The universal precursor of tetrapyrrole pigments (e.g., chlorophylls and hemes) is 5-aminolevulinic acid (ALA), which in Euglena gracilis chloroplasts is derived via the two-step C5 pathway from glutamate charged to tRNA(Glu). The first enzyme in this pathway, Glu-tRNA reductase (GluTR) catalyzes the reduction of glutamyl-tRNA(Glu) (Glu-tRNA) to glutamate 1-semialdehyde (GSA) with the release of the uncharged tRNA(Glu). The second enzyme, GSA-2,1-aminomutase, converts GSA to ALA. tRNA(Glu) is a specific cofactor for the NADPH-dependent reduction by GluTR, an enzyme that recognizes the tRNA in a sequence-specific manner. This RNA is the normal tRNA(Glu), a dual-function molecule participating both in protein and in ALA and, hence, chlorophyll biosynthesis. A chlorophyll-deficient mutant of E. gracilis (Y9ZNalL) does not synthesize ALA from glutamate, although it contains GluTR and GSA-2,1-aminomutase activity. The tRNA(Glu) isolated from the mutant can still be acylated with glutamate in vitro and in vivo. Furthermore, it supports chloroplast protein synthesis; however, it is a poor substrate for GluTR. Sequence analysis of the tRNA and of its gene revealed a C56-->U mutation in the resulting gene product. C56 is therefore an important identity element for GluTR. Thus, a point mutation in the T loop of tRNA uncouples protein from chlorophyll biosynthesis.