title: Metal requirements of the enzymes catalyzing conversion of glutamate to delta-aminolevulinic acid in extracts of Chlorella vulgaris and Synechocystis sp. PCC 6803.
abstract: In the biosynthetic conversion of glutamate to the tetrapyrrole precursor, delta-aminolevulinic acid (ALA), glutamate is activated at C-1 by glutamyl-tRNA synthetase-catalyzed ligation to tRNAGlu. 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 Mg2+) for activity, but it has not been established whether Mg2+ 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 Mg2+ and inhibited by EDTA. EDTA-pretreated Chlorella glutamyl-tRNA reductase-containing fraction had very little activity in the absence of added Mg2+, but recovered full activity in incubations containing added Mg2+. The divalent metal requirement could be met by Mg2+, Mn2+, or Ca2+. Maximum activity was reached at approximately 15 mM concentration of each of these metals, and higher concentrations were inhibitory. Zn2+ was inhibitory at micromolar concentrations. Chlorella glutamyl-tRNA synthetase showed a metal requirement that could be met by Mg2+ or Mn2+, but not Ca2+. Maximum activity was reached at approximately 15 mM Mg2+ or Mn2+. Although the presence of 10 mM Ca2+ did not affect the Mg2+ concentration optimum, Ca2+ increased the effectiveness of low concentrations of Mg2+. 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.
authors.affiliation: Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912.
authors.forename: S M
authors.initials: SM
authors.lastname: Mayer
authors.forename: S
authors.initials: S
authors.lastname: Rieble
authors.forename: S I
authors.initials: SI
authors.lastname: Beale
mesh_terms: D000445:Aldehyde Oxidoreductases / Q000187:drug effects*; D000622:Aminolevulinic Acid / Q000378:metabolism; D002413:Cations, Divalent / Q000494:pharmacology*; D002474:Cell-Free System; D002708:Chlorella / Q000201:enzymology; D000458:Cyanobacteria / Q000201:enzymology; D056890:Eukaryota / Q000201:enzymology*; D005975:Glutamate-tRNA Ligase / Q000187:drug effects*; D005971:Glutamates / Q000378:metabolism; D018698:Glutamic Acid; D019751:Intramolecular Transferases; D007535:Isomerases / Q000187:drug effects*; D011758:Pyrroles / Q000378:metabolism; D045725:Tetrapyrroles
chemical_list: D002413:Cations, Divalent; D005971:Glutamates; D011758:Pyrroles; D045725:Tetrapyrroles; D018698:Glutamic Acid; D000622:Aminolevulinic Acid; D000445:Aldehyde Oxidoreductases; C067282:glutamyl tRNA reductase; D007535:Isomerases; D019751:Intramolecular Transferases; C061049:glutamate-1-semialdehyde 2,1-aminomutase; D005975:Glutamate-tRNA Ligase