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E A Cossins

Publications and source records attributed to E A Cossins.

At least 19 recordsLinked to original sources
PubMed1998

Folylpolyglutamate synthesis in Neurospora crassa: primary structure of the folylpolyglutamate synthetase gene and elucidation of the met-6 mutation.

In Neurospora crassa, the met-6+ gene encodes folylpoly-gamma-glutamate synthetase (FPGS) which catalyzes the formation of polyglutamate forms of folate. Methionine auxotrophy of the Neurospora crassa met-6 mutant is related to a lesion affecting this enzyme. Functional complementation of the mutant strain was achieved by introducing copies of the wild-type met-6+ gene into mutant spheroplasts. The complementing sequences were found to be contained on a 3.5 kb EcoRI-BamHI restriction fragment. The nucleotide sequence of the met-6+ gene was determined and an open reading frame of 1587 bp was identified, interrupted by two introns. This open reading frame contained several AUG codons but translation beginning from either of the first two would theoretically produce a protein of appropriate size and with similarity to five other FPGS proteins. Northern blot analyses of met-6+ transcripts revealed a 2.0 kb product. The position of the transcription stop site and an intron were identified by sequencing partial cDNA clones which were truncated at the 5' end. DNA sequence analysis of the met-6 mutant allele revealed a T to C transition which would result in replacement of a highly conserved serine with a proline.

Amino Acid Sequence↗
PubMed1997

Folates and one-carbon metabolism in plants and fungi.

Folate-dependent pathways of one-carbon metabolism are essential for the synthesis of purines, formylmethionyl-tRNA, thymidylate, serine and methionine. These syntheses use a cellular source of one-carbon substituted, tetrahydrofolate polyglutamate derivatives which are the preferred substrates of most folate-dependent enzymes. In the last decade, there have been major advances in the folate biochemistry of animal, bacterial, fungal and plant systems. These have included the refinement of methods for folate isolation and characterization, basic work on key enzymes of folate biosynthesis and the detailed characterization of proteins that catalyze the generation and utilization of one-carbon substituted folates.

Folic Acid↗
PubMed1995

Isolation of dihydrofolate and folylpolyglutamate synthetase activities from Neurospora.

The possible association of dihydrofolate synthetase (DHFS) and folylpolyglutamate synthetase (FPGS) in Neurospora crassa (FGSC 853, wild type) has been examined using mycelial extracts prepared and fractionated in the presence of protease inhibitors. DHFS and FPGS were assayed by following the incorporation of labelled glutamate into dihydrofolate and methylenetetrahydrofolate polyglutamate, respectively. Both of these activities were predominately cytosolic in mycelia that were harvested 24 hr after spore inoculation of defined minimal medium. Relatively small amounts of total mycelial DHFS activity were associated with mitochondrial fractions isolated by differential centrifugation. In contrast, ca 20% of the mycelial FPGS activity was mitochondrial. Treatment of the mitochondrial fractions with Triton X-100 suggested that these activities were not latent under the assay conditions employed. Separate peaks of DHFS and FPGS activity were observed when (NH4)2SO4-fractionated protein was desalted and chromatographed on columns of either Mono Q HR, DEAE-cellulose, heparin agarose, Matrex Green A or Reactive Green 5. Gel filtration indicated average Mr values of 52 and 66 x 10(3) for DHFS and FPGS protein, respectively. Dihydrofolate synthetase protein was purified over 1000-fold by a protocol that included chromatography on DEAE-cellulose, DEAE-Sephacel, heparin agarose and Matrex Green A. The isolated protein lacked ability to glutamyl conjugate 5,10-methylene tetrahydrofolate. SDS-polyacrylamide gel electrophoresis of the Matrex Green A peak fractions revealed a major protein band of average Mr 52 x 10(3) whose concentration appeared to parallel DHFS activity. FPGS protein (average Mr 66 x 10(3)), which lacked ability to glutamyl conjugate dihydropteroate, was recovered by a similar protocol.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromatography, Ion Exchange↗
PubMed1995

Folylpolyglutamate synthesis in Neurospora crassa: transformation of polyglutamate-deficient mutants.

The methionine auxotrophy of Neurospora crassa met-6 and mac mutants is related to an inability to synthesize long-chained folylpolyglutamates. Both of these lesions affect folylpolyglutamate synthetase activity, but it is not clear whether these mutations occur in different genes or in functional domains of the same gene. To address this question, copies of the met-6+ gene have been introduced into both mutants using plasmid and cosmid vectors. Transformation to prototrophy was achieved in both mutants. The ability of these mutant and transformant strains to synthesize folylpolyglutamates was assessed by HPLC analysis of folate cleavage products. Mycelial extracts of the wild type revealed a folate pool dominated by folylhexaglutamates. These folates were also detected in the transformants but were lacking in both mutants. In the latter strains, the conjugated folates were mainly di- and triglutamates. When incubated for 24 hr with [14C]p-aminobenzoate, transformant and wild type cultures synthesized long chain folates, ca60-80% of these being hexaglutamyl derivatives. In contrast, the labelled folates of mac and met-6 were mainly mono- and diglutamyl derivatives, respectively. Polyglutamate synthesis was also studied in vitro by partial purification and characterization of mycelial folylpolyglutamate synthetase protein. Mycelial extracts of the wild type and transformant cultures utilized 5,10-methylenetetrahydrofolate monoglutamate and its diglutamate as substrates in this synthetase reaction. In contrast, extracts of met-6 and mac mycelia utilized only one of these folate substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

4-Aminobenzoic Acid↗
PubMed1994

5-Formyltetrahydropteroylpolyglutamates are the major folate derivatives in Neurospora crassa conidiospores.

5-Formyltetrahydropteroylpolyglutamate (5-CHO-H4PteGlun) is the only reduced folate derivative that is stable to oxidation and alkaline pH. However, no metabolic role has been assigned to this folate derivative, and evidence for its existence in cells has been questioned. Recently, serine hydroxymethyltransferase was shown to catalyze the formation of 5-CHO-H4PteGlun from 5,10-methenyl-H4PteGlun (Stover, P., and Schirch, V. (1990) J. Biol. Chem. 265, 14227-14233). We have proposed that 5-CHO-H4PTeGlun may serve as a storage form of reduced folates and one-carbon groups in cells that are in a dormant stage. This hypothesis was tested by determining the levels of H4PteGlun derivatives in the mycelia and conidiospores of Neurospora crassa and a mutant strain that lacks cytosolic serine hydroxymethyltransferase. N. crassa serine hydroxymethyltransferase was purified to homogeneity and characterized with respect to kinetic constants, quaternary structure, stability, and reaction specificity. A new assay for determining the concentration of the polyglutamate forms of H4PteGlun derivatives was also developed. Using this assay, it was shown that 85% of the tetrahydropteroylpolyglutamates in conidiospores was 5-CHO-H4PteGlun. After adding the spores to growth media, the 5-CHO-H4PteGlun was reduced to less than 10% of the folate pool in 20 min. Mycelia had no detectable 5-CHO-H4PteGlun. Only 10-20% of the folate pool in conidiospores from the mutant strain lacking cytosolic serine hydroxymethyltransferase was in the form of 5-CHO-H4PteGlun.

Formyltetrahydrofolates↗
PubMed1993

Effects of tetrahydrofolate polyglutamates on the kinetic parameters of serine hydroxymethyltransferase and glycine decarboxylase from pea leaf mitochondria.

Plant tissues contain highly conjugated forms of folate. Despite this, the ability of plant folate-dependent enzymes to utilize tetrahydrofolate polyglutamates has not been examined in detail. In leaf mitochondria, the glycine-cleavage system and serine hydroxymethyltransferase, present in large amounts in the matrix space and involved in the photorespiratory cycle, necessitate the presence of tetrahydrofolate as a cofactor. The aim of the present work was to determine whether glutamate chain length (one to six glutamate residues) influenced the affinity constant for tetrahydrofolate and the maximal velocities displayed by these two enzymes. The results show that the affinity constant decreased by at least one order of magnitude when the tetrahydrofolate substrate contained three or more glutamate residues. In contrast, maximal velocities were not altered in the presence of these substrates. These results are consistent with analyses of mitochondrial folates which revealed a pool of polyglutamates dominated by tetra and pentaglutamates. The equilibrium constant of the serine hydroxymethyltransferase suggests that, during photorespiration, the reaction must be permanently pushed toward the formation of serine (the unfavourable direction) to allow the recycling of tetrahydrofolate necessary for the operation of the glycine decarboxylase T-protein.

Amino Acid Oxidoreductases↗
PubMed1988

Dark Respiration during Photosynthesis in Wheat Leaf Slices.

The metabolism of [(14)C]succinate and acetate was examined in leaf slices of winter wheat (Triticum aestivum L. cv Frederick) in the dark and in the light (1000 micromoles per second per square meter photosynthetically active radiation). In the dark [1,4-(14)C]succinate was rapidly taken up and metabolized into other organic acids, amino acids, and CO(2). An accumulation of radioactivity in the tricarboxylic acid cycle intermediates after (14)CO(2) production became constant indicates that organic acid pools outside of the mitochondria were involved in the buildup of radioactivity. The continuous production of (14)CO(2) over 2 hours indicates that, in the dark, the tricarboxylic acid cycle was the major route for succinate metabolism with CO(2) as the chief end product. In the light, under conditions that supported photorespiration, succinate uptake was 80% of the dark rate and large amounts of the label entered the organic and amino acids. While carbon dioxide contained much less radioactivity than in the dark, other products such as sugars, starch, glycerate, glycine, and serine were much more heavily labeled than in darkness. The fact that the same tricarboxylic acid cycle intermediates became labeled in the light in addition to other products which can acquire label by carboxylation reactions indicates that the tricarboxylic acid cycle operated in the light and that CO(2) was being released from the mitochondria and efficiently refixed. The amount of radioactivity accumulating in carboxylation products in the light was about 80% of the (14)CO(2) release in the dark. This indicates that under these conditions, the tricarboxylic acid cycle in wheat leaf slices operates in the light at 80% of the rate occurring in the dark.

Journal Article↗
PubMed1983

Folylpolyglutamate synthetase activities of Neurospora crassa: nature of products formed by soluble and particulate enzymes in the wild type and polyglutamate-deficient mutants.

The folylpolyglutamate synthetase activities of Neurospora crassa wild type (FGSC 853) and two polyglutamate-deficient mutants (met-6, 35809, FGSC 1330 and mac, 65108, FGSC 3609) were examined using dialyzed extracts prepared during exponential mycelial growth. Enzyme assay was based on incorporation of [U-3H]glutamate in folylpolyglutamates that were separated by gradient elution from DEAE-cellulose. Extracts of the wild type produced H4PteGlu2 (15%), H4PteGlu3 (35%) and H4PteGlu6 (50%) when anaerobically incubated with glutamate, ATP, and H4PteGlu. Under these conditions, the met-6 produced only H4PteGlu2 and higher polyglutamates (H4PteGlu4 and H4PteGlu5) were not utilized. The mac mutant failed to catalyze addition of glutamate to H4PteGlu. However, H4PteGlu2 was effectively converted to the tri-, and hexaglutamates. Mixing wild type and met-6 protein stimulated the formation of tri-, and hexaglutamates. Mixing mac and met-6 extracts resulted in H4PteGlu3 and H4PteGlu6 labeling when glutamate and H4PteGlu were provided. Fractionation of wild type extracts by addition of (NH4)2SO4 or by differential centrifugation provided evidence for different synthetase activities. Protein of the 0-35% (NH4)2SO4 fraction and that associated with the mitochondrial pellet, catalyzed an H4PteGlu2 leads to H4PteGlu3 reaction. These fractions failed to utilize H4PteGlu or the corresponding tetra-, and pentagluatmates. This triglutamate-forming activity was lacking in mac and met-6. The 45-60% (NH4)2SO4 fraction of the wild type catalyzed formation of di-, tri-, and hexaglutamate from H4PteGlu and glutamate. Hexaglutamate was also formed when the folate substrate wa H4PteGlu2, H2PteGlu4, or H4PteGlu5. These activities were associated with the cytosolic fraction when crude isotonic extracts were centrifuged to remove mitochondria. The characteristic synthetase activities of met-6 and mac were associated with protein of the 45-60% (NH4)2SO4 and cytosolic fractions. It is suggested that folypolyglutamate synthesis in N. crassa involves more than one synthetase-catalyzed reaction. Production of cellular folylhexaglutamate appears to involve two steps, catalyzed by cytosolic enzymes; viz: H4PteGlu leads to H2PteGlu2 followed by H4PteGlu2 leads to H4PteGlu6. These partial reactions are lacking in mac and met-6 respectively. The mitochondrial synthetase of the wild type may not represent a mandatory step in the biosynthesis of folylhexaglutamate but could have significance in generation of compartmented folylpolyglutamates.

Kinetics↗
PubMed1976

One-carbon metabolism in Neurospora crassa wild-type and in mutants partially deficient in serine hydroxymethyltransferase.

1. The concentrations of folate-dependent enzymes in Neurospora crassa Lindegren A wild type (FGSC no. 853), Ser-l mutant, strain H605a (FGSC no. 118), and for mutant, strain C-24 (FGSC no. 9), were compared during exponential growth on defined minimal media. Both mutants were partially lacking in serine hydroxymethyltransferase, but contained higher concentrations of 10-formyltetrahydrofolate synthetase than did the wild type. Mycelia of the mutants contained higher concentrations of these enzymes when growth media were supplemented with 1mM-glycine. In the wild-type, this glycine supplement also increased the specific activities of 5,10-methylenetetrahydrofolate dehydrogenase and 5,10-methylenetetrahydrofolate reductase. 5. During growth, total folate and polyglutamyl folate concentrations were greatest in the wild-type. Methylfolates were not detected in mutant Ser-l, and were only present in the for mutant after growth in glycine-supplemented media. Exogenous glycine increased folate concentration threefold in the wild type, mainly owing to increases in unsubstituted polyglutamyl derivatives. 3. Feeding experiments using 14C-labelled substrates showed that C1 units were generated from formate, glycine and serine in the wild type. Greater incorporation of 14C occurred when mycelia were cultured in glycine-supplemented media. Formate and serine were precursors of C1 units in the mutants, but the ability to cleave glycine was slight or lacking.

Alcohol Oxidoreductases↗
PubMed1972

Localization and interconversion of tetrahydropteroylglutamates in isolated pea mitochondria.

1. Mitochondria were extracted from 4-day-old pea cotyledons and purified on a sucrose density gradient. 2. Microbiological assay of the purified mitochondrial fraction with Lactobacillus casei (A.T.C.C. 7469), Streptococcus faecalis (A.T.C.C. 8043) and Pediococcus cerevisiae (A.T.C.C. 8081) revealed a discrete pool of conjugated and unconjugated derivatives of tetrahydropteroylglutamic acid. 3. Solubilization and chromatographic studies of the mitochondrial fraction demonstrated the presence of formylated and methylated derivatives, 10-formyltetrahydropteroylmonoglutamic acid, 5-formyltetrahydropteroylmonoglutamic acid and 5-formyltetrahydropteroyldiglutamic acid being the major derivatives present. 4. The principal mitochondrial pteroylglutamates were labelled when dry seeds were allowed to imbibe [2-(14)C]pteroylglutamic acid and 5-[methyl-(14)C]-methyltetrahydropteroylmonoglutamic acid. 5. The ability of isolated mitochondria to catalyse oxidation and reduction of tetrahydropteroylglutamic acid derivatives was demonstrated in feeding experiments in which [(14)C]formaldehyde, [3-(14)C]serine, sodium [(14)C]formate, 5-[methyl-(14)C]methyltetrahydropteroylmonoglutamic acid or [2-(14)C]-glycine served as C(1) donor. In addition, (14)C was incorporated into free amino acids related to C(1) metabolism.

Carbon Isotopes↗
PubMed1972

Regulation of C metabolism by L-methionine in Saccharomyces cerevisiae.

1. The concentrations of folate derivatives in aerobic cultures of Saccharomyces cerevisiae (A.T.C.C. 9763) were determined by microbiological assay employing Lactobacillus casei (A.T.C.C. 7469) and Pediococcus cerevisiae (A.T.C.C. 8081). Cells cultured in media lacking l-methionine contained higher concentrations of folate derivatives than cells grown in the same media supplemented with 2.5mumol of l-methionine/ml. The concentrations of highly conjugated derivatives were also decreased by supplementing the growth medium with l-methionine. 2. DEAE-cellulose column chromatography of extracts prepared from cells grown under these conditions revealed that the concentrations of methylated tetrahydrofolates were drastically decreased by the methionine supplement. Smaller decreases were also observed in the concentrations of formylated and unsubstituted derivatives. 3. The concentrations of four enzymes of C(1) metabolism were compared after 6h of growth in the presence and in the absence of l-methionine (2.5mumol/ml). The specific activities of formyltetrahydrofolate synthetase, methylenetetrahydrofolate reductase and serine hydroxymethyltransferase were not altered by this treatment but that of 5-methyltetrahydrofolate-homocysteine methyltransferase was decreased by approx. 65% when l-methionine was supplied. The activities of 5-methyltetrahydrofolate-homocysteine methyltransferase, serine hydroxymethyltransferase and formyltetrahydrofolate synthetase were not appreciably altered by l-methionine in vitro. In contrast this amino acid was found to inhibit the activity of methylenetetrahydrofolate reductase. 4. Feeding experiments employing sodium [(14)C]formate indicated that cells grown in the presence of exogenous methionine, although having less ability to convert formate into methionine, readily incorporated (14)C into serine and the adenosyl moiety of S-adenosylmethionine. 5. It is suggested that exogenous l-methionine controls C(1) metabolism in Saccharomyces principally by regulation of methyl-group biogenesis within the folate pool.

Alcohol Oxidoreductases↗
PubMed1971

Pteroylglutamate derivatives in Pisum sativum L. Biosynthesis of cotyledonary tetrahydropteroylglutamates during germination.

1. The concentrations of pteroylglutamate derivatives in the cotyledons of pea (Pisum sativum L.) seedlings were determined by microbiological assay by using Lactobacillus casei (A.T.C.C. 7469), Streptococcus faecalis (A.T.C.C. 8043) and Pediococcus cerevisiae (A.T.C.C. 8081). During germination the pteroylglutamate content of the cotyledons increased rapidly from 0.2mug/g dry wt. to 4.0mug/g dry wt., the maximum values being reached approx. 120h after imbibition. 2. Approx. 50% of the pteroylglutamate pool of 3-day-old seedlings was accounted for by highly conjugated derivatives. The concentrations of such derivatives were greatest when precautions were taken to inactivate endogenous enzymes before extraction of the tissues. 3. Individual derivatives present in the tissue extracts were separated by chromatography on DEAE-cellulose. Synthesis of the major derivative, 5-methyltetrahydropteroylmonoglutamate, was inhibited by administration of 0.1mm-aminopterin and -amethopterin solutions during imbibition. Under these conditions pteroylglutamic acid accumulated in the tissues. 4. Feeding experiments employing [2-(14)C]pteroylglutamic acid and 5[(14)C]-methyltetrahydro-pteroylmonoglutamic acid revealed that both compounds were incorporated into conjugated and unconjugated derivatives of the pteroylglutamate pool.

Aminopterin↗