Purification of the glutamyl-tRNA reductase from Chlamydomonas reinhardtii involved in delta-aminolevulinic acid formation during chlorophyll biosynthesis

• Published in: The Journal of biological chemistry Vol. 265; no. 7; pp. 4058 - 4063
• Main Authors: Chen, M.W. (Shanghai Jiao Tong University, Shanghai, China), Jahn, D, O'Neill, G.P, Soll, D
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 05.03.1990
• Subjects: ACIDE GLUTAMIQUE; ACIDO GLUTAMICO; ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; ALDEHIDOS; ALDEHYDE; Aminolevulinic Acid - metabolism; Analytical, structural and metabolic biochemistry; ARN DE TRANSFERENCIA; ARN DE TRANSFERT; Biological and medical sciences; BIOSINTESIS; BIOSYNTHESE; Chlamydomonas - enzymology; Chlamydomonas reinhardtii; CHLOROPHYCEAE; Chlorophyll - biosynthesis; Chromatography, Affinity; Chromatography, DEAE-Cellulose; Chromatography, Gel; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Glutamate-tRNA Ligase - isolation & purification; Glutamate-tRNA Ligase - metabolism; Ketone Oxidoreductases - isolation & purification; Ketone Oxidoreductases - metabolism; Kinetics; Levulinic Acids - metabolism; Molecular Weight; Oxidoreductases; OXIDORREDUCTASAS; OXYDOREDUCTASE; PURIFICACION; PURIFICATION; Chlorophyll; Regulation(control); Purification; Algae; Chlamydomonas reinhardtii; Chlorophycophyta; HPLC chromatography; Biosynthesis; Biosynthesis precursor; Mechanism of action; Chloroplast; Thallophyta
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)39702-9

The formation of delta-aminolevulinic acid, the first committed precursor in porphyrin biosynthesis, occurs in certain bacteria and in the chloroplasts of plants and algae in a three-step, tRNA-dependent transformation of glutamate. Glutamyl-tRNA reductase, the second enzyme of this pathway, reduces the activated carboxyl group of glutamyl-tRNA (Glu-tRNA) in the presence of NADPH and releases glutamate 1-semialdehyde (GSA). We have purified Glu-tRNA reductase from Chlamydomonas reinhardtii by employing six different chromatographic separations. The apparent molecular mass of the protein when analyzed under both denaturing (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and nondenaturing conditions (rate zonal sedimentation on glycerol gradients) was 130,000 Da; this indicates that the active enzyme is a monomer. In the presence of NADPH Glu-tRNA reductase catalyzed the reduction to GSA of glutamate acylated to the homologous tRNA. Thus, the reductase alone is sufficient for conversion of Glu-tRNA to GSA. In the absence of NADPH, a stable complex of Glu-tRNA reductase with Glu-tRNA can be isolated