Abstract
delta-Aminolevulinic acid is the first committed precursor in the biosynthesis of hemes, phycobilins, and chlorophylls. Plants and algae synthesize delta-aminolevulinic acid from glutamate via an RNA-dependent 5-carbon pathway. Previous reports demonstrated that cyanobacteria form delta-aminolevulinic acid from glutamate in vivo. We now report the direct measurement of this activity in vitro. Three oxygenic prokaryotes were examined, the unicellular cyanobacteria Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002 (Agmenellum quadruplicatum PR-6) and the chlorophyll a- and b-containing filamentous prochlorophyte Prochlorothrix hollandica. delta-Aminolevulinic acid-forming activity was detected in soluble extracts of all three species. delta-Aminolevulinic acid formation by Synechocystis extracts was further characterized. Activity depended upon addition of reduced pyridine nucleotide, ATP, and Mg2+ to the incubation mixture. NADPH was a more effective pyridine nucleotide than NADH at low concentrations, but NADPH inhibited delta-amino-levulinic acid formation above 1 mM, whereas NADH did not. The pH optimum was about 7.6, and the ATP concentration optimum was 0.1 mM. Activity was stimulated by addition of RNA derived from Synechocystis or Chlorella, and abolished by preincubation with RNase A. After RNase inactivation, activity was restored by addition of RNasin to block further RNase action, followed by supplementation with Synechocystis RNA. Activity was inhibited by micromolar concentrations of hemin, as was previously found with plant and algal extracts. Complete dependence on added glutamate could not be achieved. Radioactivity was incorporated into delta-aminolevulinic acid when the incubation mixture contained 1-[14C]glutamate. Activity in the Synechocystis enzyme extract was stimulated by the addition of a partially purified enzyme fraction from Chlorella. It thus appears that prokaryotic oxygenic organisms share with chloroplasts the capacity for biosynthesis of photosynthetic pigments from glutamate via the RNA-dependent 5-carbon pathway.
Highlights
Tide, ATP,and M 8 +to theincubation mixture.NADPH Active extracts have been obtained from the methanogen was a more effective pyridine nucleotide thanNADH Methanobacterium thermoautotrophicum [19]
Synechocystis cells were growannd extracted, and 6-aminolevulinic acid-forming activity was determined as described in the text
In the case of Synechococcus, the activity could be detected in the highspeed supernatant and was stimulated by adding RNA isolated from Synechocystis (Table V). 6-Aminolevulinic acidforming activity in Prochlorothrix extracts was in the lowspeed supernatant after disruptioonf the cells, but was located in both the pellet and the supernatant after high-speed centrifugation
Summary
The supernatant was diluted with RNA extraction buffer (10mM Tris-HC1 (pH 7.5), 10 mM Mg(acetate)z,100 mM NaCl, 10 mM 8-mercaptoethanol) to a final volume of 4 ml/g of cells [35]. The precipitated nucleic acids were redissolved in RNA extraction buffer and chromatographed on DEAE-cellulose as previously described [35]. The fractions containing tRNAwere combined and precipitated by adding 2.5 volumesof ice-cold ethanol and cooling overnight at -25'C.Nucleic acids were deacylated by redissolving the precipitate in deacylation buffer (0.5 M Tris-HC1 (pH 8.0)), incubating at room temperature for 2 h, precipitating with ethanol, and washing twice with ethanol [35]. Extraction and Assay Procedures for 6-Aminolevulinic Acid Synthase-Cells of Synechocystis and Euglena were harvested as described above, but the buffer was changed to one of two d-aminolevulinic acid synthase incubation buffers. All other reagents were from Sigma, Fisher, or Research Organics
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