scholarly journals Purification and properties of 6-methylsalicylic acid synthase from Penicillium patulum

1992 ◽  
Vol 288 (3) ◽  
pp. 839-846 ◽  
Author(s):  
J B Spencer ◽  
P M Jordan
Keyword(s):  
Fatty Acid ◽  
Proteinase Inhibitors ◽  
Type I ◽  
Acetyl Coa ◽  
Purification Method ◽  
Methylsalicylic Acid Synthase ◽  
Acid Lactone ◽  
Penicillium Patulum ◽  
Triacetic Acid Lactone

6-Methylsalicylic acid synthase has been isolated in homogeneous form from Penicillium patulum grown in liquid culture from a spore inoculum. The enzyme is highly susceptible to proteolytic degradation in vivo and in vitro, but may be stabilized during purification by incorporating proteinase inhibitors in the buffers. The enzyme exists as a homotetramer of M(r) 750,000, with a subunit M(r) of 180,000. 6-Methylsalicyclic acid synthase also accepts acetoacetyl-CoA as an alternative starter molecule to acetyl-CoA. The enzyme also catalyses the formation of small amounts of triacetic acid lactone as an oligatory by-product of the reaction. In the absence of NADPH, triacetic acid lactone is the exclusive enzymic product, being formed at 10% of the rate of 6-methylsalicylic acid. The enzyme is inactivated by 1,3-dibromopropan-2-one, leading to the formation of cross-linked dimers similar to that observed with type I fatty acid synthases. Acetyl-CoA protects the enzyme against the inactivation and inhibits dimer formation. An adaptation of the purification method for 6-methylsalicylic acid synthase may be used for the isolation of fatty acid sythase from Penicillium patulum.

Relative Activities of Acetyl-CoA Carboxylase and Fatty Acid Synthetase in Chick Liver: Effects of Dietary Fat

1973 ◽  
Vol 51 (7) ◽  
pp. 1029-1033 ◽  
Author(s):  
Gregory I. Liou ◽  
W. E. Donaldson
Keyword(s):  
Fatty Acid ◽  
Corn Oil ◽  
Fatty Acid Synthetase ◽  
Basal Diet ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa ◽  
Carboxylase Activity ◽  
Cytosol Fraction ◽  
Activity Concentrations

The specific activities of acetyl-CoA carboxylase and fatty acid synthetase were measured in the cytosol fraction of livers from chicks fed various levels of corn oil, cottonseed oil, corn-oil free fatty acids, or crude (79%) oleic acid. Activities of both enzymes were depressed by the addition of fat to a fat-free basal diet. The ratios of synthetase to carboxylase activity were greater than unity when up to 4% fat was fed, but less than unity when 8% or higher levels of fat were fed. The depressions of the activities of these enzymes appeared to be unrelated to the dietary level of linoleate. In in vitro experiments, 2 μM concentrations of palmityl-CoA or oleoyl-CoA depressed acetyl-CoA carboxylase activity. Concentrations of 20 μM of these acyl-CoA esters did not affect the activity of fatty acid synthetase.

scholarly journals Rapid Purification and Procoagulant and Platelet Aggregating Activities of Rhombeobin: A Thrombin-Like/Gyroxin-Like Enzyme fromLachesis muta rhombeataSnake Venom

2013 ◽  
Vol 2013 ◽  
pp. 1-12 ◽  
Author(s):  
Frank Denis Torres-Huaco ◽  
Cláudio C. Werneck ◽  
Cristina Pontes Vicente ◽  
Talita Vassequi-Silva ◽  
Ana Cláudia Coelho Nery-Diez ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Proteinase Inhibitors ◽  
Ex Vivo ◽  
Serine Proteinase ◽  
Dependent Manner ◽  
Amidolytic Activity ◽  
Venom Component ◽  
Purification Method ◽  
Rapid Purification

We report a rapid purification method using one-step chromatography of SVSP Rhombeobin (LMR-47) fromLachesis muta rhombeatavenom and its procoagulant activities and effects on platelet aggregation. The venom was fractionated by a single chromatographic step in RP-HPLC on a C8 Discovery BIO Wide Pore, showing high degree of molecular homogeneity with molecular mass of 47035.49 Da. Rhombeobin showed amidolytic activity upon BAρNA, with a broad optimum pH (7–10) and was stable in solution up to 60°C. The amidolytic activity was inhibited by serine proteinase inhibitors and reducing agents, but not chelating agents. Rhombeobin showed high coagulant activity on mice plasma and bovine fibrinogen. The deduced amino acid sequence of Rhombeobin showed homology with other SVSPs, especially with LM-TL (L. m. muta) and Gyroxin (C. d. terrificus). Rhombeobin acts,in vitro, as a strong procoagulant enzyme on mice citrated plasma, shortening the APTT and PT tests in adose-dependent manner. The protein showed, “ex vivo”, a strong defibrinogenating effect with 1 µg/animal. Lower doses activated the intrinsic and extrinsic coagulation pathways and impaired the platelet aggregation induced by ADP. Thus, this is the first report of a venom component that produces a venom-induced consumptive coagulopathy (VICC).

Structural Similarities between 6-Methylsalicylic Acid Synthase fromPenicillium patulumand Vertebrate Type I Fatty Acid Synthase:  Evidence from Thiol Modification Studies†

Biochemistry ◽  
1996 ◽  
Vol 35 (38) ◽  
pp. 12267-12274 ◽  
Author(s):  
Christopher J. Child ◽  
Jonathan B. Spencer ◽  
Pamela Bhogal ◽  
Peter M. Shoolingin-Jordan
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Type I ◽  
Methylsalicylic Acid Synthase

scholarly journals Rewiring Yarrowia lipolytica toward triacetic acid lactone for materials generation

2018 ◽  
Vol 115 (9) ◽  
pp. 2096-2101 ◽  
Author(s):  
Kelly A. Markham ◽  
Claire M. Palmer ◽  
Malgorzata Chwatko ◽  
James M. Wagner ◽  
Clare Murray ◽  
...  
Keyword(s):  
Yarrowia Lipolytica ◽  
Building Blocks ◽  
Specific Productivity ◽  
Chemical Production ◽  
Conversion Yield ◽  
Acetyl Coa ◽  
Type Iii Polyketide Synthase ◽  
Chemical Catalysis ◽  
Acid Lactone ◽  
Triacetic Acid Lactone

Polyketides represent an extremely diverse class of secondary metabolites often explored for their bioactive traits. These molecules are also attractive building blocks for chemical catalysis and polymerization. However, the use of polyketides in larger scale chemistry applications is stymied by limited titers and yields from both microbial and chemical production. Here, we demonstrate that an oleaginous organism (specifically, Yarrowia lipolytica) can overcome such production limitations owing to a natural propensity for high flux through acetyl–CoA. By exploring three distinct metabolic engineering strategies for acetyl–CoA precursor formation, we demonstrate that a previously uncharacterized pyruvate bypass pathway supports increased production of the polyketide triacetic acid lactone (TAL). Ultimately, we establish a strain capable of producing over 35% of the theoretical conversion yield to TAL in an unoptimized tube culture. This strain also obtained an averaged maximum titer of 35.9 ± 3.9 g/L with an achieved maximum specific productivity of 0.21 ± 0.03 g/L/h in bioreactor fermentation. Additionally, we illustrate that a β-oxidation-related overexpression (PEX10) can support high TAL production and is capable of achieving over 43% of the theoretical conversion yield under nitrogen starvation in a test tube. Next, through use of this bioproduct, we demonstrate the utility of polyketides like TAL to modify commodity materials such as poly(epichlorohydrin), resulting in an increased molecular weight and shift in glass transition temperature. Collectively, these findings establish an engineering strategy enabling unprecedented production from a type III polyketide synthase as well as establish a route through O-functionalization for converting polyketides into new materials.

scholarly journals Inactivation of the polyketide synthase, 6-methylsalicylic acid synthase, by the specific modification of Cys-204 of the β-ketoacyl synthase by the fungal mycotoxin cerulenin

1998 ◽  
Vol 330 (2) ◽  
pp. 933-937 ◽  
Author(s):  
J. Christopher CHILD ◽  
Peter M. SHOOLINGIN-JORDAN
Keyword(s):  
Fatty Acid ◽  
Polyketide Synthase ◽  
Substrate Binding ◽  
Specific Inhibitor ◽  
Close Similarity ◽  
Type I ◽  
Specific Modification ◽  
Ketoacyl Synthase ◽  
Methylsalicylic Acid Synthase ◽  
Fatty Acid Synthases

Cerulenin, [(2S,3R)-2,3-epoxy-4-oxo-7,10-dodecadienoylamide], a mycotoxin produced by Cephalosporium caerulens, irreversibly inactivated 6-methylsalicylic acid synthase from Penicillium patulum. A combination of radiolabelling studies with [3H]cerulenin, proteolytic and chemical digestion and N-terminal sequencing of labelled peptides indicated that the site of cerulenin modification is the highly reactive substrate-binding Cys-204 of the β-ketoacyl synthase enzyme component. The thiol-specific inhibitor, iodoacetamide, was also shown to alkylate this residue. These findings are analogous with those observed for the reaction of cerulenin and iodoacetamide with type-I fatty acid synthases, demonstrating the close similarity between 6-methylsalicylic acid synthase and type-I fatty acid synthases.

Acyl carrier protein: structure–function relationships in a conserved multifunctional protein family

2007 ◽  
Vol 85 (6) ◽  
pp. 649-662 ◽  
Author(s):  
David M. Byers ◽  
Huansheng Gong
Keyword(s):  
Fatty Acid ◽  
Active Sites ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Divalent Cations ◽  
Structural Features ◽  
Carrier Protein ◽  
Type I ◽  
Prosthetic Group

Acyl carrier protein (ACP) is a universal and highly conserved carrier of acyl intermediates during fatty acid synthesis. In yeast and mammals, ACP exists as a separate domain within a large multifunctional fatty acid synthase polyprotein (type I FAS), whereas it is a small monomeric protein in bacteria and plastids (type II FAS). Bacterial ACPs are also acyl donors for synthesis of a variety of products, including endotoxin and acylated homoserine lactones involved in quorum sensing; the distinct and essential nature of these processes in growth and pathogenesis make ACP-dependent enzymes attractive antimicrobial drug targets. Additionally, ACP homologues are key components in the production of secondary metabolites such as polyketides and nonribosomal peptides. Many ACPs exhibit characteristic structural features of natively unfolded proteins in vitro, with a dynamic and flexible conformation dominated by 3 parallel α helices that enclose the thioester-linked acyl group attached to a phosphopantetheine prosthetic group. ACP conformation may also be influenced by divalent cations and interaction with partner enzymes through its “recognition” helix II, properties that are key to its ability to alternately sequester acyl groups and deliver them to the active sites of ACP-dependent enzymes. This review highlights recent progress in defining how the structural features of ACP are related to its multiple carrier roles in fatty acid metabolism.

scholarly journals Staphylococcus aureus Fatty Acid Auxotrophs Do Not Proliferate in Mice

2013 ◽  
Vol 57 (11) ◽  
pp. 5729-5732 ◽  
Author(s):  
Joshua B. Parsons ◽  
Matthew W. Frank ◽  
Jason W. Rosch ◽  
Charles O. Rock
Keyword(s):  
Fatty Acids ◽  
Staphylococcus Aureus ◽  
Fatty Acid ◽  
Fatty Acid Synthesis ◽  
Normal Growth ◽  
Acid Synthesis ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa ◽  
Content Type

ABSTRACTInactivation of acetyl-coenzyme A (acetyl-CoA) carboxylase confers resistance to fatty acid synthesis inhibitors inStaphylococcus aureuson media supplemented with fatty acids. The addition ofanteiso-fatty acids (1 mM) plus lipoic acid supports normal growth of ΔaccDstrains, but supplementation with mammalian fatty acids was less efficient. Mice infected with strain RN6930 developed bacteremia, but bacteria were not detected in mice infected with its ΔaccDderivative.S. aureusbacteria lacking acetyl-CoA carboxylase can be propagatedin vitrobut were unable to proliferate in mice, suggesting that the acquisition of inactivating mutations in this enzyme is not a mechanism for the evasion of fatty acid synthesis inhibitors.

scholarly journals Antimycobacterial action of thiolactomycin: an inhibitor of fatty acid and mycolic acid synthesis.

1996 ◽  
Vol 40 (12) ◽  
pp. 2813-2819 ◽  
Author(s):  
R A Slayden ◽  
R E Lee ◽  
J W Armour ◽  
A M Cooper ◽  
I M Orme ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Mycolic Acid ◽  
Carrier Protein ◽  
Type I ◽  
Dependent Manner ◽  
Fas Ii

Thiolactomycin (TLM) possesses in vivo antimycobacterial activity against the saprophytic strain Mycobacterium smegmatis mc2155 and the virulent strain M. tuberculosis Erdman, resulting in complete inhibition of growth on solid media at 75 and 25 micrograms/ml, respectively. Use of an in vitro murine macrophage model also demonstrated the killing of viable intracellular M. tuberculosis in a dose-dependent manner. Through the use of in vivo [1,2-14C]acetate labeling of M. smegmatis, TLM was shown to inhibit the synthesis of both fatty acids and mycolic acids. However, synthesis of the shorter-chain alpha'-mycolates of M. smegmatis was not inhibited by TLM, whereas synthesis of the characteristic longer-chain alpha-mycolates and epoxymycolates was almost completely inhibited at 75 micrograms/ml. The use of M. smegmatis cell extracts demonstrated that TLM specifically inhibited the mycobacterial acyl carrier protein-dependent type II fatty acid synthase (FAS-II) but not the multifunctional type I fatty acid synthase (FAS-I). In addition, selective inhibition of long-chain mycolate synthesis by TLM was demonstrated in a dose-response manner in purified, cell wall-containing extracts of M. smegmatis cells. The in vivo and in vitro data and knowledge of the mechanism of TLM resistance in Escherichia coli suggest that two distinct TLM targets exist in mycobacteria, the beta-ketoacyl-acyl carrier protein synthases involved in FAS-II and the elongation steps leading to the synthesis of the alpha-mycolates and oxygenated mycolates. The efficacy of TLM against M. smegmatis and M. tuberculosis provides the prospects of identifying fatty acid and mycolic acid biosynthetic genes and revealing a novel range of chemotherapeutic agents directed against M. tuberculosis.

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