δ-Aminolevulinic Acid Synthase of Euglena gracilis: Regulation of Activity

Abstract

ABSTRACTregions, which thereby supplants the necessity for ALA formation via the ALA synthase route.8-Aminolevulinic acid (ALA), a key precursor of the tetrapyrroles heme and chlorophyll, is capable of being synthesized by two different routes in cells of the unicellular green alga Euglena gracilis: from the intact carbon skeleton of glutamate, and via the condensation of glycine and succinyl CoA, mediated by the enzyme ALA synthase. The regulatory properties of ALA synthase were examined in order to establish its role in Euglena.Partially purified Euglena ALA synthase, unlike the case with the bacterial or animal-derived enzyme, does not exhibit allosteric inhibition by the tetrapyrrole pathway products heme, protoporphyrin IX, and porphobilinogen, at concentrations up to 100 micromolar.In aplastidic mutant cells, extractable ALA synthase activity is constant during exponential growth, and decreases to low levels as the cells reach the stationary state. Rapid exponential decline of ALA synthase (t1/2 = 55 min) occurs after administration of 43 micromolar cycloheximide, but not 6.2 millimolar chloramphenicol. These results suggest that, as in other eukaryotic cells, ALA synthase is synthesized on cytoplasmic ribosomes and is subject to rapid turnover in vivo.Extractable ALA synthase activity increases 2.5-fold within 6 hours after administration of 100 millimolar ethanol, a stimulator of mitochondrial development, and 4.5-fold within 12 hours after administration of I millimolar 4,6-dioxoheptanoic acid, which blocks ALA utilization, suggesting that activity is controlled in vivo by a feedback induction-repression mechanism, coupled with rapid enzyme turnover.In heterotrophically grown wild-type cells, low levels of ALA synthase rapidly increase 4.5-fold within 12 hours after cells are transferred from the light to the dark, and decrease exponentially (t1/2 = 75 min) when cells are transferred from the dark to light. The dark levels are equal to those in light-or dark-grown aplastidic mutant cells. The low level occurring in light-grown wild-type cells is not altered by the presence of 10 micromolar 3-(3,4-dichlorophenyl)-1,1-dimethylurea, which blocks photosynthetic 02 production. The decrease that occurs on dark-to-light transfer can be diminished by 12-or 24-hour prior incubation with 6.2 millimolar chloramphenicol, which also retards chlorophyll synthesis after the transfer to light.The positive relationship of ALA synthase activity to degree of mitochondrial expression, and the inverse relationship to plastid development and chlorophyll synthesis, suggests that ALA synthase functions to provide precursors to nonplastid tetrapyrroles in Euglena. In light-grown, wild-type cells, the diminished levels of ALA synthase may be due to the ability of developing plastids to export heme or a heme precursor to other cellular '

δ-Aminolevulinic Acid Formation from γ,δ-Dioxovaleric Acid in Extracts of Euglena gracilis

“…The cells used in our experiments were harvested during exponential growth under conditions which are optimal for heterotrophic growth (5,9). In our preparations, we observed stimulation of activity by pyridoxal phosphate, but no stimulation occurred when either NADPH or MgCl2, or both reagents, were added to the incubation mixture.…”

“…but serves instead to supply non-plastid tetrapyrrole precursors when the plastids are metabolically quiescent (9).…”

Light Regulation of δ-Aminolevulinic Acid Biosynthetic Enzymes and tRNA in Euglena gracilis

“…ALA synthase, on the other hand, is the sole source of mitochondrial tetrapyrrole precursors in Euglena, and may also supply other cellular tetrapyrrole end products in the dark (36). In vitro, ALA synthase activity is much higher in extracts of darkgrown cells than in light-grown cell extracts, and the extractable activity declines precipitously within the first few hours after transfer of dark-grown cells to the light (13). In this report we have determined that growth in the dark or light results in differences in the extractable activities of the three known enzymes of the five-carbon pathway, as well as the relative levels of the tRNAG"U that is required for ALA formation from glutamate.…”

Succinyl-Coenzyme A Synthetase and its Role in δ-Aminolevulinic Acid Biosynthesis in Euglena gracilis

“…ALA synthase activity is much higher in extracts of darkgrown wild-type cells than in light-grown cell extracts, and the extractable activity declines precipitously within the first few hours after transfer of dark-grown cells to the light (4,6). Certain nongreening mutant Euglena strains appear to lack the capacity to form ALA from glutamate in vivo (22).…”