Biosynthesis of 8 - Aminolevulinic Acid from Glutamate in

5-Aminolevulinic acid accumulated in the culture medium when Agmeneflum quadplicatum strain PR-6 was incubated in the presence of levulinic acid, a competitive inhibitor of 8-aminolevulinic acid dehydratase, and specifically labeled glutamate and glycine. The 8-aminolevulinic acid was purified using Dowex 50W-X8 and cleaved by periodate to yield succinic acid and formaldehyde. The distribution of radioactivity in the two fragments suggested that in blue-green algae the carbon skeleton of 8aminolevulinic acid is derived directly from glutamate. However the possibility of the pathway of 8-aminolevulinic acid synthesis, from glycine and succinyl-coenzyme A also functioning in blue-green algae was not eliminated as uptake of glycine was minimal. &-Aminolevulinic acid is the first identified biosynthetic intermediate that is unique to the tetrapyrrole pathway. ALA2 is the precursor of heme in animals (16) and bacteria (9); of Chl in bacteria (6) and plants (8); and of the phycobilins in red algae (19). In many organisms ALA is formed by the condensation of glycine and succinyl-CoA, catalyzed by ALA synthetase, a pyridoxal-requiring enzyme. The enzymatic activity was first demonstrated in photosynthetic bacteria (12) and chicken erythrocytes (7). ALA synthetase has since been reported in yeast, bacteria, and a number of animal tissues. However, workers have been unable to demonstrate conclusively ALA synthetase activity in green plants and green algae (2). In studies in which cucumber cotyledons (3), bean and barley leaves (4), maize leaves (15), and the unicellular rhodophyte Cyanidium caldarium (10) were incubated in the presence of levulinic acid and labeled compounds; glutamate, a-ketoglutarate, and glutamine were found to donate label to the ALA that accumulated, much more effectively than glycine. Furthermore, from the specific pattern of incorporation of label into ALA, it appears that ALA is formed from the intact carbon skeleton of glutamate in a manner incompatible with the ALA synthetase route (1, 5, 10, 15). Herein, we report the labeling ofALA from specifically labeled glutamate by a blue-green alga. The evidence suggests that bluegreen algae synthesize ALA from glutamate in a manner similar to that observed in green plants and green algae. MATERIALS AND METHODS Organism and Culture Conditions. The unicellular marine blue'This work was supported by Public Health Service Grant GM-23524 from the National Institute of General Medical Sciences. 2Abbreviation: ALA: 8-aminolevulinic acid. green alga, Agmenellum quadruplicatum strain PR-6, was isolated by Van Baalen (20). Broth cultures were grown in medium A (18) in a controlled temperature water bath at 39 C. Continuous agitation and CO2 were provided by bubbling 4% CO2 in air (v/v) through the cultures. Illumination consisted of four F24T12 CW/ HO fluorescent lamps. A large culture of PR-6 was grown to a concentration of 3 x 107 cells/ml. The cells were centrifuged, resuspended in fresh medium A, and divided into four 30-ml cultures. Levulinic acid was added to give a final concentration of 60 mM (13). After 2-h incubation 7 ,uCi ofradiolabeled compounds were added and incubation was continued for 6 h. The specific radioactivity of the labeled compounds was 40-60 ,Ci/,umol, therefore the concentration of the amino acids in the culture medium was about 5,M. ALA Determination. After incubation cell suspensions were centrifuged and ALA in the medium was determined using the colorimetric method of Mauzerall and Granick (14), as described in Kipe-Nolt et al. (13). Isolation of ALA. ALA was isolated using the method described by Beale et al. (5) and Jurgenson et al. (10). Twenty-five-ml aliquots of culture supernatant were added to Dowex 50W-X8 columns (5-ml bed volume in 10-ml plastic syringes) which had been washed with 10 ml 1 N NaOH and 30 ml sodium citrate buffer (pH 3.1). The columns were washed with 30 ml of the same buffer and ALA was eluted with 25 ml of sodium citrate buffer (pH 5.1). The following control experiments were done to check the isolation procedure: [14CJALA was added to spent medium A and the recovery of label in the pH 5.1 buffer wash was noted. Labeled glutamate and glycine was also added to spent medium A and the recoveries in the pH 5.1 buffer wash noted. Periodate Cleavage of ALA. The ALA in the pH 5.1 buffer wash from the Dowex 50W-X8 columns was cleaved with periodate under alkaline conditions as described by Beale et al. (5). The cleavage products are formaldehyde (C5) and succinic acid (CiC4) (17). The formaldehyde fragment was precipitated as the dimedone derivative, collected on Millipore filters and assayed for radioactivity (10). The succinic acid fragment was extracted from the resulting filtrate using three 30-ml volumes of diethyl ether. The diethyl ether was evaporated to dryness and the residue was assayed for radioactivity. The procedure was checked by cleaving [4-"CIALA and [5-14CIALA and noting recoveries in the formaldehyde and succinic acid fractions. The recovery of label from glutamate and glycine in the two fractions was also checked. Uptake of Radiolabeled Compounds. One-ml samples, from cultures of PR-6 containing the specifically labeled glutamate and glycine, were removed at 1-h intervals. The samples were centrifuged and the cells washed with two 8-ml volumes of water. The cells were digested with 1 ml Protosol, decolorized with 0.2 ml 20o benzoyl peroxide in toluene (w/v), and counted in Aquasol2. Chemicals. Levulinic acid, periodic acid, and 5,5-dimethyl-1,3-