type	Journal Article
authors	Jahn, D.; Chen, M. W.; Soll, D.
title	Purification and functional characterization of glutamate-1- semialdehyde aminotransferase from Chlamydomonas reinhardtii
journal	J Biol Chem
Activity	5.4.3.8
Family	5.4.3.8
ui	91093119
year	(1991)
volume	266
number	1
pages	161-7.
keywords	Aminooxyacetic Acid/pharmacology
abstract	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., Schon, A., O'Neill, G. P., and Soll, D. (1990) J. Biol. Chem. 265, 4054-4057 and Chen, M.- W., Jahn, D., O'Neill, G. P., and Soll, 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.
last changed	2008/05/21 16:26