In vivo and in vitro effects of thiolactomycin on fatty acid biosynthesis in Streptomyces collinus.

The fatty acids palmitic, palmitoleic, stearic, and oleic have been isolated from rabbit testis and evidence for the synthesis of palmitic and stearic acids de novo from acetate-1-C14is presented. ICSH did not produce demonstrable stimulation of the synthesis of these acids in vitro although the hormone stimulated the production of testosterone-C14by the same tissue. Adrenal tissue was shown to contain palmitic, stearic, and oleic acids, and ACTH did not increase the incorporation of acetate-1-C14into a fatty acid fraction extracted following incubation of adrenal tissue in the presence of this substrate. Fatty acid biosynthesis, therefore, is probably not influenced by the mechanisms by which tropic hormones increase steroid formation.

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In Vitro Inhibition of the Mycobacterium tuberculosis β-Ketoacyl-Acyl Carrier Protein Reductase MabA by Isoniazid

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.1.242-249.2004 ◽

2004 ◽

Vol 48 (1) ◽

pp. 242-249 ◽

Cited By ~ 37

Author(s):

Stéphanie Ducasse-Cabanot ◽

Martin Cohen-Gonsaud ◽

Hedia Marrakchi ◽

Michel Nguyen ◽

Didier Zerbib ◽

...

Keyword(s):

Fatty Acid ◽

Fatty Acid Biosynthesis ◽

Dynamic Equilibrium ◽

Acyl Carrier Protein ◽

Multidrug Resistant ◽

Carrier Protein ◽

Ligand Complex ◽

Fas Ii

ABSTRACT The first-line specific antituberculous drug isoniazid inhibits the fatty acid elongation system (FAS) FAS-II involved in the biosynthesis of mycolic acids, which are major lipids of the mycobacterial envelope. The MabA protein that catalyzes the second step of the FAS-II elongation cycle is structurally and functionally related to the in vivo target of isoniazid, InhA, an NADH-dependent enoyl-acyl carrier protein reductase. The present work shows that the NADPH-dependent β-ketoacyl reduction activity of MabA is efficiently inhibited by isoniazid in vitro by a mechanism similar to that by which isoniazid inhibits InhA activity. It involves the formation of a covalent adduct between MnIII-activated isoniazid and the MabA cofactor. Liquid chromatography-mass spectrometry analyses revealed that the isonicotinoyl-NADP adduct has multiple chemical forms in dynamic equilibrium. Both kinetic experiments with isolated forms and purification of the enzyme-ligand complex strongly suggested that the molecules active against MabA activity are the oxidized derivative and a major cyclic form. Spectrofluorimetry showed that the adduct binds to the MabA active site. Modeling of the MabA-adduct complex predicted an interaction between the isonicotinoyl moiety of the inhibitor and Tyr185. This hypothesis was supported by the fact that a higher 50% inhibitory concentration of the adduct was measured for MabA Y185L than for the wild-type enzyme, while both proteins presented similar affinities for NADP+. The crystal structure of MabA Y185L that was solved showed that the substitution of Tyr185 induced no significant conformational change. The description of the first inhibitor of the β-ketoacyl reduction step of fatty acid biosynthesis should help in the design of new antituberculous drugs efficient against multidrug-resistant tubercle bacilli.

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Engineered Fatty Acid Biosynthesis inStreptomyces by Altered Catalytic Function of β-Ketoacyl-Acyl Carrier Protein Synthase III

Journal of Bacteriology ◽

10.1128/jb.183.7.2335-2342.2001 ◽

2001 ◽

Vol 183 (7) ◽

pp. 2335-2342 ◽

Cited By ~ 23

Author(s):

Natalya Smirnova ◽

Kevin A. Reynolds

Keyword(s):

Fatty Acid ◽

Type Strain ◽

Wild Type Strain ◽

Fatty Acid Biosynthesis ◽

Acyl Carrier Protein ◽

Chain Fatty Acid ◽

Wild Type ◽

Acid Biosynthesis ◽

Branched Chain

ABSTRACT The Streptomyces glaucescens β-ketoacyl-acyl carrier protein (ACP) synthase III (KASIII) initiates straight- and branched-chain fatty acid biosynthesis by catalyzing the decarboxylative condensation of malonyl-ACP with different acyl-coenzyme A (CoA) primers. This KASIII has one cysteine residue, which is critical for forming an acyl-enzyme intermediate in the first step of the process. Three mutants (Cys122Ala, Cys122Ser, Cys122Gln) were created by site-directed mutagenesis. Plasmid-based expression of these mutants in S. glaucescens resulted in strains which generated 75 (Cys122Ala) to 500% (Cys122Gln) more straight-chain fatty acids (SCFA) than the corresponding wild-type strain. In contrast, plasmid-based expression of wild-type KASIII had no effect on fatty acid profiles. These observations are attributed to an uncoupling of the condensation and decarboxylation activities in these mutants (malonyl-ACP is thus converted to acetyl-ACP, a SCFA precursor). Incorporation experiments with perdeuterated acetic acid demonstrated that 9% of the palmitate pool of the wild-type strain was generated from an intact D3 acetyl-CoA starter unit, compared to 3% in a strain expressing the Cys122Gln KASIII. These observations support the intermediacy of malonyl-ACP in generating the SCFA precursor in a strain expressing this mutant. To study malonyl-ACP decarboxylase activity in vitro, the KASIII mutants were expressed and purified as His-tagged proteins in Escherichia coli and assayed. In the absence of the acyl-CoA substrate the Cys122Gln mutant and wild-type KASIII were shown to have comparable decarboxylase activities in vitro. The Cys122Ala mutant exhibited higher activity. This activity was inhibited for all enzymes by the presence of high concentrations of isobutyryl-CoA (>100 μM), a branched-chain fatty acid biosynthetic precursor. Under these conditions the mutant enzymes had no activity, while the wild-type enzyme functioned as a ketoacyl synthase. These observations indicate the likely upper and lower limits of isobutyryl-CoA and related acyl-CoA concentrations within S. glaucescens.

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Regulation of gene transcription by thyroid hormone receptor β agonists in clinical development for the treatment of non-alcoholic steatohepatitis (NASH)

PLoS ONE ◽

10.1371/journal.pone.0240338 ◽

2020 ◽

Vol 15 (12) ◽

pp. e0240338

Author(s):

Xuan G. Luong ◽

Sarah K. Stevens ◽

Andreas Jekle ◽

Tse-I Lin ◽

Kusum Gupta ◽

...

Keyword(s):

Fatty Acid ◽

Thyroid Hormone ◽

Hormone Receptor ◽

Fatty Acid Biosynthesis ◽

Thyroid Hormone Receptor ◽

Density Lipoprotein ◽

Liver Fat ◽

Alcoholic Steatohepatitis

Thyroid hormones are important modulators of metabolic activity in mammals and alter cholesterol and fatty acid levels through activation of the nuclear thyroid hormone receptor (THR). Currently, there are several THRβ agonists in clinical trials for the treatment of non-alcoholic steatohepatitis (NASH) that have demonstrated the potential to reduce liver fat and restore liver function. In this study, we tested three THRβ-agonism-based NASH treatment candidates, GC-1 (sobetirome), MGL-3196 (resmetirom), and VK2809, and compared their selectivity for THRβ and their ability to modulate the expression of genes specific to cholesterol and fatty acid biosynthesis and metabolism in vitro using human hepatic cells and in vivo using a rat model. Treatment with GC-1 upregulated the transcription of CPT1A in the human hepatocyte-derived Huh-7 cell line with a dose-response comparable to that of the native THR ligand, triiodothyronine (T3). VK2809A (active parent of VK2809), MGL-3196, and VK2809 were approximately 30-fold, 1,000-fold, and 2,000-fold less potent than T3, respectively. Additionally, these relative potencies were confirmed by quantification of other direct gene targets of THR, namely, ANGPTL4 and DIO1. In primary human hepatocytes, potencies were conserved for every compound except for VK2809, which showed significantly increased potency that was comparable to that of its active counterpart, VK2809A. In high-fat diet fed rats, a single dose of T3 significantly reduced total cholesterol levels and concurrently increased liver Dio1 and Me1 RNA expression. MGL-3196 treatment resulted in concentration-dependent decreases in total and low-density lipoprotein cholesterol with corresponding increases in liver gene expression, but the compound was significantly less potent than T3. In conclusion, we have implemented a strategy to rank the efficacy of THRβ agonists by quantifying changes in the transcription of genes that lead to metabolic alterations, an effect that is directly downstream of THR binding and activation.

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Evolution and Functional Characteristics of the Novel Elovl8 That Play Pivotal Roles in Fatty Acid Biosynthesis

Genes ◽

10.3390/genes12081287 ◽

2021 ◽

Vol 12 (8) ◽

pp. 1287

Author(s):

Shouxiang Sun ◽

Yumei Wang ◽

Pei-Tian Goh ◽

Mónica Lopes-Marques ◽

L. Filipe C. Castro ◽

...

Keyword(s):

Fatty Acid ◽

Fatty Acid Biosynthesis ◽

The Novel ◽

Acid Biosynthesis ◽

Long Chain Fatty Acid ◽

Rate Limiting Step ◽

Gene Expression Quantification ◽

Rate Limiting ◽

Zebrafish Liver

Elongation of very long-chain fatty acid (Elovl) proteins are key enzymes that catalyze the rate-limiting step in the fatty acid elongation pathway. The most recently discovered member of the Elovl family, Elovl8, has been proposed to be a fish-specific elongase with two gene paralogs described in teleosts. However, the biological functions of Elovl8 are still to be elucidated. In this study, we showed that in contrast to previous findings, elovl8 is not unique to teleosts, but displays a rather unique and ample phylogenetic distribution. For functional determination, we generated elovl8a (elovl8a−/−) and elovl8b (elovl8b−/−) zebrafish using CRISPR/Cas9 technology. Fatty acid composition in vivo and zebrafish liver cell experiments suggest that the substrate preference of Elovl8 overlapped with other existing Elovl enzymes. Zebrafish Elovl8a could elongate the polyunsaturated fatty acids (PUFAs) C18:2n-6 and C18:3n-3 to C20:2n-6 and C20:3n-3, respectively. Along with PUFA, zebrafish Elovl8b also showed the capacity to elongate C18:0 and C20:1. Gene expression quantification suggests that Elovl8a and Elovl8b may play a potentially important role in fatty acid biosynthesis. Overall, our results provide novel insights into the function of Elovl8a and Elovl8b, representing additional fatty acid elongases not previously described in chordates.

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Cellularity and in Vitro Fatty Acid Biosynthesis in Adipose Tissue of Meal-Fed and Nibbling Rats

Experimental Biology and Medicine ◽

10.3181/00379727-139-36255 ◽

1972 ◽

Vol 139 (3) ◽

pp. 868-871 ◽

Cited By ~ 1

Author(s):

D. R. Romsos ◽

G. A. Leveille

Keyword(s):

Adipose Tissue ◽

Fatty Acid ◽

Fatty Acid Biosynthesis ◽

Acid Biosynthesis

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Studies on the response of cholesterol biogenesis in feeding in rats: evidence against the existence of diurnal rhythms

Biochemical Journal ◽

10.1042/bj1580053 ◽

1976 ◽

Vol 158 (1) ◽

pp. 53-60 ◽

Cited By ~ 20

Author(s):

R Fears ◽

B Morgan

Keyword(s):

Fatty Acid Biosynthesis ◽

Diurnal Rhythms ◽

Cholesterol Feeding ◽

Acid Biosynthesis ◽

Accurate Representation ◽

Ad Libitum ◽

Hepatic Cholesterogenesis ◽

Stimulation Of

1. The biosynthesis of cholesterol was studied, by using various precursors, in rats subjected to several dietary regimes. 2. The use of 3H2O as a substrate to demonstrate differences in cholesterogenesis under various conditions was validated by using rats fed on cholesterol or cholestyramine. Cholesterol feeding resulted in decreased cholesterogenesis, whereas cholestyramine caused an increase. 3. With acetate as precursor, the biosynthesis of both digitonin-precipitable sterol and fatty acids was increased in vitro in response to a meal. 4. In rats fed ad libitum, hepatic cholesterogenesis was increased at midnight relative to mid-morning as measured by using acetate precursor in vitro. However, no such difference was found by using 3H2O in vivo. 5. The lipogenic response was measured in meal-fed rats by using 3H2O or octanoate in vivo. In contrast with findings with acetate in vitro, no postprandial stimulation of cholesterogenesis was seen with either 3H2O or octanoate as precursor, whereas fatty acid biosynthesis from either substrate was increased. 6. These findings are discussed with respect to current theories about the circadian rhythm of cholesterogenesis. Such theories are based on experiments using isolated enzyme measurements or non-physiological precursors such as acetate. 7. It is considered that results obtained with 3H2O give an accurate representation of cholesterogenesis under various conditions, and it is therefore suggested that hepatic cholesterogenesis in rats is not subjected to the same degree of diurnal rhythm as has previously been believed.

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SMb20651 is another acyl carrier protein from Sinorhizobium meliloti

Microbiology ◽

10.1099/mic.0.022079-0 ◽

2009 ◽

Vol 155 (1) ◽

pp. 257-267 ◽

Cited By ~ 9

Author(s):

Ana Laura Ramos-Vega ◽

Yadira Dávila-Martínez ◽

Christian Sohlenkamp ◽

Sandra Contreras-Martínez ◽

Sergio Encarnación ◽

...

Keyword(s):

Fatty Acid ◽

Sinorhizobium Meliloti ◽

Fatty Acid Biosynthesis ◽

Acyl Carrier Protein ◽

Fatty Acyl ◽

Acyl Carrier Proteins ◽

Coa Ligase ◽

Acyl Chains

Acyl carrier proteins (ACPs) are small acidic proteins that carry growing acyl chains during fatty acid or polyketide synthesis. In rhizobia, there are four different and well-characterized ACPs: AcpP, NodF, AcpXL and RkpF. The genome sequence of Sinorhizobium meliloti 1021 reveals two additional ORFs that possibly encode additional ACPs. One of these, smb20651, is located on the plasmid pSymB as part of an operon. The genes of the operon encode a putative asparagine synthetase (AsnB), the predicted ACP (SMb20651), a putative long-chain fatty acyl-CoA ligase (SMb20650) and a putative ammonium-dependent NAD+ synthetase (NadE1). When SMb20651 was overexpressed in Escherichia coli, [3H]β-alanine, a biosynthetic building block of 4′-phosphopantetheine, was incorporated into the protein in vivo. The purified SMb20651 was modified with 4′-phosphopantetheine in the presence of S. meliloti holo-ACP synthase (AcpS). Also, holo-SMb20651 was modified in vitro with a malonyl group by malonyl CoA-ACP transacylase. In E. coli, coexpression of SMb20651 together with other proteins such as AcpS and SMb20650 led to the formation of additional forms of SMb20651. In this bacterium, acylation of SMb20651 with C12 : 0 or C18 : 0 fatty acids was detected, demonstrating that this protein is involved in fatty acid biosynthesis or transfer. Expression of SMb20651 was detected in S. meliloti as holo-SMb20651 and acyl-SMb20651.

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