Plant Physiol. (1971) 48, 316-319 Studies on the Biosynthesis and Metabolism of a-Aminolevulinic Acid in Chlorellal Received for publication February 10, 1971 SAMUEL I. BEALE2 Department of Botanical Sciences, University of California, Los Angeles, California 90024 ABSTRACT glycine and succinyl-CoA, ALA synthetase, has been found in extracts from bacteria (13) and from animal sources (9), but The regulation of chlorophyll synthesis in Chlorella was ex- ALA synthetase activity has not been detected in extracts from amined at the level of the formation and metabolism of 5- any plant, including algae (6). Metabolic control of the bac- aminolevulinic acid. 8-Aminolevulinic acid synthetase activity terial enzyme activity has been shown to be exerted by various could not be detected in broken cell preparations, and exoge- tetrapyrroles, particularly ferrous and ferric protoporphyrin nously supplied 8-aminolevulinic acid was taken up only in the IX, possibly acting as negative feedback inhibitors (3). presence of dimethylsulfoxide, with a corresponding production An alternate route of ALA synthesis has been found in of porphobilinogen. Chlorella (8), involving a transamination of DOVA. The sig- The 8-aminolevulinic acid dehydratase of Chlorella has a pH is of and at this the Michaelis constant for 8- nificance of the transamination mode of ALA synthesis not optimum 7.8 pH known at present, but several factors argue against this route: aminolevulinic acid is 0.13 millimolar. DOVA has not been reported to be present in Chlorella; the 5-Aminolevulinic acid excretion in the presence of levulinic of DOVA acid, a competitive inhibitor of 5-aminolevulinic acid dehy- activity transaminase does not follow rates of chloro- dratase, allowed measurement of the relative rates of 5-amino- phyll synthesis (8), as would be expected for a key reaction, the levulinic acid synthesis under various growth conditions. A first step unique to a metabolic pathway; and the tetrapyrroles mutant which requires light for chlorophyll synthesis also re- formed from ALA synthesized in this manner would not have quires light for 8-aminolevulinic acid accumulation in the the labeling patterns found in those synthesized from succinate presence of levulinic acid. This same mutant has 40% of the and glycine. On the other hand, there is some doubt concern- 8-aminolevulinic acid dehydratase activity of the wild-type ing certain labeling patterns in chlorophyll from specifically Chlorella during growth in the dark on glucose. labeled glycine, in particular the incorporation of the carboxyl The necessity for protein synthesis during chlorophyll syn- carbon (4, 22, 23), and, again, ALA synthetase has not been thesis is due primarily to the requirement for protein synthesis found in plants, so the DOVA transaminase route of ALA bio- during 8-aminolevulinic acid formation. synthesis in plants cannot presently be excluded. It is concluded that the rate of chlorophyll formation and the The immediate fate of ALA in porphyrin synthesis is the cellular chlorophyll content are regulated by the relative rates of condensation of two molecules to form one of the monopyrrole synthesis and breakdown of an enzyme responsible for 8- PBG, with the loss of two water molecules. The enzyme re- aminolevulinic acid biosynthesis and that this enzyme has an sponsible for this condensation, ALA dehydratase, has been in vivo lifetime of about 30 minutes. found in liver (10), bacteria (3), and plant tissue (12, 25, 28), where it appears to be a constitutive enzyme, since its activity is high even under conditions of low chlorophyll synthesis (27). In a previous report (1), use was made of LA, a competitive inhibitor of ALA dehydratase, to demonstrate the in vivo syn- thesis of ALA in Chlorella. In the present report, ALA bio- synthesis and LA-induced ALA accumulation are further in- vestigated, and ALA biosynthesis is correlated with chlorophyll The key role of ALA3 in the biosynthesis of tetrapyrroles was synthesis under a variety of conditions. first established by Shemin and Russell (24). A condensation of succinate or succinyl-CoA with glycine would form a-amino- /3-ketoadipic acid, which, after loss of the carbon atom derived METHODS from the carboxyl group of glycine, would yield ALA. ALA Chlorella vulgaris Beijerinck was grown photoautotrophi- thus formed could yield tetrapyrroles which have predicted cally as previously described (2). In order to maintain condi- label positions exactly matching those found in protoporphyrin tions of constant culture pH, 15 mm urea was substituted for formed from specifically labeled glycine or succinate (for a re- 30 mM KNO3 as a nitrogen source in some experiments. When view of tetrapyrrole biosynthesis, see Ref. 18). this was done, sterile filtered urea was added to nitrogen-free An enzyme which catalyzes the formation of ALA from autoclaved medium. PC volume and chlorophyll were measured as previously de- 1 scribed (2), the former by centrifugation in Hopkins vaccine Supported in part by National Institutes of Health Predoctoral and Fellowship GM-28419. tubes the latter by extraction into 90% methanol (17). aPresent address: Department of Biology, Brookhaven National ALA was measured by the method of Mauzerall and Granick Laboratory, Upton, N. Y. 11973. (19), which involves a condensation with ethyl acetoacetate and $ Abbreviations: ALA: 8-aminolevulinic acid; DOVA: a,8-di- then reaction with modified Ehrlich reagent. The absorbance oxovaleric acid (a-ketoglutaraldehyde); LA: levulinic acid; PBG: of the colored product was measured at 553 nm in a Zeiss spec- porphobilinogen; PC: packed cells. trophotometer. 316 Plant Physiol. Vol. 48, 1971 BIOSYNTHESIS AND METABOLISM OF ALA 317 Attempts to show ALA synthetase activity were made by the method of Burnham and Lascelles (3) and by modifications of this method which will be described in "Results and Discus- sion." ALA dehydratase activity was measured by the method of Burnham and Lascelles (3) on Chlorella cells which were broken in a modified French pressure cell (29). The method 2.00 consists, basically, of incubation of the cell extract with ALA, 2-mercaptoethanol, and buffer and measurement of the ac- cumulation of PBG by the method of Mauzerall and Granick -1A = -7.9/ (19), a spectrophotometric determination of the reaction prod- 1.00 uct with modified Ehrlich reagent. More specific details are Y- - .125 .M presented in "Results and Discussion." ALA and LA were separated in the following way. A solu- 1 1 tion containing both was passed through a cation exchange L 6 8 10 12 column (BioRad AG5OW-X8), which was previously converted zhr. ALA to the H+ form by washing with acid and then water. Water FIG. 2. Lineweaver-Burk plot of ALA dehydratase from Chlo- was passed through the column until all of the LA was washed rella. The assays were run at 37 C for 30 min as described in the off, and then 0.25 M glycine-KOH buffer, pH 9.5, was passed text. The results are plotted according to the method of Line- through until the ALA was eluted off the column. weaver and Burk (16). The line was found by the method of least Levulinic acid (K and K Laboratories) and 1 ,4-14C-levulinic squares analysis. acid (New England Nuclear) were freed of cationic material by passage through a cation exchange column (BioRad AG5OW- sample. With this method, I could detect the formation of X8) in the H+ form. 0.05 /moles of ALA per ml of PC-hr, or about Yhooth of that Cycloheximide (actidione) was purchased from Calbiochem, necessary for the observed rates of chlorophyll synthesis. and chloramphenicol, ALA, ATP, and CoA were purchased To investigate the possibility that an inhibitor of ALA syn- from Sigma Chemical Co. Dimethylsulfoxide was from Mathe- thetase was present, an extract of R. spheroides was added to son Coleman and Bell. the incubation mixture containing broken Chlorella cells. The Succinyl-CoA was prepared by the method of Simon and amount of ALA formed was about equal to that formed in Shemin (26) or was generated in situ from succinic acid and incubation mixtures containing R. spheroides extract alone. CoA with an extract of Rhodopseudomonas spheroides mutant A mutant of R. spheroides, H-5, has no ALA synthetase but H-5, which lacks ALA synthetase (15). can form succinyl-CoA. When an extract of this mutant was substituted for the wild-type cell extract in the incubation mix- RESULTS AND DISCUSSION ture containing broken Chlorella cells, no ALA was detected. Effects of Exogenously Supplied ALA. One mm ALA sup- Lack of Detectable ALA Synthetase Activity in Chlorella plied to the culture medium had no effects on growth or chloro- Extracts. Chlorella cells were broken in the presence of 100 mm phyll synthesis. No PBG or fluorescent material accumulated glycine, 100 mm sodium succinate, 1O mm MgCl2, 1 mm 2- in the medium, and the ALA concentration did not decrease mercaptoethanol, 250 ,uM pyridoxal phosphate, 580 [M CoA, after 72 hr of growth. 7.5 mM ATP, 1 mM EDTA, and 50 mm tris-HCl buffer, pH Dimethylsulfoxide has been used to increase cellular per- 7.8, according to the method of Burnham and Lascelles (3). meability to substrates and products in order to study enzymatic After 30 or 60 min of incubation at 30 or 37 C, 5% trichloro- activity in unbroken cells (5). When Chlorella was grown in acetic acid was added, the reaction mixture was centrifuged, the presence of both 1% dimethylsulfoxide and 1 mm ALA, and the supernatant was assayed for ALA. Alternatively, suc- growth and chlorophyll synthesis were only slightly affected, cinyl-CoA was substituted for sodium succinate, ATP, and and PBG was found in the culture medium at a concentration CoA in the incubation mixture. No ALA was detected in any of 10 nm. These results suggest that, under normal conditions, growing cells are impermeable to exogenously supplied ALA.