Abstract
delta-Aminolevulinic acid is the universal precursor for all tetrapyrroles including hemes, chlorophylls, and bilins. In plants, algae, cyanobacteria, and many other bacteria, delta-aminolevulinic acid is synthesized from glutamate in a reaction sequence that requires three enzymes, ATP, NADPH, and tRNA(Glu). The three enzymes have been characterized as glutamyl-tRNA synthetase, glutamyl-tRNA reductase, and glutamate-1-semialdehyde aminotransferase. All three enzymes have been separated and partially characterized from plants and algae. In prokaryotic phototrophs, only the glutamyl-tRNA synthetase and glutamate-1-semialdehyde aminotransferase have been decribed. We report here the purification and some properties of the glutamyl-tRNA reductase from extracts of the unicellular cyanobacterium, Synechocystis sp. PCC 6803. The glutamyl-tRNA reductase has been purified over 370-fold to apparent homogeneity. Its native molecular mass was determined to be 350 kDa by glycerol density gradient centrifugation, and its subunit size was estimated to be 39 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence was determined for 42 residues. Much higher activity occurred with NADPH than with NADH as the reduced pyridine nucleotide substrate. Half-maximal rates occurred at 5 microM NADPH, whereas saturation was not reached even at 10 mM NADH. Purified Synechocystis glutamyl-tRNA reductase was inhibited 50% by 5 microM heme. Activity was unaffected by 10 microM 3-amino-2,3-dihydrobenzoic acid. No flavin, pyridine nucleotide, or other light-absorbing prosthetic group was detected on the purified enzyme. The catalytic turnover number of purified Synechocystis glutamyl-tRNA reductase is comparable to those of prokaryotic and plastidic glutamyl-tRNA synthetases.
Highlights
Nonphototrophiclower eukaryotes, centrifugation, and its subunit size was estimated atnod cbeertain bacteria
The N-terminal amino acid sequence nonsulfur bacteria
Half-maximal rates oc- Species utilizing the five-carbon pathway include all higher curred at 5 FM NADPH, whereas saturation was not plants (IO), green [11] and red [12] algae, and oxygenic reached even a1t0mM NADH
Summary
Glutamyl-tRNA reductase has been the least-well characterized of the threeknown enzymes required to catalyzAe LA synthesis from glutamate via the five-carbon pathway. Full size photocopies are included in the microfilm ocystis glutamyl-tRNA reductase was 6.33 pmol of product/ 51 ng of protein in the 5-min incubation period This value corresponds to a catalytic turnover number of 8.7 product molecules/min for each 350-kDa native enzyme molecule, or edition of the Journal that isavailable from Waverly Press. These values are comparable to theturnover numbers calculated from the specific activities reported for glutamyl-tRNA synthetase from B. subtilis [55], Chlamydomonas [56], and wheat plastids [57],which range from 3 to 9.
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