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., 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.
Highlights
Theformation of b-aminolevulinic acid, the first by a three-step reaction from the 5-carbon skeleton of glutacommitted precursor of chlorophyll biosynthesis, oc- mate.Glutamate, activated pathway, a three-step, tRNA-dependent transforma- by formation of Glu-tRNAG'"by Glu-tRNA synthetase
In the presenceof pyridoxal 5'-phosphate, GSA aminotransferases from barley chloroplasts and from purified glutamate-1-semialdehyde aminotransferase Synechcoccus have been purified (Grimm et al, 1989).Invesconverts synthetic glutamate 1-semialdehyde bt-oami- tigation of the enzyme structure andof cofactor requirements nolevulinic acid.The enzyme is inhibited by gabaculine showed clear differences between the two enzymes.While the and aminooxyacetate, both typical inhibitors of ami- barley enzyme is a dimer of 80 kDa molecular mass, the notransferases
We present the purification of the GSA aminotransferase from C. reinhardtii and itsinitial characterization
Summary
0 1991 by The American Society for Biochemistry and Molecular Biology, Inc. Vol 266,No 1, Issue of January 5 , pp. 161-167,1991 Printed in U.S A. Approximately 43,000 Da as analyzed by denaturing Transaminasesarea well studied class of enzymes (resodium dodecyl sulfate-polyacrylamide gel electropho- viewed in Christen andMetzler, 1985).It is generally accepted resis, by nondenaturing rate zonal sedimentation on that inpyridoxal 5"phosphate-dependent enzymes the cofacglycerol gradients, andby gel filtration By these cri- tor PLP is bound by Schiff base formation with the amino teria, the enzyme in its active form is a monomer of group of a lysine in the active site of the enzyme. 43,000 Da. In the presenceof pyridoxal 5'-phosphate, GSA aminotransferases from barley chloroplasts and from purified glutamate-1-semialdehyde aminotransferase Synechcoccus have been purified (Grimm et al, 1989).Invesconverts synthetic glutamate 1-semialdehyde bt-oami- tigation of the enzyme structure andof cofactor requirements nolevulinic acid.The enzyme is inhibited by gabaculine showed clear differences between the two enzymes.While the and aminooxyacetate, both typical inhibitors of ami- barley enzyme is a dimer of 80 kDa molecular mass, the notransferases. Fast proteinliquid chromatography; HPLC, high pressure liquid chromatography; gabaculine, 3-amino-2,3-dihydrobenzoicacid; Glu-
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