Formation of the chlorophyll precursor delta-aminolevulinic acid in cyanobacteria requires aminoacylation of a tRNAGlu species

Abstract

In the chloroplasts of higher plants and algae, the biosynthesis of the chlorophyll precursor 8-aminolevulinic acid (ALA) involves at least three enzymes and a tRNA species. Here we demonstrate that in cell extracts of the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 ALA was formed from glutamate in a series of reactions in which activation of glutamate by glutamyl-tRNAGIu formation was the first step. The activated glutamate was reduced by a dehydrogenase which displayed tRNA sequence specificity. Fractionation of strain 6803 tRNA by reverse-phase chromatography and polyacrylamide gel electrophoresis yielded two pure tRNAGIU species which stimulated ALA synthesis in vitro. These tRNAs had identical primary sequences but differed in the nucleotide modification of their anticodon. The 6803 tRNAGIU was similar to the sequences of tRNAGI, species or tRNAGI, genes from Escherichia coli and from chloroplasts of Euglena grailis and higher plants. Southern blot analysis revealed at least two tRNAGIU gene copies in the 6803 chromosome. A glutamate-1-semialdehyde aminotransferase, the terminal enzyme in the conversion of glutamate to ALA in chloroplasts, was detected in 6803 cell extracts by the conversion of glutamate-l-semialdehyde to ALA and by the inhibition of this reaction by gabaculin.

The genes aroA and trnQ are located upstream of psbO in the chromosome of Synechocystis 6803

“…Consequently this 07'-type element appears to be the prime candidate for the trnQ promoter. Supporting this speculation, it has been noted that trnE, which is the only other tm gene so far sequenced from Synechocystis 6803, also possesses an upstream 07'-like sequence motif [26].…”

“…In these organelles and bacteria, tRNAG'" is firstly 'mischarged' with glutamate to form Glu-tRNAG'", which is then converted to Gln-tRNAG'" by a specific amidotransferase that requires ATP, Mg*+ and a suitable amide donor, such as glutamine or asparagine [31]. Two distinct Glu-tRNAG'" species (tRNAG:" and tRNAGp) have been resolved from Synechocystis 6803 by reverse-phase chromatography [26,29]. Therefore the conclusion that trnQ is single-copy in Synechocystis 6803 argues that in this organism both resolvable tRNAG1" species are likely to be derived from the same trnQ gene by post-transcriptional modification.…”

Glutamyl-tRNA in Bacteria. Multiple Identities for Multiple Functions

“…The function of this complex would be to channel GSA between the two enzymes preventing its degradation by side reactions with water. 64,69−71 Glu-tRNA Glu , but not Glu-tRNA Gln , is a substrate of GluTR 14,72,73 which may represent a protective mechanism that prevents the use of Gln-tRNA…”

delta-Aminolevulinic acid dehydratase deficiency can cause delta-aminolevulinate auxotrophy in Escherichia coli

“…The fact that a hemA hemL double mutant still was able to produce siroheme led to the suggestion that there are two pathways of ALA formation in S. typhimurium (14). (30,31). ALA dehydratase activity in the supernatant fraction was determined by assaying the conversion of ALA to porphobilinogen and detection of the formed porphobilinogen by using Ehrlich reagent (16).…”

“…When grown aerobically, the hem-201 mutant GE1360 displayed a strict dependence for aerobic growth on the addition of exogenous ALA, suggesting that the defect was in one of the components of the C5 pathway. The ability of GE1360 cells and extracts to transform ['4C]glutamate into ALA was analyzed by using the in vivo and in vitro assays for ALA formation described in Materials and Methods (2,30,31). Cells and cell extracts were prepared from log-phase cultures of GE1360 grown in M9 medium (29) Cloning and DNA sequence of the hem-201 gene.…”