Edwin A. Cossins

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.

Some properties of a higher plant alcohol dehydrogenase

Abstract The alcohol dehydrogenase (EC 1.1.1.1) from germinating pea cotyledons has been purified approximately twenty-five fold. Preparations of the enzyme were found to be very unstable in the absence of sulphydryl reagents after chromatography on DEAE-Sephadex or after treatment with calcium phosphate gel. The enzyme was found to display broad specificity towards aliphatic alcohols but was specific for NAD and NADH. The Michaelis constants for ethanol and acetaldehyde were found to vary with pH and the reversibility characteristics of the reaction indicated that ethanol formation was favoured. On the basis of gel filtration studies, the molecular weight was found to be approximately 60,000. Enzyme activity was markedly inhibited by iodoacetate, iodoacetamide, p -chloromercuribenzoate, heavy metal ions and a number of chelating agents. The inhibition by iodoacetate and iodoacetamide was competitive with respect to ethanol concentration but non-competitive with respect to NAD concentration. Inhibition by o -phenanthroline was competitive with respect to NAD concentration and non-competitive with respect to ethanol concentration. It is concluded that the enzyme possesses certain catalytic properties in common with the alcohol dehydrogenases of horse-liver and yeast and also that the enzyme has importance in ethanol utilization in germinating tissues.

Methionine biosynthesis in isolated Pisum sativum mitochondria

Abstract Homocysteine-dependent transmethylases utilizing 5-methyltetrahydropteroylglutamic acid and S -adenosylmethionine as methyl donors have been examined using ammonium sulphate fractions prepared from isolated mitochondria of pea cotyledons. Substantial levels of a 5-rnethyltetrahydropteroylglutamate transmethylase were detected, the catalytic properties of this enzyme being found similar to those of a previously reported enzyme present in cotyledon extracts. The mitochondrial 5-CH 3 -H 4 PteGlu transmethylase had an apparent K m of 25 μM for the methyl donor, was saturated with homocysteine at 1 mM and was inhibited 50% by l -methionine at 2.5 mM. At similar concentrations of methyl donor the mitochondrial S -adenosylmethionine methyltransferase was not saturated. Mitochondrial preparations were found capable of synthesizing substantial amounts of S -adenosylmethionine but lacked ability to form S -methylmethionine. Significant levels of β-cystathionase, cystathionine-γ-synthase, l -homoserine transacetylase and l -homoserine transsuccinylase were detected in the isolated mitochondria. The activity of the enzymes of homocysteine biosynthesis was not affected by l -methionine in vitro . It is concluded that pea mitochondria have ability to catalyze the synthesis of methionine de novo .

The biosynthesis of glycine and serine by isolated chloroplasts

Abstract Chloroplasts were isolated non-aqueously from the leaves of 15-day-old pea plants. After sonication aqueous extracts were assayed for ability to synthesize glycine and serine. Such extracts were found to contain glyoxylate aminotransferase activity which was stimulated by addition of pyridoxal-5′-phosphate. Boiled enzyme controls indicated that the reaction was mainly enzymic. Chloroplast extracts also catalyzed the synthesis of serine from glycine. The reaction displayed requirements for tetrahydrofolate (H 4 PteGlu) and pyridoxal-5′-phosphate and was further stimulated by additions of formaldehyde. Sonicated chloroplasts also catalyzed the incorporation of [ 14 C]-formaldehyde into serine in a reaction having requirements for glycine, H 4 PteGlu and pyridoxal-5′-phosphate. [3- 14 C]-serine was readily converted into [ 14 C]-formaldehyde by chloroplasts contain a serine hydroxymethyltransferase ( l -serine:tetrahydrofolate-5,10-hydroxymethyltransferase EC 2.1.2.1).

Studies of the particulate and soluble aspartate aminotransferases in germinating pea cotyledons

Abstract The soluble and particulate aspartate aminotransferases (E.C. 2.6.1.1) present in germinating pea cotyledons were isolated by differential centrifugation and partially purified by ammonium sulphate fractionation and gel filtration. The activities of both enzymes varied during the first 5 days of germination, the soluble enzyme accounting for more than 90 per cent of the total extractable aspartate aminotransferase activity. Particulate enzyme activity was solubilized by various treatments including osmotic shock, sonication and by addition of deoxycholate. The general catalytic properties of both enzymes were compared and kinetic experiments suggested that both have a binary mode of action. The ability of various vitamin B 6 derivatives to reconstitute the soluble apoenzyme demonstrated that only pyridoxal-5′-phosphate and pyridoxamine-5′-phosphate were effective. These latter compounds were both tightly bound in the reconstituted enzyme as prolonged dialysis did not appreciably alter the specific enzyme activity. Studies of the rates of reconstitution of the soluble apoenzyme with these coenzymes showed that pyridoxal-5′-phosphate activated the enzyme more readily than equimolar amounts of pyridoxamine-5′-phosphate.

The polyglutamate nature of plant folates

Abstract Folylpolyglutamates were extracted from a variety of higher plant tissues and cleaved to p-aminobenzoyl polyglutamates by acid-base and ZN-HCl treatments. After purification as azo dyes and reconversion to p-aminobenzoylpolyglutamates these derivatives were separated according to their glutamyl chain lengths by HPLC on Partisil SAX. Polyglutamate concentrations were calculated by reference to standard calibration curves. Tomato leaf extracts contained relatively high folate contents (7.55 nmol/g fr. wt) and these were mainly hexa- (23%) and heptaglutamates (30%). Carrot root extracts had low folate contents (0.22 nmol/g fr. wt) with diglutamates accounting for 69% of the folate recovered after chromatography. Carboxypeptidase treatments of broccoli floret extracts indicated that the folylpolyglutamates principally contained γ-glutamyl linkages. [3H]Glutamate and [14C] p-aminobenzoate were incorporated into the polyglutamates of broccoli seedlings but equilibrium of the label with the endogenous pools was not attained after a 48 hr pulse feeding. Difrerential, oxidative cleavage of broccoli leaf folates into three polyglutamate pools showed that the formyl- and methylfolates were mainly (80%) diglutamyl derivatives. The methylene and unsubstituted folates of this tissue were principally (78%) hexaglutamates.

A 5,10-methylenetetrahydrofolate dehydrogenase: 5,10-methenyltetrahydrofolate cyclohydrolase protein from Pisum sativum

Abstract A cytosolic protein, that exhibits NADP-dependent 5,10-methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) and 5,10-methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9) activities but lacks 10-formyltetrahydrofolate synthetase (EC 6.3.4.3) activity, was isolated from extracts of pea ( Pisum sativum L.) cotyledons and leaves. Dehydrogenase: cyclohydrolase activities co-purified when protein was fractionated by (NH 4 ) 2 SO 4 precipitation, followed by chromatography on Sephacryl S-300, Matrex Green A and heparin agarose. The resulting protein ( M r 38 500) was apparently homogeneous after SDS-polyacrylamide gel electrophoresis and silver staining. Purified enzyme preparations lacked methylenetetrahydrofolate dehydrogenase activity when NAD replaced NADP in the reaction system. Attempts to sequence this protein suggested that it may be blocked at the N -terminus. Tryptic digestion of the purified protein resulted in coordinate losses of dehydrogenase and cyclohydrolase activity. The NADP provided significant protection of both enzyme activities during short-term treatments with trypsin but additions of 5,10-methylenetetrahydrofolate appeared to accentuate the proteolytic loss of dehydrogenase activity. The apparent Michaelis constants for NADP (11 μM) and methylenetetrahydrofolate (21 μM) in the dehydrogenase reaction were not changed significantly when the folylpentaglutamate substrate was provided (8 and 25 μM, respectively). The dehydrogenase and cyclohydrolase activities were competitively inhibited by dihydrofolates. The K i values of the dehydrogenase reaction indicated that the pentaglutamate derivative was a more potent inhibitor than dihydrofolate monoglutamate. The ELISA and Western blot analyses, using rabbit polyclonal antibodies raised against the purified enzyme, revealed cross-reactivity with proteins of similar molecular size in leaf extracts of wheat, barley, corn, bean and pea. Chromatography of these leaf extracts on Matrex Green A, in the presence of protease inhibitors, showed that 10-formyltetrahydrofolate synthetase was readily separated from protein with dehydrogenase and cyclohydrolase activity.

Folylpolyglutamate synthetase activities of Neurospora

Abstract The folylpolyglutamate synthetase (FPGS) 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 after growth in defined media. Extracts of the wild type produced H4PteGlu6 (60 %), H4PteGlu3 (35 %) and H4PteGlu2 (15 %). Met-6 extracts formed H4PteGlu2 but lacked the ability to utilize H4PteGlu4 or H4PteGlu5. The mac mutant failed to catalyse glutamate addition to H4PteGlu but H4PteGlu2 was an effective substrate for tri- and hexaglutamate synthesis. These polyglutamates were also formed by reaction systems containing mixtures of met-6 and mac protein or heterokaryon protein derived from mycelial fusions of met-6 and mac. Extract fractionations and heat treatments provided evidence for more than one FPGS activity in the wild type. A mitochondrial FPGS catalysed the H4PteGlu2 → H4PteGlu3 reaction but a cytosolic fraction synthesized di-, tri- and hexaglutamates when incubated with H4PteGlu and glutamate. The latter system contained a temperature-sensitive diglutamate-forming activity and a relatively stable H4PteGlu2 → H4PteGlu6 activity. Polyglutamate synthesis in N. crassa appears to involve more than one step, H4PteGlu → H4PteGlu2 followed by H4PteGlu2 → H4PteGlu6, in addition to the mitochondrial activity. These partial activities are lacking in mac and met-6 respectively. Consequently, these mutants are unable to form the folylhexaglutamates that predominate the folate pool of the wild type.

Relationships between glycollate and folate metabolism in Euglena gracilis

Abstract When division synchronized cultures of Euglena gracilis Klebs (strain Z) were aerated with 5% CO 2 in air the specific activity of glycollate dehydrogenase was only 13% of that in cultures receiving unsupplemented air. The concentrations of 10-formyltetrahydrofolate synthetase (EC 6.3.4.3) and formylfolate derivatives were also lowered by this treatment. In contrast, the specific activity of serine hydroxymethyltransferase (EC 2.1.2.1) and the concentration of methylfolates were raised by supplying CO 2 -supplemented air. These effects on enzyme levels were reversed when air was supplied following a period of CO 2 treatment. The levels of glycollate dehydrogenase, 10-formyl-tetrahydrofolate synthetase and formylfolate derivatives were decreased when cells were aerated in media containing 5 mM α-hydroxy-2-pyridinemethane sulphonate. Cell free extracts had the ability to decarboxylate glyoxylate, producing ca equal amounts of CO 2 and formate from C-1 and C-2 respectively. Cells receiving 5% CO 2 in air had a decreased ability to incorporate formate-[ 14 C] into serine and methionine. It is concluded that during growth at low CO 2 concentrations glycollate metabolism will provide substrate for the formyltetrahydrofolate synthetase reaction.

Folate derivatives of photosynthetic tissues

Abstract An examination has been conducted of folate derivatives in extracts of spinach, pea, and wheat leaves and the cotyledons of 6-day-old radish seedlings using Lactobacillus casei. These tissues contained folate activity equivalent to 28, 15, 14 and 30 μg of pteroylglutamic acid per g dry wt of tissue respectively, and the folate derivatives were shown to consist mainly of formyl and methyl tetrahydropteroylpolyglutamates, conjugated with more than three glutamic acid moieties. During germination the folate content of both green and etiolated radish cotyledons increased rapidly to a maximum at 6–8 days and then decreased. Etiolated tissues contained half the folate levels found in normal tissues, but the proportion of methyl derivatives was higher. A comparison of the levels of folates present in radish cotyledons harvested in the dark and light phases of a 13:11 hr light:dark cycle revealed significant differences in formyl and unsubstituted derivatives. In light-harvested tissues, formyl derivatives decreased by 50% while unsubstituted derivatives increased by 600%, methyl derivatives being unchanged. Treatment of radish cotyledons with aminopterin resulted in a rapid depletion of methyl tetrahydropteroylglutamates while formyl and unsubstituted derivatives declined less abruptly. Pteroylglutamic acid accumulated in these treatment.