Purification and functional characterization of glutamate-1-semialdehyde aminotransferase from Chlamydomonas reinhardtii.
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.
Jahn
D
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Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06511.
Chen
M W
MW
Söll
D
D
D000625:Aminooxyacetic Acid / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D002696:Chlamydomonas / Q000201:enzymology*; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003509:Cyclohexanecarboxylic Acids / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D019751:Intramolecular Transferases; D007535:Isomerases / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D011732:Pyridoxal Phosphate / Q000494:pharmacology
D003509:Cyclohexanecarboxylic Acids; D000625:Aminooxyacetic Acid; C012987:gabaculine; D011732:Pyridoxal Phosphate; D007535:Isomerases; D019751:Intramolecular Transferases; C061049:glutamate-1-semialdehyde 2,1-aminomutase