delta-Aminolevulinic Acid Biosynthesis from Glutamatein Euglena gracilis: Photocontrol of Enzyme Levels in a Chlorophyll-Free Mutant

Wild-type Euglena gracillis cells synthesize the key chlorophyll precursor, δ-aminolevulinic acid (ALA), from glutamate in their plastids. The synthesis requires transfer RNA^Glu (tRNA^Glu) and the three enzymes, glutamyl-tRNA synthetase, glutamyl-tRNA reductase, and glutamate-1-semialdehyde aminotransferase. Non-greening mutant Euglena strain W₁₄ZNaIL does not synthesize ALA from glutamate and is devoid of the required tRNA^Glu. Other cellular tRNA^Glus 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 tRNA^Glu 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 tRNA^Glu, and that the activity of all three enzymes is regulated by light. Because the absence of plastid tRNA^Glu 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.