–activity relationship (SAR) study stimulated by the knowledge that OSB-AMS can adopt two isomeric forms in which the OSB side chain exists either as an open-chain keto acid or a cyclic lactol. These studies revealed that negatively charged analogues of the keto-acid form bind, while neutral analogues do not, consistent with the hypothesis that the negatively-charged keto-acid form of OSB-AMS is the active isomer. X-ray crystallography and site-directed mutagenesis confirm the importance of a conserved arginine for binding the OSB carboxylate. Although most lactol isomers tested were inactive, a novel difluoroindanediol inhibitor (11) with improved antibacterial activity was discovered, providing a pathway toward the development of optimized MenE inhibitors in the future. PMID:26394156

Mechanism of MenE inhibition by acyl-adenylate analogues and discovery of novel antibacterial agents.

PubMed

Matarlo, Joe S; Evans, Christopher E; Sharma, Indrajeet; Lavaud, Lubens J; Ngo, Stephen C; Shek, Roger; Rajashankar, Kanagalaghatta R; French, Jarrod B; Tan, Derek S; Tonge, Peter J

2015-10-27

MenE is an o-succinylbenzoyl-CoA (OSB-CoA) synthetase in the bacterial menaquinone biosynthesis pathway and is a promising target for the development of novel antibacterial agents. The enzyme catalyzes CoA ligation via an acyl-adenylate intermediate, and we have previously reported tight-binding inhibitors of MenE based on stable acyl-sulfonyladenosine analogues of this intermediate, including OSB-AMS (1), which has an IC50 value of ≤25 nM for Escherichia coli MenE. Herein, we show that OSB-AMS reduces menaquinone levels in Staphylococcus aureus, consistent with its proposed mechanism of action, despite the observation that the antibacterial activity of OSB-AMS is ∼1000-fold lower than the IC50 for enzyme inhibition. To inform the synthesis of MenE inhibitors with improved antibacterial activity, we have undertaken a structure-activity relationship (SAR) study stimulated by the knowledge that OSB-AMS can adopt two isomeric forms in which the OSB side chain exists either as an open-chain keto acid or a cyclic lactol. These studies revealed that negatively charged analogues of the keto acid form bind, while neutral analogues do not, consistent with the hypothesis that the negatively charged keto acid form of OSB-AMS is the active isomer. X-ray crystallography and site-directed mutagenesis confirm the importance of a conserved arginine for binding the OSB carboxylate. Although most lactol isomers tested were inactive, a novel difluoroindanediol inhibitor (11) with improved antibacterial activity was discovered, providing a pathway toward the development of optimized MenE inhibitors in the future.

Molecular Recognition of Fluorine Impacts Substrate Selectivity in the Fluoroacetyl-CoA Thioesterase FlK

PubMed Central

2015-01-01

The fluoroacetate-producing bacterium Streptomyces cattleya has evolved a fluoroacetyl-CoA thioesterase (FlK) that exhibits a remarkably high level of discrimination for its cognate substrate compared to the cellularly abundant analogue acetyl-CoA, which differs only by the absence of the fluorine substitution. A major determinant of FlK specificity derives from its ability to take advantage of the unique properties of fluorine to enhance the reaction rate, allowing fluorine discrimination under physiological conditions where both substrates are likely to be present at saturating concentrations. Using a combination of pH–rate profiles, pre-steady-state kinetic experiments, and Taft analysis of wild-type and mutant FlKs with a set of substrate analogues, we explore the role of fluorine in controlling the enzyme acylation and deacylation steps. Further analysis of chiral (R)- and (S)-[2H1]fluoroacetyl-CoA substrates demonstrates that a kinetic isotope effect (1.7 ± 0.2) is observed for only the (R)-2H1 isomer, indicating that deacylation requires recognition of the prochiral fluoromethyl group to position the α-carbon for proton abstraction. Taken together, the selectivity for the fluoroacetyl-CoA substrate appears to rely not only on the enhanced polarization provided by the electronegative fluorine substitution but also on molecular recognition of fluorine in both formation and breakdown of the acyl-enzyme intermediate to control active site reactivity. These studies provide insights into the basis of fluorine selectivity in a naturally occurring enzyme–substrate pair, with implications for drug design and the development of fluorine-selective biocatalysts. PMID:24635371

Lipoproteins of slow-growing Mycobacteria carry three fatty acids and are N-acylated by Apolipoprotein N-Acyltransferase BCG_2070c

PubMed Central

2013-01-01

Background Lipoproteins are virulence factors of Mycobacterium tuberculosis. Bacterial lipoproteins are modified by the consecutive action of preprolipoprotein diacylglyceryl transferase (Lgt), prolipoprotein signal peptidase (LspA) and apolipoprotein N- acyltransferase (Lnt) leading to the formation of mature triacylated lipoproteins. Lnt homologues are found in Gram-negative and high GC-rich Gram-positive, but not in low GC-rich Gram-positive bacteria, although N-acylation is observed. In fast-growing Mycobacterium smegmatis, the molecular structure of the lipid modification of lipoproteins was resolved recently as a diacylglyceryl residue carrying ester-bound palmitic acid and ester-bound tuberculostearic acid and an additional amide-bound palmitic acid. Results We exploit the vaccine strain Mycobacterium bovis BCG as model organism to investigate lipoprotein modifications in slow-growing mycobacteria. Using Escherichia coli Lnt as a query in BLASTp search, we identified BCG_2070c and BCG_2279c as putative lnt genes in M. bovis BCG. Lipoproteins LprF, LpqH, LpqL and LppX were expressed in M. bovis BCG and BCG_2070c lnt knock-out mutant and lipid modifications were analyzed at molecular level by matrix-assisted laser desorption ionization time-of-flight/time-of-flight analysis. Lipoprotein N-acylation was observed in wildtype but not in BCG_2070c mutants. Lipoprotein N- acylation with palmitoyl and tuberculostearyl residues was observed. Conclusions Lipoproteins are triacylated in slow-growing mycobacteria. BCG_2070c encodes a functional Lnt in M. bovis BCG. We identified mycobacteria-specific tuberculostearic acid as further substrate for N-acylation in slow-growing mycobacteria. PMID:24093492

Lipoproteins of slow-growing Mycobacteria carry three fatty acids and are N-acylated by apolipoprotein N-acyltransferase BCG_2070c.

PubMed

Brülle, Juliane K; Tschumi, Andreas; Sander, Peter

2013-10-05

Lipoproteins are virulence factors of Mycobacterium tuberculosis. Bacterial lipoproteins are modified by the consecutive action of preprolipoprotein diacylglyceryl transferase (Lgt), prolipoprotein signal peptidase (LspA) and apolipoprotein N- acyltransferase (Lnt) leading to the formation of mature triacylated lipoproteins. Lnt homologues are found in Gram-negative and high GC-rich Gram-positive, but not in low GC-rich Gram-positive bacteria, although N-acylation is observed. In fast-growing Mycobacterium smegmatis, the molecular structure of the lipid modification of lipoproteins was resolved recently as a diacylglyceryl residue carrying ester-bound palmitic acid and ester-bound tuberculostearic acid and an additional amide-bound palmitic acid. We exploit the vaccine strain Mycobacterium bovis BCG as model organism to investigate lipoprotein modifications in slow-growing mycobacteria. Using Escherichia coli Lnt as a query in BLASTp search, we identified BCG_2070c and BCG_2279c as putative lnt genes in M. bovis BCG. Lipoproteins LprF, LpqH, LpqL and LppX were expressed in M. bovis BCG and BCG_2070c lnt knock-out mutant and lipid modifications were analyzed at molecular level by matrix-assisted laser desorption ionization time-of-flight/time-of-flight analysis. Lipoprotein N-acylation was observed in wildtype but not in BCG_2070c mutants. Lipoprotein N- acylation with palmitoyl and tuberculostearyl residues was observed. Lipoproteins are triacylated in slow-growing mycobacteria. BCG_2070c encodes a functional Lnt in M. bovis BCG. We identified mycobacteria-specific tuberculostearic acid as further substrate for N-acylation in slow-growing mycobacteria.

Genetic basis for correction of very-long-chain acyl-coenzyme A dehydrogenase deficiency by bezafibrate in patient fibroblasts: toward a genotype-based therapy.

PubMed

Gobin-Limballe, S; Djouadi, F; Aubey, F; Olpin, S; Andresen, B S; Yamaguchi, S; Mandel, H; Fukao, T; Ruiter, J P N; Wanders, R J A; McAndrew, R; Kim, J J; Bastin, J

2007-12-01

Very-long-chain acyl-coenzyme A dehydrogenase (VLCAD) deficiency is an inborn mitochondrial fatty-acid beta-oxidation (FAO) defect associated with a broad mutational spectrum, with phenotypes ranging from fatal cardiopathy in infancy to adolescent-onset myopathy, and for which there is no established treatment. Recent data suggest that bezafibrate could improve the FAO capacities in beta-oxidation-deficient cells, by enhancing the residual level of mutant enzyme activity via gene-expression stimulation. Since VLCAD-deficient patients frequently harbor missense mutations with unpredictable effects on enzyme activity, we investigated the response to bezafibrate as a function of genotype in 33 VLCAD-deficient fibroblasts representing 45 different mutations. Treatment with bezafibrate (400 microM for 48 h) resulted in a marked increase in FAO capacities, often leading to restoration of normal values, for 21 genotypes that mainly corresponded to patients with the myopathic phenotype. In contrast, bezafibrate induced no changes in FAO for 11 genotypes corresponding to severe neonatal or infantile phenotypes. This pattern of response was not due to differential inductions of VLCAD messenger RNA, as shown by quantitative real-time polymerase chain reaction, but reflected variable increases in measured VLCAD residual enzyme activity in response to bezafibrate. Genotype cross-analysis allowed the identification of alleles carrying missense mutations, which could account for these different pharmacological profiles and, on this basis, led to the characterization of 9 mild and 11 severe missense mutations. Altogether, the responses to bezafibrate reflected the severity of the metabolic blockage in various genotypes, which appeared to be correlated with the phenotype, thus providing a new approach for analysis of genetic heterogeneity. Finally, this study emphasizes the potential of bezafibrate, a widely prescribed hypolipidemic drug, for the correction of VLCAD deficiency and

Miscibility and Phase Behavior of N-Acylethanolamine/Diacylphosphatidylethanolamine Binary Mixtures of Matched Acyl Chainlengths (n = 14, 16)

PubMed Central

Kamlekar, Ravi Kanth; Satyanarayana, S.; Marsh, Derek; Swamy, Musti J.

2007-01-01

The miscibility and phase behavior of hydrated binary mixtures of two N-acylethanolamines (NAEs), N-myristoylethanolamine (NMEA), and N-palmitoylethanolamine (NPEA), with the corresponding diacyl phosphatidylethanolamines (PEs), dimyristoylphosphatidylethanolamine (DMPE), and dipalmitoylphosphatidylethanolamine (DPPE), respectively, have been investigated by differential scanning calorimetry (DSC), spin-label electron spin resonance (ESR), and 31P-NMR spectroscopy. Temperature-composition phase diagrams for both NMEA/DMPE and NPEA/DPPE binary systems were established from high sensitivity DSC. The structures of the phases involved were determined by 31P-NMR spectroscopy. For both systems, complete miscibility in the fluid and gel phases is indicated by DSC and ESR, up to 35 mol % of NMEA in DMPE and 40 mol % of NPEA in DPPE. At higher contents of the NAEs, extensive solid-fluid phase separation and solid-solid immiscibility occur depending on the temperature. Characterization of the structures of the mixtures formed with 31P-NMR spectroscopy shows that up to 75 mol % of NAE, both DMPE and DPPE form lamellar structures in the gel phase as well as up to at least 65°C in the fluid phase. ESR spectra of phosphatidylcholine spin labeled at the C-5 position in the sn-2 acyl chain present at a probe concentration of 1 mol % exhibit strong spin-spin broadening in the low-temperature region for both systems, suggesting that the acyl chains pack very tightly and exclude the spin label. However, spectra recorded in the fluid phase do not exhibit any spin-spin broadening and indicate complete miscibility of the two components. The miscibility of NAE and diacyl PE of matched chainlengths is significantly less than that found earlier for NPEA and dipalmitoylphosphatidylcholine, an observation that is consistent with the notion that the NAEs are most likely stored as their precursor lipids (N-acyl PEs) and are generated only when the system is subjected to membrane stress. PMID

Cloning and Characterization of Unusual Fatty Acid Desaturases from Anemone leveillei: Identification of an Acyl-Coenzyme A C20 Δ5-Desaturase Responsible for the Synthesis of Sciadonic Acid1

PubMed Central

Sayanova, Olga; Haslam, Richard; Venegas Caleron, Monica; Napier, Johnathan A.

2007-01-01

The seed oil of Anemone leveillei contains significant amounts of sciadonic acid (20:3Δ5,11,14; SA), an unusual non-methylene-interrupted fatty acid with pharmaceutical potential similar to arachidonic acid. Two candidate cDNAs (AL10 and AL21) for the C20 Δ5cis-desaturase from developing seeds of A. leveillei were functionally characterized in transgenic Arabidopsis (Arabidopsis thaliana) plants. The open reading frames of both Δ5-desaturases showed some similarity to presumptive acyl-coenzyme A (CoA) desaturases found in animals and plants. When expressed in transgenic Arabidopsis, AL21 showed a broad range of substrate specificity, utilizing both saturated (16:0 and 18:0) and unsaturated (18:2, n-6 and 18:3, n-3) substrates. In contrast, AL10 did not show any activity in wild-type Arabidopsis. Coexpression of AL10 or AL21 with a C18 Δ9-elongase in transgenic Arabidopsis plants resulted in the production of SA and juniperonic fatty acid (20:4Δ5,11,14,17). Thus, AL10 acted only on C20 polyunsaturated fatty acids in a manner analogous to “front-end” desaturases. However, neither AL10 nor AL21 contain the cytochrome b5 domain normally present in this class of enzymes. Acyl-CoA profiling of transgenic Arabidopsis plants and developing A. leveillei seeds revealed significant accumulation of Δ5-unsaturated fatty acids as acyl-CoAs compared to the accumulation of these fatty acids in total lipids. Positional analysis of triacylglycerols of A. leveillei seeds showed that Δ5-desaturated fatty acids were present in both sn-2 and sn-1 + sn-3 positions, although the majority of 16:1Δ5, 18:1Δ5, and SA was present at the sn-2 position. Our data provide biochemical evidence for the A. leveillei Δ5-desaturases using acyl-CoA substrates. PMID:17384161

Analysis of protein prenylation and S-acylation using gas chromatography-coupled mass spectrometry.

PubMed

Sorek, Nadav; Akerman, Amir; Yalovsky, Shaul

2013-01-01

Lipid modifications play a key role in protein targeting and function. The two Arabidopsis Gγ subunits, AGG1 and AGG2, have been shown to undergo prenylation (AGG1) and S-acylation (AGG2). Prenylation involves covalent nonreversible attachment of either farnesyl (15 carbons) or geranylgeranyl (20 carbons) isoprenoids to conserved cysteine residues at or near the C-terminus of proteins. S-acylation, frequently referred to as palmitoylation, involves the attachment of acyl fatty acids to thiol groups of cysteine residues through a reversible thioester bond. The procedures described below allow direct analysis of the prenyl and acyl moieties using gas chromatography-coupled mass spectrometry (GC-MS). These methods are based on (1) cleavage of prenyl groups with the Raney nickel catalyst and (2) analysis of protein S-acylation following cleavage of the acyl fatty acids from proteins by hydrogenation with platinum (IV) oxide. The hydrogenation under these conditions causes an acid transesterification of the acyl moieties, adding an ethyl group to the carboxyl head of the fatty acid. The addition of the ethyl group reduces the polarity of the fatty acids, allowing their efficient separation by gas chromatography.

Chemically Modified N-Acylated Hyaluronan Fragments Modulate Proinflammatory Cytokine Production by Stimulated Human Macrophages*

PubMed Central

Babasola, Oladunni; Rees-Milton, Karen J.; Bebe, Siziwe; Wang, Jiaxi; Anastassiades, Tassos P.

2014-01-01

Low molecular mass hyaluronans are known to induce inflammation. To determine the role of the acetyl groups of low molecular mass hyaluronan in stimulating the production of proinflammatory cytokines, partial N-deacetylation was carried out by hydrazinolysis. This resulted in 19.7 ± 3.5% free NH2 functional groups, which were then acylated by reacting with an acyl anhydride, including acetic anhydride. Hydrazinolysis resulted in bond cleavage of the hyaluronan chain causing a reduction of the molecular mass to 30–214 kDa. The total NH2 and N-acetyl moieties in the reacetylated hyaluronan were 0% and 98.7 ± 1.5% respectively, whereas for butyrylated hyaluronan, the total NH2, N-acetyl, and N-butyryl moieties were 0, 82.2 ± 4.6, and 22.7 ± 3.8%, respectively, based on 1H NMR. We studied the effect of these polymers on cytokine production by cultured human macrophages (THP-1 cells). The reacetylated hyaluronan stimulated proinflammatory cytokine production to levels similar to LPS, whereas partially deacetylated hyaluronan had no stimulatory effect, indicating the critical role of the N-acetyl groups in the stimulation of proinflammatory cytokine production. Butyrylated hyaluronan significantly reduced the stimulatory effect on cytokine production by the reacetylated hyaluronan or LPS but had no stimulatory effect of its own. The other partially N-acylated hyaluronan derivatives tested showed smaller stimulatory effects than reacetylated hyaluronan. Antibody and antagonist experiments suggest that the acetylated and partially butyrylated lower molecular mass hyaluronans exert their effects through the TLR-4 receptor system. Selectively N-butyrylated lower molecular mass hyaluronan shows promise as an example of a novel semisynthetic anti-inflammatory molecule. PMID:25053413

Lipase-catalyzed acylation of microbial mannosylerythritol lipids (biosurfactants) and their characterization.

PubMed

Recke, Verena K; Beyrle, Catharina; Gerlitzki, Melanie; Hausmann, Rudolf; Syldatk, Christoph; Wray, Victor; Tokuda, Harukuni; Suzuki, Nobutaka; Lang, Siegmund

2013-05-24

Culturing Pseudozyma aphidis on glucose as main carbon source and soybean oil as co-substrate the mannosylerythritol lipids MEL-A and MEL-B were produced. Based on their excellent surface/interfacial active behavior they possess a high potential among all known biosurfactants. The components of a microbial MEL mixture were purified by medium pressure liquid chromatography (MPLC) and were used as substrates for in vitro enzymatic modifications. Lipase-catalyzed acylations of MEL-A and MEL-B with uncommon fatty acids from other microbial glycolipids-3-hydroxydecanoic acid from rhamnolipids and 17-hydroxyoctadecanoic acid from classical sophorolipids-yielded functionalized products at the C-1 position of the erythritol. The novel products were purified by MPLC and their structures elucidated by (1)H and (13)C nuclear magnetic resonance spectroscopy and mass spectrometry. In physicochemical characterization experiments two of the three new glycoconjugates lowered the surface tension of water from 72 mN m(-1) to 27-38 mN m(-1). Moreover the novel compounds inhibited the growth of gram-positive bacteria and showed a potential for anti-tumor-promoting activity. Copyright © 2013 Elsevier Ltd. All rights reserved.

Exploring the Mechanism of β-Lactam Ring Protonation in the Class A β-lactamase Acylation Mechanism Using Neutron and X-ray Crystallography.

PubMed

Vandavasi, Venu Gopal; Weiss, Kevin L; Cooper, Jonathan B; Erskine, Peter T; Tomanicek, Stephen J; Ostermann, Andreas; Schrader, Tobias E; Ginell, Stephan L; Coates, Leighton

2016-01-14

The catalytic mechanism of class A β-lactamases is often debated due in part to the large number of amino acids that interact with bound β-lactam substrates. The role and function of the conserved residue Lys 73 in the catalytic mechanism of class A type β-lactamase enzymes is still not well understood after decades of scientific research. To better elucidate the functions of this vital residue, we used both neutron and high-resolution X-ray diffraction to examine both the structures of the ligand free protein and the acyl-enzyme complex of perdeuterated E166A Toho-1 β-lactamase with the antibiotic cefotaxime. The E166A mutant lacks a critical glutamate residue that has a key role in the deacylation step of the catalytic mechanism, allowing the acyl-enzyme adduct to be captured for study. In our ligand free structures, Lys 73 is present in a single conformation, however in all of our acyl-enzyme structures, Lys 73 is present in two different conformations, in which one conformer is closer to Ser 70 while the other conformer is positioned closer to Ser 130, which supports the existence of a possible pathway by which proton transfer from Lys 73 to Ser 130 can occur. This and further clarifications of the role of Lys 73 in the acylation mechanism may facilitate the design of inhibitors that capitalize on the enzyme's native machinery.

Exploring the Mechanism of β-Lactam Ring Protonation in the Class A β-lactamase Acylation Mechanism Using Neutron and X-ray Crystallography

DOE PAGES

Vandavasi, Venu Gopal; Weiss, Kevin L.; Cooper, Jonathan B.; ...

2015-12-02

The catalytic mechanism of class A beta-lactamases is often debated due in part to the large number of amino acids that interact with bound beta-lactam substrates. The role and function of the conserved residue Lys 73 in the catalytic mechanism of class A type beta-lactamase enzymes is still not well understood after decades of scientific research. To better elucidate the functions of this vital residue, we used both neutron and high-resolution X-ray diffraction to examine both the structures of the ligand free protein and the acyl-enzyme complex of perdeuterated E166A Toho-1 beta-lactamase with the antibiotic cefotaxime. The E166A mutant lacksmore » a critical glutamate residue that has a key role in the deacylation step of the catalytic mechanism, allowing the acyl-enzyme adduct to be captured for study. In our ligand free structures, Lys 73 is present in a single conformation, however in all of our acyl-enzyme structures, Lys 73 is present in two different conformations, in which one conformer is closer to Ser 70 while the other conformer is positioned closer to Ser 130, which supports the existence of a possible pathway by which proton transfer from Lys 73 to Ser 130 can occur. This and further clarifications of the role of Lys 73 in the acylation mechanism may facilitate the design of inhibitors that capitalize on the enzymes native machinery.« less

Exploring the Mechanism of β-Lactam Ring Protonation in the Class A β-lactamase Acylation Mechanism Using Neutron and X-ray Crystallography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vandavasi, Venu Gopal; Weiss, Kevin L.; Cooper, Jonathan B.

The catalytic mechanism of class A beta-lactamases is often debated due in part to the large number of amino acids that interact with bound beta-lactam substrates. The role and function of the conserved residue Lys 73 in the catalytic mechanism of class A type beta-lactamase enzymes is still not well understood after decades of scientific research. To better elucidate the functions of this vital residue, we used both neutron and high-resolution X-ray diffraction to examine both the structures of the ligand free protein and the acyl-enzyme complex of perdeuterated E166A Toho-1 beta-lactamase with the antibiotic cefotaxime. The E166A mutant lacksmore » a critical glutamate residue that has a key role in the deacylation step of the catalytic mechanism, allowing the acyl-enzyme adduct to be captured for study. In our ligand free structures, Lys 73 is present in a single conformation, however in all of our acyl-enzyme structures, Lys 73 is present in two different conformations, in which one conformer is closer to Ser 70 while the other conformer is positioned closer to Ser 130, which supports the existence of a possible pathway by which proton transfer from Lys 73 to Ser 130 can occur. This and further clarifications of the role of Lys 73 in the acylation mechanism may facilitate the design of inhibitors that capitalize on the enzymes native machinery.« less

Head-group acylation of monogalactosyldiacylglycerol is a common stress response, but the acyl-galactose acyl composition varies with the plant species and applied stress

USDA-ARS?s Scientific Manuscript database

Head group acylation of monogalactosyldiacylglycerol is a plant lipid modification occurring during bacterial infection. Little is known about the range of stresses that induce this lipid modification, the molecular species induced, and the function of the modification. Lipidomic analysis using trip...

Acyl transfer from membrane lipids to peptides is a generic process.

PubMed

Dods, Robert H; Bechinger, Burkhard; Mosely, Jackie A; Sanderson, John M

2013-11-15

The generality of acyl transfer from phospholipids to membrane-active peptides has been probed using liquid chromatography-mass spectrometry analysis of peptide-lipid mixtures. The peptides examined include melittin, magainin II, PGLa, LAK1, LAK3 and penetratin. Peptides were added to liposomes with membrane lipid compositions ranging from pure phosphatidylcholine (PC) to mixtures of PC with phosphatidylethanolamine, phosphatidylserine or phosphatidylglycerol. Experiments were typically conducted at pH7.4 at modest salt concentrations (90 mM NaCl). In favorable cases, lipidated peptides were further characterized by tandem mass spectrometry methods to determine the sites of acylation. Melittin and magainin II were the most reactive peptides, with significant acyl transfer detected under all conditions and membrane compositions. Both peptides were lipidated at the N-terminus by transfer from PC, phosphatidylethanolamine, phosphatidylserine or phosphatidylglycerol, as well as at internal sites: lysine for melittin; serine and lysine for magainin II. Acyl transfer could be detected within 3h of melittin addition to negatively charged membranes. The other peptides were less reactive, but for each peptide, acylation was found to occur in at least one of the conditions examined. The data demonstrate that acyl transfer is a generic process for peptides bound to membranes composed of diacylglycerophospholipids. Phospholipid membranes cannot therefore be considered as chemically inert toward peptides and by extension proteins. © 2013. Published by Elsevier Ltd. All rights reserved.

Preparation of holo- and malonyl-[acyl-carrier-protein] in a manner suitable for analog development.

PubMed

Marcella, Aaron M; Jing, Fuyuan; Barb, Adam W

2015-11-01

The fatty acid biosynthetic pathway generates highly reduced carbon based molecules. For this reason fatty acid synthesis is a target of pathway engineering to produce novel specialty or commodity chemicals using renewable techniques to supplant molecules currently derived from petroleum. Malonyl-[acyl carrier protein] (malonyl-ACP) is a key metabolite in the fatty acid pathway and donates two carbon units to the growing fatty acid chain during each step of biosynthesis. Attempts to test engineered fatty acid biosynthesis enzymes in vitro will require malonyl-ACP or malonyl-ACP analogs. Malonyl-ACP is challenging to prepare due to the instability of the carboxylate leaving group and the multiple steps of post-translational modification required to activate ACP. Here we report the expression and purification of holo- and malonyl-ACP from Escherichia coli with high yields (>15 mg per L of expression). The malonyl-ACP is efficiently recognized by the E. coli keto-acyl synthase enzyme, FabH. A FabH assay using malonyl-ACP and a coumarin-based fluorescent reagent is described that provides a high throughput alternative to reported radioactive assays. Copyright © 2015 Elsevier Inc. All rights reserved.

The structure of (3R)-hydroxyacyl-acyl carrier protein dehydratase (FabZ) from Pseudomonas aeruginosa.

PubMed

Kimber, Matthew S; Martin, Fernando; Lu, Yingjie; Houston, Simon; Vedadi, Masoud; Dharamsi, Akil; Fiebig, Klaus M; Schmid, Molly; Rock, Charles O

2004-12-10

Type II fatty acid biosynthesis systems are essential for membrane formation in bacteria, making the constituent proteins of this pathway attractive targets for antibacterial drug discovery. The third step in the elongation cycle of the type II fatty acid biosynthesis is catalyzed by beta-hydroxyacyl-(acyl carrier protein) (ACP) dehydratase. There are two isoforms. FabZ, which catalyzes the dehydration of (3R)-hydroxyacyl-ACP to trans-2-acyl-ACP, is a universally expressed component of the bacterial type II system. FabA, the second isoform, as has more limited distribution in nature and, in addition to dehydration, also carries out the isomerization of trans-2- to cis-3-decenoyl-ACP as an essential step in unsaturated fatty acid biosynthesis. We report the structure of FabZ from the important human pathogen Pseudomonas aeruginosa at 2.5 A of resolution. PaFabZ is a hexamer (trimer of dimers) with the His/Glu catalytic dyad located within a deep, narrow tunnel formed at the dimer interface. Site-directed mutagenesis experiments showed that the obvious differences in the active site residues that distinguish the FabA and FabZ subfamilies of dehydratases do not account for the unique ability of FabA to catalyze isomerization. Because the catalytic machinery of the two enzymes is practically indistinguishable, the structural differences observed in the shape of the substrate binding channels of FabA and FabZ lead us to hypothesize that the different shapes of the tunnels control the conformation and positioning of the bound substrate, allowing FabA, but not FabZ, to catalyze the isomerization reaction.

Acyl guanidine inhibitors of β-secretase (BACE-1): optimization of a micromolar hit to a nanomolar lead via iterative solid- and solution-phase library synthesis.

PubMed

Gerritz, Samuel W; Zhai, Weixu; Shi, Shuhao; Zhu, Shirong; Toyn, Jeremy H; Meredith, Jere E; Iben, Lawrence G; Burton, Catherine R; Albright, Charles F; Good, Andrew C; Tebben, Andrew J; Muckelbauer, Jodi K; Camac, Daniel M; Metzler, William; Cook, Lynda S; Padmanabha, Ramesh; Lentz, Kimberley A; Sofia, Michael J; Poss, Michael A; Macor, John E; Thompson, Lorin A

2012-11-08

This report describes the discovery and optimization of a BACE-1 inhibitor series containing an unusual acyl guanidine chemotype that was originally synthesized as part of a 6041-membered solid-phase library. The synthesis of multiple follow-up solid- and solution-phase libraries facilitated the optimization of the original micromolar hit into a single-digit nanomolar BACE-1 inhibitor in both radioligand binding and cell-based functional assay formats. The X-ray structure of representative inhibitors bound to BACE-1 revealed a number of key ligand:protein interactions, including a hydrogen bond between the side chain amide of flap residue Gln73 and the acyl guanidine carbonyl group, and a cation-π interaction between Arg235 and the isothiazole 4-methoxyphenyl substituent. Following subcutaneous administration in rats, an acyl guanidine inhibitor with single-digit nanomolar activity in cells afforded good plasma exposures and a dose-dependent reduction in plasma Aβ levels, but poor brain exposure was observed (likely due to Pgp-mediated efflux), and significant reductions in brain Aβ levels were not obtained.

The insulin receptor substrate-1-related 4PS substrate but not the interleukin-2R gamma chain is involved in interleukin-13-mediated signal transduction.

PubMed

Wang, L M; Michieli, P; Lie, W R; Liu, F; Lee, C C; Minty, A; Sun, X J; Levine, A; White, M F; Pierce, J H

1995-12-01

Interleukin-13 (IL-13) induced a potent mitogenic response in IL-3-dependent TF-1 cells and DNA synthesis to a lesser extent in MO7E and FDC-P1 cells. IL-13 stimulation of these lines, like IL-4 and insulin-like growth factor-1 (IGF-1), resulted in tyrosine phosphorylation of a 170-kD substrate. The tyrosine-phosphorylated 170-kD substrate strongly associated with the 85-kD subunit of phosphoinositol-3 (PI-3) kinase and with Grb-2. Anti-4PS serum readily detected the 170-kD substrate in lysates from both TF-1 and FDC-P1 cells stimulated with IL-13 or IL-4. These data provide evidence that IL-13 induces tyrosine phosphorylation of the 4PS substrate, providing an essential interface between the IL-13 receptor and signaling molecules containing SH2 domains. IL-13 and IL-4 stimulation of murine L cell fibroblasts, which endogenously express the IL-4 receptor (IL-4R alpha) and lack expression of the IL-2 receptor gamma subunit (IL-2R gamma), resulted in tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1)/4PS. Enhanced tyrosine phosphorylation of IRS-1/4PS was observed in response to IL-4, but not IL-13 treatment of L cells transfected with the IL-2R gamma chain. These results indicate that IL-13 does not use the IL-2R gamma subunit in its receptor complex and that expression of IL-2R gamma enhances, but is not absolutely required for mediating IL-4-induced tyrosine phosphorylation of IRS-1/4PS.

Total and acylated ghrelin in liver cirrhosis: correlation with clinical and nutritional status.

PubMed

El-Shehaby, Amal M; Obaia, Eman M; Alwakil, Sahar S; Hiekal, Ahmed A

2010-07-01

The pathogenesis of anorexia in cirrhotic patients is complex and the appetite-modulating hormone ghrelin could be involved. Acylated ghrelin is the biologically active form that modifies insulin sensitivity and body composition. The aim of the present study was to compare acylated and total ghrelin concentration in patients with liver cirrhosis and to investigate the possible relationship between ghrelin and clinical and nutritional parameters. Sixty patients with viral liver cirrhosis who did not have hepatocellular carcinoma or acute infections were studied. Twenty healthy volunteers were recruited after matching for age, gender, and body mass index with the patients and served as controls. Fasting levels of total, acylated ghrelin, leptin, TNF-alpha and insulin were measured in all subjects, in addition, clinical and nutrition parameters were assessed. In cirrhotic patients, plasma levels of both acylated and total ghrelin were significantly higher than those in the controls. The mean plasma acylated ghrelin levels were significantly higher in Child C cirrhosis compared to Child A and B. Ghrelin (total and acylated) were negatively correlated with leptin in cirrhotic patients confirming the fact that leptin acts as a physiological counterpart of ghrelin. Nutritional and metabolic abnormalities in cirrhotic patients may be dependent on the changes in the ghrelin/leptin systems, mainly the acylated form of ghrelin.