Synthesis Of 6-methylsalicylic Acid Research Articles

Targeting mitochondrial metabolite transporters in Penicillium expansum for reducing patulin production

There is an increasing need of alternative treatments to control fungal infection and consequent mycotoxin accumulation in harvested fruits and vegetables. Indeed, only few biological targets of antifungal agents have been characterized and can be used for limiting fungal spread from decayed fruits/vegetables to surrounding healthy ones during storage. On this concern, a promising target of new antifungal treatments may be represented by mitochondrial proteins due to some species-specific functions played by mitochondria in fungal morphogenesis, drug resistance and virulence.One of the most studied mycotoxins is patulin produced by several species of Penicillium and Aspergillus genera. Patulin is toxic to many biological systems including bacteria, higher plants and animalia. Although precise biochemical mechanisms of patulin toxicity in humans are not completely clarified, its high presence in fresh and processed apple fruits and other apple-based products makes necessary developing a strategy for limiting its presence/accumulation. Patulin biosynthetic pathway consists of an enzymatic cascade, whose first step is represented by the synthesis of 6-methylsalicylic acid, obtained from the condensation of one acetyl-CoA molecule with three malonyl-CoA molecules. The most abundant acetyl-CoA precursor is represented by citrate produced by mitochondria. In the present investigation we report about the possibility to control patulin production through the inhibition of mitochondrial/peroxisome transporters involved in the export of acetyl-CoA precursors from mitochondria and/or peroxisomes, with specific reference to the predicted P. expansum mitochondrial Ctp1p, DTC, Sfc1p, Oac1p and peroxisomal PXN carriers.

A Synthetic Malonyl-CoA Metabolic Oscillator in Komagataella phaffii.

Malonyl-CoA is a key metabolic molecule that participates in a diverse range of physiological responses and can act as a building block for a variety of value-added pharmaceuticals and chemicals. The cytosolic malonyl-CoA concentration is usually very low, and thus dynamic metabolic control of malonyl-CoA variation will aid its stable formation and efficient consumption. We developed a synthetic malonyl-CoA metabolic oscillator in yeast. A synthetic regulatory protein, Prm1-FapR, was constructed by fusing a yeast transcriptional activator, Prm1, with a bacterial malonyl-CoA-sensitive transcription repressor, FapR. Two oppositely regulated biosensors were then engineered. A total of 18 hybrid promoter variants were designed, each carrying the operator sequence (fapO) of FapR and the core promoter of PAOX1 (cPAOX1), which is naturally regulated by Prm1. The promoter activities of these variants, regulated by Prm1-FapR, were tested. Through this process, a sensor for Prm1-FapR/(-52)fapO-PAOX1 carrying an activation/deactivation regulation module was built. Meanwhile, 24 promoter variants of PGAP with fapO inserted were designed and tested using the fusion regulator, giving a sensor for Prm1-FapR/PGAP-(+22) fapO that contained a repression/derepression regulation module. Both sensors were subsequently integrated into a single cell, which exhibited correct metabolic switching of eGFP and mCherry reporters following manipulation of cytosolic malonyl-CoA levels. The Prm1-FapR/(-52)fapO-PAOX1 and the Prm1-FapR/PGAP-(+22)fapO were also used to control the malonyl-CoA source and sink pathways, respectively, for the synthesis of 6-methylsalicylic acid. This finally led to an oscillatory metabolic mode of cytosolic malonyl-CoA. Such a metabolator is useful in exploring potential industrial and biomedical applications not limited by natural cellular behavior.

6-Methylsalicylic acid synthetase from Penicillium patulum. Some catalytic properties of the enzyme and its relation to fatty acid synthetase.

1. The specificity of 6-methylsalicylic acid synthetase with respect to the priming substrate was studied. If acetyl-CoA was replaced by propionyl-CoA 6-ethylsalicylic acid was synthesized. The rate of this synthesis was about 13% that of 6-methylsalicylic acid synthesis in presence of acetyl-CoA. 2. 6-Methylsalicylic acid synthetase contains an acetyl transferase activity as demonstrated by the transfer of acetyl residues from acetyl-CoA to pantetheine. This transferase also catalyses propionyl transfer but only with an about 13 times slower rate. 3. Analogous to fatty acid synthetase, treatment with iodoacetamide converts 6-methylsalicylic acid synthetase into a malonyl-CoA decarboxylase. Under certain conditons the iodoacetamide-treated enzyme could catalyse 6-methylsalicylic acid formation from malonyl CoA and NADPH without the external addition of acetyl CoA.

Inhibition of 6-methylsalicyclic acid synthesis by the antibiotic cerulenin.

Cerulenin, a specific inhibitor of beta-ketoacyl-(acyl carrier protein) synthetase [EC 2.3.1.41] and 3-hydroxy-3-methylglutaryl-CoA synthetase [EC 4.1.3.5], was studied to determine whether it inhibits 6-methylsalicylic acid synthesis, in which so-called "polyketide" formation, a condensation step similar to that in fatty acid synthesis, is involved. In fact, 100 mug/ml (4.5 X 10(-4) M) of cerulenin inhibited 60% of 6-methylsalicylic acid synthetase activity and 68% of fatty acid synthetase activity of Penicillium urticae.

Enzymic Synthesis of an Aromatic Ring from Acetate Units

Flavanone synthase was isolated and purified about 300-fold from fermenter-grown, light-induced cell suspension cultures of Petroselinum hortense. The enzyme catalyzed the formation of the flavanone naringenin from p-coumaroyl-CoA and malonyl-CoA. Trapping experiments with an enzyme preparation, which was free of chalcone isomerase activity, revealed that in fact the flavanone and not the isomeric chalcone was the immediate product of the synthase reaction. Thus the enzyme is not a chalcone synthase as previously assumed. No coafactors were required for flavanone synthase activity. The enzyme was strongly inhibited by the two reaction products naringenin and CoASH, by the antibiotic cerulenin, by acetyl-CoA, and by several compounds reacting with sulfhydryl groups. Optimal enzyme activity was found at pH 8.0, at 30 degrees C, and at an ionic strength of 0.1--0.3 M potassium phosphate. EDTA, Mg2+, Ca2+, or Fe2+ at concentrations of about 0.7 muM did not affect the enzyme activity. Apparent molecular weights of approx. 120 000, 50 000, and 70 000, respectively, were determined for flavanone synthase and two metabolically related enzymes, chalcone isomerase and malonyl-CoA: flavonoid glycoside malonyl transferase. The partially purified flavanone synthase efficiently catalyzed the formation of malonyl pantetheine from malonyl-CoA and pantetheine. This malonyl transferase activity, and a general similarity with the condensation steps involved in the mechanisms of fatty acid and 6-methylsalicylic acid synthesis from "acetate units", are the basis for a hypothetical scheme which is proposed for the sequence of reactions catalyzed by the multifunctional flavanone synthase.

Biosynthesis of polyketides the synthesis of 6-methylsalicylic acid and triacetic acid lactone in Penicillium patulum

An enzyme fraction from P. patulum has been prepared which will synthesize 6-MSA 5 5 The abbreviations used are: NAC, N-acetylcysteamine; 6-MSA, 6-methylsalicylic acid; TAL, triacetic acid lactone. and fatty acids from acetyl CoA, malonyl CoA and NADPH. The 6-MSA synthetase was purified threefold by ammonium sulphate fractionation and catalyses the formation of 0.86 nmoles of 6-MSA per minute per mg of protein. Chemical degradation of 14C-6-MSA biosynthesized from 1,3- 14C-malonyl CoA, acetyl CoA and NADPH gave a ratio of 2.39 1 for the radioactivity in the aromatic ring to that in the terminal carboxyl group. This is consistent with the acetatepolymalonate origin of 6-MSA; the deviation from the theoretical value of 2 1 is probably due to malonyl CoA decarboxylase activity leading to the formation of 1- 14C-acetyl CoA. The rate of synthesis of 6-MSA was generally about ten times faster than the rate of fatty acid synthesis, but this ratio varied with the preparation. The 6-MSA synthetase is inhibited by isodoacetamide and N-ethymaleimide, which suggests that the synthetase activity contains sulphydryl groups in the active sites. The 6-MSA synthetase activity is inhibited by the following acetylenic thioesters: 3-pentynoyl-NAC, 3-hexynoyl-NAC and 2-hexynoyl-NAC. The activity is not inhibited by 3-hexynoic acid. The synthesis of 6-MSA can be abolished at a concentration of 10 −4 M 3-hexynoyl-NAC without affecting the synthesis of free palmitic and stearic acid. The synthesis of 6-MSA by P. patulum under in vivo conditions is also inhibited by 3-hexynoyl-NAC. The syntheses of 6-MSA and TAL (in the presence of NADPH) are inhibited to comparable extents by 5 × 10 −5 M 3-hexynoyl-NAC while the synthesis of fatty acids is not inhibited below 10 −4 M. The mechanism of 6-MSA synthesis is discussed in relation to the formation of TAL and to the possible mode of action of the acetylenic thioester inhibitors.

6-Methylsalicylic acid synthesis by isolated barley chloroplasts.

1 Chloroplasts isolated from dark grown barley leaves greened for 6 h rapidly incorporate [1-14C]acetate into 6-methylsalicylic acid. Etioplasts synthesize little 6-methylsalicylic acid and mature chloroplasts lack this activity. 2 Carbon dioxide and CoA are essential cofactors for the synthesis. While light increases the synthesis of 6-methylsalicylic acid, a considerable amount of the acid is formed in the dark. Externally supplied NADPH has no effect on the incorporation of [1-14C]acetate into 6-methylsalicylic acid by isolated chloroplasts. 3 The biosynthesized 6-methylsalicylic acid has been purified by thin layer chromatography. Characterization of the labeled compound is done by radio gas liquid chromatography, by repeated crystallization to constant specific radioactivity and by identification of its nitration product as 3-methyl-2,4,6-trinitrophenol. 4 Since 75% of the radioactivity of 6-methylsalicylic acid synthesized by isolated chloroplasts is confined to the aromatic nucleus, a mechanism of biosynthesis involving head to tail condensation of acetyl CoA with malonyl CoA is indicated.

Biosynthesis of 6-methylsalicylic acid

An extract was prepared from Penicillium patulum grown in submerged culture, which converted acetyl‐CoA and malonyl‐CoA into 6‐methylsalicylic acid in the presence of NADPH. The enzyme was very labile in the crude extract, but appeared stable after acetone precipitation. A 100‐fold purification was achieved by ammonium sulfate fractionation, sedimentation in the ultracentrifuge, and sucrose‐density gradient centrifugation. The enzyme was a single particle with a molecular weight of 1.1–1.5 × 106 as determined by gradient centrifugation with catalase and fatty acid synthetase as standards. In the first steps of purification, fatty acid synthetase from Penicillium patulum migrated with the methylsalicylic acid synthetase complex. A separation of the two enzyme systems was accomplished by sucrose‐density gradient centrifugation. The purified enzyme represents a new multienzyme complex which shares several common properties with fatty acid synthetase. Methylsalicylic acid synthetase is inhibited by iodoacetamide and N‐ethylmaleinimide. The incubation of enzyme with acetyl‐CoA and malonyl‐CoA in the absence of NADPH led to triacetic acid lacton. This indicates that in 6‐methylsalicylic acid synthesis reduction occurs at the triacetic acid level before the final condensation with malonyl‐CoA. Triacetic acid lacton was produced by the enzyme at 10% of the rate of 6‐methylsalicylic acid synthesis in the complete system. This rate of lacton synthesis, however, is an order of magnitude greater than that catalysed by fatty acid synthetase. A possible explanation for this difference in behavior is given. Covalent linkage of substrates and intermediates to SH‐groups of the protein during the synthesis of 6‐methylsalicyslic acid is suggested. A reaction scheme leading to 6‐methylsalicylic acid is proposed and the partial reactions of this synthesis are discussed with respect to similar reactions of fatty acid biosynthesis.

Inhibition and possible feedback by 6-methylsalicylic acid.

WHEN commenting generally on secondary metabolic phenomena and on the particular example of the synthesis of 6-methylsalicylic acid (6-MS) in Penicillium urticae Bainier (≡P. patulum Bainier, etc.), we referred1 to unpublished evidence suggesting that 6-MS could exert feedback inhibition on its own synthesis. Recently, Arihood and Light have described2 investigations of this problem, and have reported that the addition of 6-MS, or of certain related compounds, inhibits the incorporation into 6-MS of carbon-14 from exogenous 1-14C-acetate in intact mycelium. Our own work, however, revealed unsuspected complexities in this type of experiment. These have been summarized elsewhere3, but the publication of Arihood and Light prompts us to record some of our evidence in more detail. The basic experimental techniques and the general dynamics of 6-MS synthesis have already been described4.

Inhibition of 6-Methylsalicylic Acid Synthesis in Penicillium patulum

VARIOUS reports1–3 have indicated that the biosynthesis of 6-methylsalicylic acid (2,6-cresotic acid; 6-MSA) may be patterned after fatty acids, involving the condensation of one acetyl and three malonyl derivatives (presumably derivatives of coenzyme A or of a protein analogous to the acyl carrier protein4 in E. coli). Bu'Lock and Ryan5 and later Tanenbaum and Bassett6 showed that 6-MSA is metabolized by Penicillium patulum to form patulin and possibly other compounds as well. These compounds, which have been designated secondary metabolites7, have no apparent function in the organism. While the formation of 6-MSA is limited to a few species, the phenomenon of secondary metabolite formation is widespread in micro-organisms.