Robley J. Light

6-Methylsalicylic acid decarboxylase from Penicillium patulum

Abstract 1. 1. A crude extract catalyzing the decarboxylation of 6-methylsalicyclic acid has been isolated from Penicillium patulum. 2. 2. Culture conditions which lead to the formation of decarboxylase activity are described. 3. 3. No other activity metabolizing 6-methylsalicylic acid can be detected in vitro with extracts containing the decarboxylase. 4. 4. The decarboxylase has a broad pH optimum between 7 and 8, and the K m for 6-methylsalicylic acid is 7 μM. Decarboxylase activity is eluted from a Bio-Gel A5m column at a position corresponding to a Stokes radius of about 30 A.

Structure and composition of hydrocarbons and fatty acids from a marine blue-green alga, Synechococcus sp.

The major hydrocarbons of Synechococcus sp., a marine blue-green alga isolated from the Gulf of Mexico, were identified as 1-nonadecene and 1,14-nonadecadiene. The content of 1-nonadecene increased with culture age from about 0.5 mg/g dry weight in young cells to about 2.3 mg/g dry weight in old cells, while the content of 1,14-nonadecadiene remained constant with culture age at about 1 mg/g dry weight. Both [1-14C]acetate and [2-14C]acetate were incorporated to equal extents into the hydrocarbons. [1-14C]Stearate was incorporated into the hydrocarbons, but [3H]arachidate was not. The fatty acids of Synechococcus sp. were typical of blue-green algae, consisting of C16:0, C16:1, C16:1, C16:0, C18:0, C18:1, C18:2 and C18:3 fatty acids were detected. The hexadecenoic acid was shown to be 9-C16:1 while the octadecenoic acid was a mixture of 93% 11-C18:1 and 7% 9-C18:1. The fatty acid content increased during the first 4 days of growth and then decreased slightly.

Molecular parameters of 6-methylsalicylic acid synthetase from gel filtration and sucrose density gradient centrifugation.

Abstract Molecular parameters for 6-methylsalicylic acid (MSA) synthetase have been estimated by using a combination of Sephadex G-200 gel filtration and sucrose density gradient centrifugation on crude extracts of Penicillium patulum. A value of 85A for the Stokes radius from gel filtration and an s 20.w of 10.4S from sucrose density gradient centrifugation leads to a molecular weight of about 3.7 × 10 5 and a frictional ratio ( f f 0 ) of 1.8 when a partial specific volume of 0.725 cm 3 is assumed. The synthesis of MSA therefore appears to be carried out by a molecular complex of enzymes similar to the fatty acid synthetase complex. The MSA synthetase activity is stable in lyophilized mycelium for at least several weeks, but the activity rapidly decays in crude extracts. While enough stabilization is obtained in high ionic strength buffer to permit the characterization reported here, instability has so far prevented purification of the crude extracts.

Effects of cycloheximide and amino acid analogues on biosynthesis of 6-methylsalicylic acid in Penicillium patulum☆

Abstract The effect of several inhibitors on formation of 6-methylsalicylic acid (MSA) synthetase activity in two isolates of Penicillium patulum NRRL 2159A has been studied. Synthetase activity was measured in crude extracts by the incorporation of 14 C-malonyl-CoA into MSA. The two isolates differ in the time of appearance of MSA synthetase after transfer from a germinating medium to Czapek-Dox medium. Cycloheximide at a concentration of 0.3 μg/ml stimulates the formation of synthetase activity in both strains, but inhibits formation of the activity at a concentration of 10 μg/ml. In comparing this effect with the inhibition of protein synthesis by cyclo-heximide, it was found that a concentration of 10 μg/ml is sufficient to block in vivo incorporation of leucine- 14 C into trichloroacetic acid-insoluble material, while 0.3 μg/ml only partially inhibits this incorporation. dl - p -Fluorophenylalanine and dl -4-methyltryptophan also stimulate formation of MSA synthetase activity at a concentration of 10–100 μg/ml and partially inhibit formation at 1 mg/ml. dl -5-Methyltryptophan shows slight inhibition but little stimulation. dl -Ethionine at 100μg/ml partially inhibits MSA synthetase appearance. It is suggested that the inhibitors produce metabolic changes similar to those found in older cultures when MSA synthesis is prevalent. The possibility of applying this stimulatory effect to increase production yields of some antibiotics is mentioned.

Acetate metabolism in Penicillium griseofulvum. Incorporation of 1-C14-2-H3-acetate into 6-methylsalicylic acid and fatty acids.

Abstract Twelve-day cultures of Penicillium griseofulvum (ATCC 11885) were supplied with a pulse of 1-C 14 -2-H 3 acetate for 6 hours. The fatty acids were isolated from the mycelium and their methyl esters were separated by combining silicic acid chromatography of mercuric acetate adducts with gas-liquid chromatography. The unsaturated methyl esters were degraded by permanganate-periodate oxidation, and the oxidation products were isolated. 6-Methylsalicylic acid was isolated from the growth medium, diluted with carrier, and degraded by bromination and by Kuhn-Roth oxidation. Specific activities and H 3 C 14 ratios in the isolated compounds and their degraded products provide information on the distribution of isotope in the molecules. Of the three equivalents of tritium present in the starting acetate, 0.75 equivalent appears to be retained at the even carbon atoms of the fatty acid chain, and 0.68 equivalent appears to be retained in the corresponding ring positions of the 6-methylsalicylic acid molecule.

Applications of high-performance liquid chromatography to quantitation of metabolites and enzymes of the patulin pathway from Penicillium patulum.

Conditions for extraction and high-performance liquid chromatographic (HPLC) analysis for fourteen of the patulin pathway metabolites from Penicillium patulum are described which allow quantitation of the metabolite content of cultures at hourly intervals. The HPLC analysis is more sensitive than gas-liquid chromatographic analysis and is more quantitative than thin-layer chromatographic analysis. Separations on a preparative column allow for the collection and identification of new metabolites. The column elution program can be varied to optimize analysis time for individual metabolites, allowing individual enzymes of the pathway to be assayed by following the conversion of substrate to product. Analysis of product formation in crude enzyme mixtures can be used to assay an enzyme in the presence of subsequent enzymes of the pathway and to establish the pathway reaction sequence.

Identification and analysis of arachidonic acid in Plexaura homomalla, Var. R And Var. S

Abstract The content of eicosatetraenoic (C20:4) acid in Plexaura homomalla lipids was determined by transesterification with sodium methoxide and gas-liquid chromatography of the resulting fatty acid methyl esters. The C20:4 methyl ester constituted 22.6 % of the non-polar fatty acid esters of variant R lipids, 33.9 % of the non-polar fatty acid esters of variant S lipids, and 0.4% of the dry weight in both variants. The position and stereochemistry of the double bonds in the C20:4 acid was determined by partial reduction to a C20:1 acid mixture, separation of cis and trans C20:1 methyl esters on AgNO3 impregnated silicic acid thin layer plates, and oxidative cleavage of the C20:1 methyl ester mixture with permanganate-periodate. The double bonds were shown to be at Positions 5, 8, 11, and 14 with less than 2.4 % of the molecules having a trans double bond. Therefore, more than 97% of the C20:4 acid in the lipids of both variant S and variant R of P. homomalla is arachidonic acid, and the difference in stereo-chemistry at Position 15 of the P. homomalla prostaglandins ((15R) for variant R, (15S) for variant S) cannot be reasonably explained by a stereochemical difference of a C20:4 precursor fatty acid.

Biosynthesis of hydrocarbon in anabaena variabilis in vivo incorporation of [18-14c]stearate

Abstract Cell-free extracts of Anabaena variabilis which are capable of incorporating the methyl group of S -[methyl- 14 C ] adenosylmethionine into branch methylheptadecanes (Fehler, S.W.G. and Light, R.J. (1972) Biochem-istry 11, 2411–2416) have now been shown to incorporate [18- 14 C]stearate into alkane. The rate of stearate incorporation is only about 0.1% that of the methyl group incorporation, and only about 10% the rate of stearate decarboxylation, as measured by conversion of [1- 14 C]stearate to carbon dioxide. Oxygen stimulates the stearate incorporation into alkanes but inhibits the methyl group incorporation.

Liver fatty acids of Rana grylio tadpoles and frogs

Abstract 1. 1. Liver fatty acids of Rana grylio before, during and after the early stages of metamorphosis have been analyzed by GLC (gas-liquid chromatography). Of the twenty-six peaks discernible in the fatty acids spectra, eight are present in quantities greater than 5%. On the basis of relative retention times and polarity of mercuric acetate adducts, these acids have been identified as C 16 : 0, C 16 : 0,C 18 : 0, C 18 : 1, C 18 : 2, C 18 : ⩾3, C 20 : ⩾3, and C 21 : ⩾3 (chain length: number of double bonds). 2. 2. There seems to be no appreciable difference in either the quantity or types of liver fatty acids which can be associated with metamorphosis.