Purification and functional characterization of glutamate-1-semialdehyde aminotransferase from Chlamydomonas reinhardtii

• Published in: The Journal of biological chemistry Vol. 266; no. 1; pp. 161 - 167
• Main Authors: Jahn, D, Chen, M W, Söll, D
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 05.01.1991; American Society for Biochemistry and Molecular Biology
• Subjects: ACIDE ORGANIQUE; ACIDOS ORGANICOS; AMINOLEVULINIC ACID; Aminooxyacetic Acid - pharmacology; AMINOTRANSFERASAS; AMINOTRANSFERASE; AMINOTRANSFERASES; Analytical, structural and metabolic biochemistry; BIOCHEMICAL PATHWAYS; Biological and medical sciences; Cell Membrane - enzymology; CHEMICOPHYSICAL PROPERTIES; Chlamydomonas - enzymology; CHLOROPHYCEAE; chromatography; Chromatography, DEAE-Cellulose; Chromatography, Gel; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Cyclohexanecarboxylic Acids - pharmacology; Electrophoresis, Polyacrylamide Gel; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; gel electrophoresis; glutamate-1-semialdehyde aminotransferase; Intramolecular Transferases; ISOLATION; Isomerases - isolation & purification; Isomerases - metabolism; Kinetics; membrane proteins; Molecular Weight; ORGANIC ACIDS; PROPIEDADES FISICO-QUIMICAS; PROPRIETE PHYSICOCHIMIQUE; PURIFICACION; PURIFICATION; Pyridoxal Phosphate - pharmacology; Transferases; VIA BIOQUIMICA DEL METABOLISMO; VOIE BIOCHIMIQUE DU METABOLISME; Purification; Enzymatic activity; Enzyme; Reconstituted system; Ion exchange chromatography; Ultraviolet visible spectrometry
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)52416-9

The formation of delta-aminolevulinic acid, the first committed precursor of chlorophyll biosynthesis, occurs in the chloroplast of plants and algae by the C5-pathway, a three-step, tRNA-dependent transformation of glutamate. Previously, we reported the purification and characterization of the first two enzymes of this pathway, glutamyl-tRNA synthetase and Glu-tRNA reductase from the green alga Chlamydomonas reinhardtii (Chen, M.-W., Jahn, D., Schön, A., O'Neill, G. P., and Söll, D. (1990) J. Biol. Chem. 265, 4054-4057 and Chen, M.-W., Jahn, D., O'Neill, G. P., and Söll, D. (1990) J. Biol. Chem. 265, 4058-4063). Here we present the purification of the third enzyme of the pathway, the glutamate-1-semialdehyde aminotransferase from C. reinhardtii. The enzyme was purified from the membrane fraction of a whole cell extract employing four different chromatographic separations. The apparent molecular mass of the protein was approximately 43,000 Da as analyzed by denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, by nondenaturing rate zonal sedimentation on glycerol gradients, and by gel filtration. By these criteria, the enzyme in its active form is a monomer of 43,000 Da. In the presence of pyridoxal 5‘-phosphate, purified glutamate-1-semialdehyde aminotransferase converts synthetic glutamate 1-semialdehyde to delta-aminolevulinic acid. The enzyme is inhibited by gabaculine and aminooxyacetate, both typical inhibitors of aminotransferases. The purified glutamate-1-semialdehyde aminotransferase successfully reconstitutes the whole C5-pathway in vitro from glutamate in the presence of purified glutamyl-tRNA synthetase, glutamyl-tRNA reductase, Mg2+, ATP, NADPH, tRNA, and pyridoxal 5‘-phosphate.