Metal Requirements of the Enzymes Catalyzing Conversion of Glutamate to δ-Aminolevulinic Acid in Extracts of Chlorella vulgaris and Synechocystis sp. PCC-6803

Abstract

In the biosynthetic conversion of glutamate to the tetrapyrrole precursor, δ-aminolevulinic acid (ALA), glutamate is activated at C-1 by glutamyl-tRNA synthetase-catalyzed ligation to tRNA Glu. Glutamyl-tRNA reductase next catalyzes reduction of the activated glutamate to glutamate-1-semialdehyde (GSA), which is then converted to ALA by GSA aminotransferase. Glutamyl-tRNA synthetase is known to require a divalent metal (usually Mg 2+) for activity, but it has not been established whether Mg 2+ or another metal ion is also required for glutamyl-tRNA reductase or GSA aminotransferase, because these enzymes have previously been assayed in combined incubations containing all factors required for conversion of glutamate to ALA. We now report the metal requirements individually for each of the three enzyme reactions. Glutamyl-tRNA reductase activity in extracts from both Chlorella vulgaris and Synechocystis sp. PCC 6803 was stimulated by Mg 2+ and inhibited by EDTA. EDTA-pretreated Chlorella glutamyl-tRNA reductase-containing fraction had very little activity in the absence of added Mg 2+, but recovered full activity in incubations containing added Mg 2+. The divalent metal requirement could be met by Mg 2+, Mn 2+, or Ca 2+. Maximum activity was reached at approximately 15 mM concentration of each of these metals, and higher concentrations were inhibitory. Zn 2+ was inhibitory at micromolar concentrations. Chlorella glutamyl-tRNA synthetase showed a metal requirement that could be met by Mg 2+ or Mn 2+ but not Ca 2+. Maximum activity was reached at approximately 15 mM Mg 2+ or Mn 2+. Although the presence of 10 mM Ca 2+ did not affect the Mg 2+ concentration optimum, Ca 2+ increased the effectiveness of low concentrations of Mg 2+. In contrast to glutamyl-tRNA synthetase and glutamyl-tRNA reductase, Chlorella GSA aminotransferase did not show a metal requirement or inhibition by EDTA. However, EDTA decreased nonenzymatic transformation of GSA to ALA.