Fatty Acid Synthetase from Pythium debaryanum

1973 ◽  
Vol 51 (3) ◽  
pp. 241-248 ◽  
Author(s):  
Simon W. T. Law ◽  
David N. Burton
Keyword(s):  
Fatty Acid ◽  
De Novo ◽  
Acyl Carrier Protein ◽  
Fatty Acid Synthetase ◽  
Apparent Size ◽  
Supernatant Fraction ◽  
Acetyl Coa ◽  
Ph Optimum ◽  
Malonyl Coa ◽  
Long Chain

The presence of a fatty acid synthetase, capable of de novo synthesis of long chain fatty acids from acetyl-CoA, malonyl-CoA, and NADPH has been demonstrated in the simple fungus Pythium debaryanum (ATCC 9998), a member of the class Oomycetes. The enzyme is found in the supernatant fraction after centrifugation at 123 000 g for 35 min.The fatty acid synthetase was demonstrated to be of the multienzyme complex type, showing no dependence on acyl carrier protein for activity. The fatty acid products were identified as principally a mixture of free and esterified palmitic and stearic acids by gas-radiochromatography, and were shown to be synthesized de novo by the ratio of incorporation of acetyl- and malonyl-CoA, the Schmidt decarboxylation, and the lack of activity of medium and long chain acyl-CoA derivatives as acceptors of two carbon units in an elongation reaction. The pH optimum for the reaction was 6.8, and the Km for acetyl-CoA was 3.3 μM.The molecular weight of the fatty acid synthetase was estimated by gel filtration on Sepharose-4B as being of the order of 4 × 106. Attempts to reduce the apparent size of the enzyme by treatment with detergents and various enzymes were unsuccessful.

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.

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 Effect of phenylpyruvate on enzymes involved in fatty acid synthesis in rat brain

1973 ◽  
Vol 134 (2) ◽  
pp. 545-555 ◽  
Author(s):  
John M. Land ◽  
John B. Clark
Keyword(s):  
Fatty Acid ◽  
Citrate Synthase ◽  
Mitochondrial Protein ◽  
Fatty Acid Synthetase ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa ◽  
Adult Rats ◽  
Malonyl Coa ◽  
Old Rats ◽  
The Brain

1. The activities of, and the effects of phenylpyruvate on, citrate synthase (EC 4.1.3.7), acetyl-CoA carboxylase (EC 6.4.1.2) and fatty acid synthetase derived from the brains of 14-day-old and adult rats were investigated. 2. The brain citrate synthase from 14-day-old rats had a Km for oxaloacetate of 2.38μm and for acetyl-CoA of 16.9μm, and a Vmax. of 838nmol of acetyl-CoA incorporation/min per mg of mitochondrial protein. From adult rat brain this enzyme had a Km for oxaloacetate of 2.5μm and for acetyl-CoA of 16.6μm and a Vmax. of 1070nmol of acetyl-CoA incorporated/min per mg of mitochondrial protein. Phenylpyruvate inhibited the enzyme from adult and young rat brains in a competitive fashion with respect to acetyl-CoA, with a Ki of 700μm. 3. The brain acetyl-CoA carboxylase from 14-day-old rats had a Km for acetyl-CoA of 21μm and a Vmax. of 0.248nmol/min per mg of protein, and from adult rats a Km for acetyl-CoA of 21μm and a Vmax. of 0.173nmol/min per mg of protein. The enzyme from young and adult rats required citrate (Ka=3mm) for activation and were inhibited non-competitively by phenylpyruvate, with a Ki of 10mm. 4. The brain fatty acid synthetase from 14-day-old rats had a Km for acetyl-CoA of 7.58μm and a Vmax. of 1.1 nmol of malonyl-CoA incorporated/min per mg of protein, and from adult rats a Km for acetyl-CoA of 4.9μm and a Vmax. of 0.48nmol of malonyl-CoA incorporated/min per mg of protein. Phenylpyruvate acted as a competitive inhibitor with respect to acetyl-CoA with a Ki of 250μm for the enzyme from 14-day-old rats. 5. These results are discussed with respect to phenylketonuria, and it is suggested that the inhibition of the brain fatty acid synthetase and possibly the citrate synthetase by phenylpyruvate could explain the defective myelination characteristic of this condition.

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 Isolation of a Saccharomyces cerevisiae long chain fatty acyl:CoA synthetase gene (FAA1) and assessment of its role in protein N-myristoylation.

1992 ◽  
Vol 117 (3) ◽  
pp. 515-529 ◽  
Author(s):  
R J Duronio ◽  
L J Knoll ◽  
J I Gordon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fatty Acid ◽  
De Novo ◽  
Fatty Acid Synthetase ◽  
Acid Synthesis ◽  
Single Copy ◽  
Synthetase Activity ◽  
Acid Activation ◽  
The Right ◽  
Long Chain

Regulation of myristoylCoA pools in Saccharomyces cerevisiae plays an important role in modulating the activity of myristoylCoA:protein N-myristoyltransferase (NMT), an essential enzyme with an ordered Bi Bi reaction that catalyzes the transfer of myristate from myristoylCoA to greater than or equal to 12 cellular proteins. At least two pathways are available for generating myristoylCoA: de novo synthesis by the multifunctional, multisubunit fatty acid synthetase complex (FAS) and activation of exogenous myristate by acylCoA synthetase. The FAA1 (fatty acid activation) gene has been isolated by genetic complementation of a faal mutant. This single copy gene, which maps to the right arm of chromosome XV, specifies a long chain acylCoA synthetase of 700 amino acids. Analyses of strains containing NMT1 and a faal null mutation indicated that FAA1 is not essential for vegetative growth when an active de novo pathway for fatty acid synthesis is present. The role of FAA1 in cellular lipid metabolism and protein N-myristoylation was therefore assessed in strains subjected to biochemical or genetic blockade of FAS. At 36 degrees C, FAA1 is required for the utilization of exogenous myristate by NMT and for the synthesis of several phospholipid species. This requirement is not apparent at 24 or 30 degrees C, suggesting that S. cerevisiae contains another acylCoA synthetase activity whose chain length and/or temperature optima may differ from Faalp.

scholarly journals Inhibition of Very-Long-Chain Fatty Acid Formation by Indanofan, 2-[2-(3-Chlorophenyl)oxiran-2-ylmethyl]-2-ethylindan-1,3-dione, and Its Relatives

2002 ◽  
Vol 57 (1-2) ◽  
pp. 72-74 ◽  
Author(s):  
Hideomi Takahashi ◽  
Jochen Schmalfuß ◽  
Aiko Ohki ◽  
Akemi Hosokawa ◽  
Akira Tanaka ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
De Novo ◽  
Allium Porrum ◽  
Inhibitory Effects ◽  
Strong Activity ◽  
Oxirane Group ◽  
Malonyl Coa ◽  
High Concentrations ◽  
Long Chain

Indanofan and its analogs inhibited the elongation of stearoyl- or arachidoyl-CoA by [2-14C]-malonyl-CoA in leek microsomes from Allium porrum. Although the precise mode of interaction of indanofan at the molecular level is not completely clarified by the present study, it is concluded that indanofan and analogs act as inhibitor of the elongase enzyme involved in de novo biosynthesis of fatty acids with an alkyl chain longer than C18, called very-long-chain fatty acids (VLCFAs). For a strong inhibition of VLCFA formation chloro substituents at the benzene ring and the oxirane group were necessary. Furthermore, the greenhouse test showed strong activity for indanofan and its analogs, and the scores coincided with cell-free elongation inhibition. The cell-free assay, however, failed to indicate any activity for an analog having a methylene instead of the oxirane group, while both Digitaria ciliaris and Echinochloa oryzicola were killed with 1 kg a.i./ha. This finding cannot be discussed because the applied use rate of 1 kg a.i./ha is too high to allow for a score differentiation. For high concentrations of this compound additional unknown inhibitory effects may be involved besides fatty acid elongation.

On the purification of a fatty acid synthetase complex from an insect

1976 ◽  
Vol 54 (8) ◽  
pp. 1397-1399 ◽  
Author(s):  
S. N. Thompson ◽  
J. S. Barlow
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Column Chromatography ◽  
De Novo ◽  
Gel Filtration ◽  
Fatty Acid Synthetase ◽  
De Novo Synthesis ◽  
Anion Exchange Column ◽  
Malonyl Coa ◽  
Salt Fractionation

The fatty acid synthetase complex of the blowfly, Lucilia sericata, catalyzing the de novo synthesis of fatty acids from acetyl- and malonyl-CoA (EC 2.3.1.9 and EC 2.3.1.39 respectively) and requiring NADPH, was isolated from whole-insect homogenates by ultracentrifugation. Purification was carried out by salt fractionation, anion exchange column chromatography on ECTEOLA (epichlorohydrin triethanolamine) cellulose, and gel filtration column chromatography on Sephadex®.

scholarly journals Kinetic studies of the fatty acid synthetase multienzyme complex from Euglena gracilis variety bacillaris

1981 ◽  
Vol 199 (2) ◽  
pp. 383-392 ◽  
Author(s):  
T A Walker ◽  
Z L Jonak ◽  
L M S Worsham ◽  
M L Ernst-Fonberg
Keyword(s):  
Fatty Acid ◽  
Euglena Gracilis ◽  
Substrate Inhibition ◽  
Fatty Acid Synthetase ◽  
Synthetase Activity ◽  
Multienzyme Complex ◽  
Acetyl Coa ◽  
Malonyl Coa ◽  
Ping Pong ◽  
Wide Range

A fatty acid synthetase multienzyme complex was purified from Euglena gracilis variety bacillaris. The fatty acid synthetase activity is specifically inhibited by antibodies against Escherichia coli acyl-carrier protein. The Euglena enzyme system requires both NADPH and NADH for maximal activity. An analysis was done of the steady-state kinetics of the reaction catalysed by the fatty acid synthetase multienzyme complex. Initial-velocity studies were done in which the concentrations of the following pairs of substrates were varied: malonyl-CoA and acetyl-CoA, NADPH and acetyl-CoA, malonyl-CoA and NADPH. In all three cases patterns of the Ping Pong type were obtained. Product-inhibition studies were done with NADP+ and CoA. NADP+ is a competitive inhibitor with respect to NADPH, and uncompetitive with respect to malonyl-CoA and acetyl-CoA. CoA is uncompetitive with respect to NADPH and competitive with respect to malonyl-CoA and acetyl-CoA. When the concentrations of acetyl-CoA and malonyl-CoA were varied over a wide range, mutual competitive substrate inhibition was observed. When the fatty acid synthetase was incubated with radiolabelled acetyl-CoA or malonyl-CoA, labelled acyl-enzyme was isolated. The results are consistent with the idea that fatty acid synthesis proceeds by a multisite substituted-enzyme mechanism involving Ping Pong reactions at the following enzyme sites: acetyl transacylase, malonyl transacylase, beta-oxo acyl-enzyme synthetase and fatty acyl transacylase.

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.

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