scholarly journals Chemoenzymatic Generation of Phospholipid Membranes Mediated by Type I Fatty Acid Synthase

2021 ◽  
Author(s):  
Satyam Khanal ◽  
Roberto Javier Brea Fernandez ◽  
Michael Burkart ◽  
Neal Devaraj
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
De Novo ◽  
Type I ◽  
Phospholipid Membrane ◽  
Acetyl Coa ◽  
Chemical Ligation ◽  
Malonyl Coa ◽  
Synthetic Cell ◽  
Membrane Bound

The de novo formation of lipid membranes from minimal reactive precursors is a major goal in synthetic cell research. In nature, the synthesis of membrane phospholipids is orchestrated by numerous enzymes, including fatty acid synthases and membrane-bound acyltransferases. However, these enzymatic pathways are difficult to fully reproduce in vitro. As such, the reconstitution of phospholipid membrane synthesis from simple metabolic building blocks remains a challenge. Here, we describe a chemoenzymatic strategy for lipid membrane generation that utilizes a soluble bacterial fatty acid synthase (cgFAS I) to synthesize palmitoyl-CoA in situ from acetyl-CoA and malonyl-CoA. The fatty acid derivative spontaneously reacts with a cysteine-modified lysophospholipid by native chemical ligation (NCL), affording a non-canonical amidophospholipid that self-assembles into micron-sized membrane-bound vesicles. To our knowledge, this is the first example of reconstituting phospholipid membrane formation directly from acetyl-CoA and malonyl-CoA precursors. Our results demonstrate that combining the specificity and efficiency of a type I fatty acid synthase with a highly selective bioconjugation reaction provides a biomimetic route for the de novo formation of membrane-bound vesicles.

Effect of Thiolactomycin on de novo Fatty Acid Biosynthesis in Plants

1990 ◽  
Vol 45 (5) ◽  
pp. 518-520 ◽  
Author(s):  
Manfred Focke ◽  
Andrea Feld ◽  
Hartmut K. Lichtenthaler
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Fatty Acid Biosynthesis ◽  
De Novo ◽  
Fatty Acid Synthetase ◽  
Acetate Incorporation ◽  
Acid Biosynthesis ◽  
Acetyl Coa ◽  
Malonyl Coa ◽  
Total Fatty Acids

Thiolactomycin was shown to be a potent inhibitor of de novo fatty acid biosynthesis in intact isolated chloroplasts (measured as [14C]acetate incorporation into total fatty acids). In our attempt to further localize the inhibition site we confirmed the inhibition with a fatty acid synthetase preparation, measuring the incorporation of [14C]malonyl-CoA into total fatty acids. From the two proposed enzymic targets of the fatty acid synthetase by thiolactomycin we could exclude the acetyl-CoA: ACP transacetylase. It appears that the inhibition by thiolactomycin occurs on the level of the condensing enzymes, i.e. the 3-oxoacyl-ACP synthases. We also demonstrated that the two starting enzymes of de novo fatty acid biosynthesis, the acetyl-CoA synthetase and the acetyl-CoA carboxylase, are not affected by thiolactomycin.

scholarly journals Stromal concentrations of coenzyme A and its esters are insufficient to account for rates of chloroplast fatty acid synthesis: evidence for substrate channelling within the chloroplast fatty acid synthase

1997 ◽  
Vol 327 (1) ◽  
pp. 267-273 ◽  
Author(s):  
P. Grattan ROUGHAN
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthesis ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Acid Synthesis ◽  
Acetyl Coa ◽  
Minimal Processing ◽  
Malonyl Coa ◽  
Substrate Channelling ◽  
Isolated Chloroplasts

Concentrations of total CoAs in chloroplasts freshly isolated from spinach and peas were 10–20 μM, assuming a stromal volume of 66 μl per mg of chlorophyll. Acetyl-CoA and CoASH constituted at least 90% of the total CoA in freshly isolated chloroplasts. For a given chloroplast preparation, the concentration of endogenous acetyl-CoA was the same when extractions were performed using HClO4, trichloroacetic acid, propan-2-ol or chloroform/methanol, and the extracts analysed by quantitative HPLC after minimal processing. During fatty acid synthesis from acetate, concentrations of CoASH within spinach and pea chloroplasts varied from less than 0.1 to 5.0 μM. Malonyl-CoA concentrations were also very low (< 0.1–3.0 μM) during fatty acid synthesis but could be calculated from radioactivity incorporated from [1-14C]acetate. Concentrations of CoASH in chloroplasts synthesizing fatty acids could be doubled in the presence of Triton X-100, suggesting that the detergent stimulates fatty acid synthesis by increasing the turnover rate of acyl-CoA. However, although taken up, exogenous CoASH (1 μM) did not stimulate fatty acid synthesis by permeabilized spinach chloroplasts. Calculated rates for acetyl-CoA synthetase, acetyl-CoA carboxylase and malonyl-CoA–acyl-carrier-protein transacylase reactions at the concentrations of metabolites measured here are < 0.1–4% of the observed rates of fatty acid synthesis from acetate by isolated chloroplasts. The results suggest that CoA and its esters are probably confined within, and channelled through, the initial stages of a fatty acid synthase multienzyme complex.

scholarly journals Malonyl-CoA and the regulation of fatty acid oxidation in soleus muscle

1998 ◽  
Vol 334 (1) ◽  
pp. 233-241 ◽  
Author(s):  
Nasreen ALAM ◽  
E. David SAGGERSON
Keyword(s):  
Skeletal Muscle ◽  
Fatty Acid ◽  
Soleus Muscle ◽  
Fatty Acid Synthase ◽  
Fatty Acyl ◽  
Acid Oxidation ◽  
Type I ◽  
Tissue Content ◽  
Malonyl Coa ◽  
Long Chain

1. Rat soleus strips were incubated with 5 mM glucose, after which tissue metabolites were measured. Alternatively, muscle strips were incubated with 5 mM glucose and 0.2 mM palmitate, and the formation of 14CO2 from exogenous palmitate or from fatty acids released from prelabelled glycerolipids was measured. 2. Etomoxir, which inhibits the mitochondrial overt form of carnitine palmitoyltransferase (CPT1), increased the tissue content of long-chain fatty acyl-CoA esters and decreased the ratio of fatty acylcarnitine to fatty acyl-CoA, suggesting that such changes could be a diagnostic for the inhibition of CPT1. 3. Over a range of incubation conditions there was a positive correlation between the tissue contents of malonyl-CoA and long-chain fatty acyl-CoA esters. Under conditions in which these two metabolites increased in content (i.e. with insulin or with 3 mM dichloroacetate) there was a corresponding decrease in the ratio of fatty acylcarnitine to fatty acyl-CoA and a decrease in β-oxidation. Isoprenaline or palmitate (0.5 mM) opposed the effect of insulin, decreasing the contents of malonyl-CoA and long-chain fatty acyl-CoA, increasing the ratio of fatty acylcarnitine to fatty acyl-CoA and increasing β-oxidation. These findings are consistent with the notion that all of these agents can cause the acute regulation of CPT1 in Type I skeletal muscle. 4. The addition of 5-amino-4-imidazolecarboxamide ribonucleoside (AICAriboside) to cause activation of the AMP-activated protein kinase decreased the tissue content of malonyl-CoA. AICAriboside also had an antilipolytic effect in the muscle strips. 5. Measurements were made of the activities of ATP-citrate lyase, acetyl-CoA carboxylase, fatty acid synthase and malonyl-CoA decarboxylase in soleus muscle and in representative Type IIa and Type IIb muscles. A cytosolic activity of malonyl-CoA decarboxylase would seem to offer a feasible route for the disposal of malonyl-CoA in skeletal muscle.

scholarly journals Development of a Medium-Throughput Targeted LCMS Assay to Detect Endogenous Cellular Levels of Malonyl-CoA to Screen Fatty Acid Synthase Inhibitors

2015 ◽  
Vol 21 (2) ◽  
pp. 111-116 ◽  
Author(s):  
Philip J. Hopcroft ◽  
David I. Fisher
Keyword(s):  
Fatty Acid ◽  
Mammalian Cells ◽  
Fatty Acid Synthase ◽  
De Novo ◽  
Acid Synthesis ◽  
Structure Activity ◽  
Malonyl Coa ◽  
Scale Normalization ◽  
Development Structure ◽  
Screening Cascade

The fatty acid synthase (FAS) enzyme in mammalian cells is a large multidomain protein responsible for de novo synthesis of fatty acids. The steps catalyzed by FAS involve the condensation of acetyl-CoA and malonyl-CoA moieties in the presence of NADPH until palmitate is formed. Inhibition of FAS causes an accumulation of intracellular malonyl-CoA, as this metabolite is essentially committed to fatty acid synthesis once formed. Detection of intracellular metabolites for screening can be problematic due to a lack of appropriate tools, but here we describe a targeted liquid chromatography–mass spectroscopy (LCMS) method to directly measure endogenous levels of malonyl-CoA to drive a drug development structure–activity relationship (SAR) screening cascade. Our process involves preparation of samples at 96-well scale, normalization postpermeabilization via use of a whole-well imaging platform, and the LCMS detection methodology. The assay is amenable to multiplexing cellular endpoints, has a typical Z′ of >0.6, and has high reproducibility of EC50 values.

Single Particle Reconstruction of Human Fatty Acid Synthase

2000 ◽  
Vol 6 (S2) ◽  
pp. 270-271
Author(s):  
J. Brink ◽  
S.J. Ludtke ◽  
C.-Y. Yang ◽  
Z.-W. Gu ◽  
S. Wakil ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
De Novo ◽  
Acyl Carrier Protein ◽  
Cancer Cell Line ◽  
Prosthetic Group ◽  
Active Centers ◽  
Acetyl Coa ◽  
Single Particle Reconstruction ◽  
C Terminus

Fatty acid synthase (FAS) is the enzyme responsible for de novo synthesis of fatty acids from acetyl-CoA, malonyl-CoA and NADPH. FAS (550 kDa) is a homodimer of two multifunctional polypeptides, each with seven distinct catalytic activities and a site for the prosthetic group, 4’- phosphopantetheine, acyl carrier protein (ACP). These domains are organized from the N- to the C-terminus as follows: keto acylsynthase, acetyl/malonyl transacetylase, dehydratase, the interdomain, enoyl reductase, ketoreductase, ACP and thioesterase. The two polypeptides are held together through the interdomain and oriented in an anti-parallel manner, each contributing complementary half sites and giving rise to two independently active centers for palmitate synthesis. Interest in FAS arises from its involvement in human disorders, such as obesity, hyperlipidemia and carcinogenesis.Human FAS purified from a breast cancer cell line, ZR75-1, was vitrified at 50-70 μg/ml on holey grids in the presence of NADPH and acetyl-CoA and kept at -166°C in a Gatan 626 cryo-holder.

Notes: Inhibition of the Acetyl-CoA Carboxylase of Barley Chloroplasts by Cycloxydim and Sethoxydim

1987 ◽  
Vol 42 (11-12) ◽  
pp. 1361-1363 ◽  
Author(s):  
Manfred Focke ◽  
Hartmut K. Lichtenthaler
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Enzyme Preparation ◽  
Fatty Acid Biosynthesis ◽  
De Novo ◽  
Acetyl Coa Carboxylase ◽  
Acid Biosynthesis ◽  
Hordeum Vulgare L ◽  
Acetyl Coa ◽  
Malonyl Coa

The effect of the three cyclohexane-1,3-dione derivatives cycloxydim, sethoxydim and clethodim on the incorpora­tion of 14C-labelled acetate, malonate. acctyl-CoA or malonyl-CoA into fatty acids was studied in an enzyme preparation isolated from barley chloroplasts (Hordeum vulgare L. var. “Alexis”). The herbicides cycloxydim, clethodim and sethoxydim block the de novo fatty acid biosynthesis from [2-14C]acetatc and [1-14C]acetyl-CoA, whereas that of [2-14C]malonatc and [2-14C)malonyl-CoA is not affected. The data indicate that the mode of action of the cyclohexane-1,3-dione derivatives in the sensitive bar­ley plant consists in the inhibition of de novo fatty acid biosynthesis by blocking the acetyl-CoA carboxylase (EC 6.4.1.2.).

scholarly journals Type I fatty acid synthase (FAS) trapped in the octanoyl-bound state

2019 ◽  
Author(s):  
Alexander Rittner ◽  
Karthik S. Paithankar ◽  
Aaron Himmler ◽  
Martin Grininger
Keyword(s):  
Fatty Acid ◽  
Domain Structure ◽  
Fatty Acid Synthase ◽  
De Novo ◽  
Substrate Binding ◽  
Conformational Dynamics ◽  
Bound State ◽  
Type I ◽  
List Type ◽  
Substrate Promiscuity

AbstractDe novo fatty acid biosynthesis in humans is accomplished by a multidomain protein, the type I fatty acid synthase (FAS). Although ubiquitously expressed in all tissues, fatty acid synthesis is not essential in normal healthy cells due to sufficient supply with fatty acids by the diet. However, FAS is overexpressed in cancer cells and correlates with tumor malignancy, which makes FAS an attractive selective therapeutic target in tumorigenesis. Herein, we present a crystal structure of the condensing part of murine FAS, highly homologous to human FAS, with octanoyl moieties covalently bound to the transferase (MAT) and the condensation (KS) domain. The MAT domain binds the octanoyl moiety in a novel (unique) conformation, which reflects the pronounced conformational dynamics of the substrate binding site responsible for the MAT substrate promiscuity. In contrast, the KS binding pocket just subtly adapts to the octanoyl moiety upon substrate binding. Besides the rigid domain structure, we found a positive cooperative effect in the substrate binding of the KS domain by a comprehensive enzyme kinetic study. These structural and mechanistic findings contribute significantly to our understanding of the mode of action of FAS and may guide future rational inhibitor designs.HighlightsThe X-ray structure of the KS-MAT didomain of murine type I FAS is presented in an octanoyl-bound state.Multiple conformations of the MAT domain and a dynamic active site pocket explain substrate promiscuity.The rigid domain structure and minor structural changes upon acylation are in line with the strict substrate specificity of the KS domain.Enzyme kinetics reveals cooperativity in the KS-mediated transacylation step.

Studies on the Inhibition of Biotin-Containing Carboxylases by Acetyl-CoA Carboxylase Inhibitors

1993 ◽  
Vol 48 (3-4) ◽  
pp. 294-300 ◽  
Author(s):  
Anja Motel ◽  
Simone Günther ◽  
Martin Clauss ◽  
Klaus Kobek ◽  
Manfred Focke ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Biosynthesis ◽  
De Novo ◽  
Higher Plants ◽  
Acetate Incorporation ◽  
Acetyl Coa Carboxylase ◽  
Acid Biosynthesis ◽  
Acetyl Coa ◽  
Malonyl Coa ◽  
Key Enzyme

In higher plants the biosynthetic machinery of de novo fatty acid biosynthesis, measured as [14C]acetate incorporation into fatty acids, is predominantly located in plastids. A key enzyme in this pathway is the biotin-containing acetyl-CoA carboxylase (ACC , EC 6.4.1.2) which catalyzes the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. The ACC from Poaceae is very efficiently blocked by two herbicide classes, the cyclohexane-1,3-diones (e.g. sethoxydim, cycloxydim) and the aryloxyphenoxy-propionic acids (e.g. diclofop, fluazifop). It is shown that within the Poaceae not only different species but also different varieties exist which exhibit an altered sensitivity and tolerance towards both herbicide classes, which points to a mutation of the target enzyme ACC. In purifying the ACC we extended our research to the possible presence of other biotin-containing plant enzymes. In protein preparations from maize, oat, barley, pea and lentil we were able to demonstrate the carboxylation of acetyl-CoA, propionyl-CoA and methylcrotonyl-CoA. The two herbicide classes not only block the ACC, but also the activity of the propionyl-CoA carboxylase (PCC ), whereas the methylcrotonyl- CoA carboxylase (MCC ), a distinct biotin-containing enzyme from mitochondria, is not affected. MCC may play a role in isoprenoid catabolism. Whether PCC is a separate plastid enzyme or only a side activity of ACC is under current investigation. The efficiency of the graminicides in sensitive Poaceae is then not only determined by the inhibition of ACC, malonyl-CoA and fatty acid biosynthesis, but also by the exclusion of the PCC-catalyzed metabolic pathways of the plant cell.

scholarly journals The fractionation of the fatty acid synthetase activities of avocado mesocarp plastids

1975 ◽  
Vol 146 (2) ◽  
pp. 439-445 ◽  
Author(s):  
P J Weaire ◽  
R G O Kekwick
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
De Novo ◽  
Acyl Carrier Protein ◽  
Fatty Acid Synthetase ◽  
Carrier Protein ◽  
Acetyl Coa ◽  
Principal Product ◽  
Absolute Requirement ◽  
Malonyl Coa

1. The range of fatty acids formed by preparations of ultrasonically ruptured avocado mesocarp plastids was dependent on the substrate. Whereas [1-14C]palmitate and [14C]oleate were the major products obtained from [-14C]acetate and [1-14C]acetyl-CoA, the principal product from [2-14C]malonyl-CoA was [14-C]stearate. 2. Ultracentrifugation of the ruptured plastids at 105000g gave a supernatant that formed mainly stearate from [2-14C]malonyl-CoA and to a lesser extent from [1-14C]acetate. The incorporation of [1-14C]acetate into stearate by this fraction was inhibited by avidin. 3. The 105000g precipitate of the disrupted plastids incorporated [1-14C]acetate into a mixture of fatty acids that contained largely [14C]plamitate and [14C]oleate. The formation of [14C]palmitate and [14C]oleate by disrupted plastids was unaffected by avidin. 4. The soluble fatty acid synthetase was precipitated from the 105000g supernatant in the 35-65%-saturated-(NH4)2SO4 fraction and showed an absolute requirement for acyl-carrier protein. 5. Both fractions synthesized fatty acids de novo.

scholarly journals Characterization of the inactivation of rat fatty acid synthase by C75: inhibition of partial reactions and protection by substrates

2005 ◽  
Vol 388 (3) ◽  
pp. 895-903 ◽  
Author(s):  
Alan R. RENDINA ◽  
Dong CHENG
Keyword(s):  
Fatty Acid ◽  
Rat Liver ◽  
Room Temperature ◽  
Fatty Acid Synthase ◽  
Enzyme Inhibitor ◽  
Acetyl Coa ◽  
Enoyl Reductase ◽  
Malonyl Coa ◽  
Ketoacyl Synthase

C75, a synthetic inhibitor of FAS (fatty acid synthase), has both anti-tumour and anti-obesity properties. In this study we provide a detailed kinetic characterization of the mechanism of in vitro inhibition of rat liver FAS. At room temperature, C75 is a competitive irreversible inhibitor of the overall reaction with regard to all three substrates, i.e. acetyl-CoA, malonyl-CoA and NADPH, exhibiting pseudo-first-order kinetics of the complexing type, i.e. a weak non-covalent enzyme–inhibitor complex is formed before irreversible enzyme modification. C75 is a relatively inefficient inactivator of FAS, with a maximal rate of inactivation of 1 min−1 and an extrapolated KI (dissociation constant for the initial complex) of approx. 16 mM. The apparent second-order rate constants calculated from these values are 0.06 mM−1·min−1 at room temperature and 0.21 mM−1·min−1 at 37 °C. We also provide experimental evidence that C75 inactivates the β-ketoacyl synthase (3-oxoacyl synthase) partial activity of FAS. Unexpectedly, C75 also inactivates the enoyl reductase and thioesterase partial activities of FAS with about the same rates as for inactivation of the β-ketoacyl synthase. In contrast with the overall reaction, the β-ketoacyl synthase activity and the enoyl reductase activity, substrates do not protect the thioesterase activity of rat liver FAS from inactivation by C75. These results differentiate inactivation by C75 from that by cerulenin, which only inactivates the β-ketoacyl synthase activity of FAS, by forming an adduct with an active-site cysteine. Interference by dithiothreitol and protection by the substrates, acetyl-CoA, malonyl-CoA and NADPH, further distinguish the mechanism of C75-mediated inactivation from that of cerulenin. The most likely explanation for the multiple effects observed with C75 on rat liver FAS and its partial reactions is that there are multiple sites of interaction between C75 and FAS.

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