δ-Aminolevulinic Acid Biosynthesis from Glutamatein Euglena gracilis

Abstract

Wild-type Euglena gracilis cells synthesize the key chlorophyll precursor, 5-aminolevulinic acid (ALA), from glutamate in their plastids. The synthesis requires transfer RNAG'U (tRNAG'U) and the three enzymes, glutamyl-tRNA synthetase, glutamyl-tRNA reductase, and glutamate-1-semialdehyde aminotransferase. Nongreening mutant Euglena strain W14ZNalL does not synthesize ALA from glutamate and is devoid of the required tRNAGIU. Other cellular tRNAG'Us present in the mutant cells were capable of being charged with glutamate, but the resulting glutamyl-tRNAs did not support ALA synthesis. Surprisingly, the mutant cells contain all three of the enzymes, and their cell extracts can convert glutamate to ALA when supplemented with tRNAGlU obtained from wild-type cells. Activity levels of the three enzymes were measured in extracts of cells grown under a number of light conditions. All three activities were diminished in extracts of cells grown in complete darkness, and full induction of activity required 72 hours of growth in the light. A light intensity of 4 microeinsteins per square meter per second was sufficient for full induction. Blue light was as effective as white light, but red light was ineffective, in inducing extractable enzyme activity above that of cells grown in complete darkness, indicating that the light control operates via the nonchloroplast blue light receptor in the mutant cells. Of the three enzyme activities, the one that is most acutely affected by light is glutamate-1-semialdehyde aminotransferase, as has been previously shown for wild-type Euglena cells. These results indicate that the enzymes required for ALA synthesis from glutamate are present in an active form in the nongreening mutant cells, even though they cannot participate in ALA formation in these cells because of the absence of the required tRNAG'U, and that the activity of all three enzymes is regulated by light. Because the absence of plastid tRNAGIU precludes the synthesis of proteins within the plastids, the three enzymes must be synthesized in the cytoplasm and their genes encoded in the nucleus in Euglena. Pchl(ide). Exposure to light brings about the conversion of these pigments to Chl(ide), and in continuous light this is followed by a phase of rapid Chl accumulation (17) and transformation of the developing plastids into photosynthetically functional chloroplasts (37).The source of plastid tetrapyrrole pigments in Euglena, as in higher plants, algae, cyanobacteria, and other oxygenic organisms, is ALA2 that is formed from glutamate via the tRNA-dependent five-carbon pathway (26, 39). The fivecarbon pathway begins with activation of glutamate by ligation to tRNAGIU, catalyzed by glutamyl-tRNA synthetase, followed by reduction of the activated glutamate to GSA, catalyzed by glutamyl-tRNA reductase, and transamination of the GSA, catalyzed by GSA aminotransferase, to yield ALA (2).Unlike other oxygenic organisms, Euglena also has the ability to form ALA via the route used by animals, yeasts, and certain bacterial groups,...

A point mutation in Euglena gracilis chloroplast tRNA(Glu) uncouples protein and chlorophyll biosynthesis.

“…However, as with other nongreening Euglena mutants thought to be aplastidic and later found to have some plastid DNA sequences (29), W14ZNalL contains an intact tRNAGIu gene. Thus, the lack of chloroplast tRNAGlU in this strain (30) implies that the expression and processing of this gene depend on some product of chloroplast protein synthesis. Although the level of tRNAGIU in the chloroplast of the mutant Y9 strain was only 20%6 of that of the wild-type organelle, we are convinced that the Y9 phenotype is not merely a function of the low abundance of this particular tRNA.…”

Pigment Biosynthesis: Chlorophylls, Heme, and Carotenoids

“…In higher plants, ALA formation is thought to be regulated in part by a low fluence phytochrome response (Huang et al, 1989). Both red and blue light treatments were found to be effective inducers of ALA formation in dark-grown wild-type E. gracilis cells (Mayer and Beale, 1990), but only blue light treatment was required to induce the enzymes involved in ALA formation in an achlorophyllous mutant of this alga (Mayer and Beale, 1991). In Chlamydomonas, expression of the nuclear genes encoding GSA-AT (designated Gsa) and PEGS (designated Alad) were shown to be regulated by a carotenoid-type blue-light photoreceptor system rather than by phytochrome, rhodopsin, or a protochlorophyllide-based photoreceptor (Matters and Beale, 1995b).…”

Photo and Nutritional Regulation of Euglena Organelle Development

“…All of the enzymes required for the synthesis of chlorophyll from glutamate are found in the chloroplast and they appear to be synthesized on cytoplasmic ribosomes (Mayer and Beale 1991;Richard and Nigon 1973;Schwartzbach et al 1976b). ALA dehydratase, the first enzyme required for the conversion of ALA to chlorophyll , and porphobilinogen (PBG) deaminase (Shashidhara and Smith 1991), an enzyme required for porphyrin synthesis, are also made on cytoplasmic ribosomes.…”