Biosynthesis of Δ-aminolevulinate in Cyanidium caldarium: Characterization of tRNAGlu, ligase, dehydrogenase and glutamate 1-semialdehyde aminotransferase

Abstract

The unicellular red algae Cyanidium caldarium converts the intact carbon skeleton of glutamate to δ-aminolevulinate. The components required for this conversion are: tRNAGlu, ligase, dehydrogenase, glutamate 1-semialdehyde aminotransferase, ATP, NADPH and Mg2+. The enzymes and the tRNA involved in δ-aminolevulinate synthesis were isolated from Cyanidium caldarium and characterised. Soluble proteins from greening cells were separated by gel filtration on Sephacryl S-300 and passed sequentially through Blue Sepharose, Matrex gel Red A and chlorophyllin Sepharose. The ligase and the dehydrogenase activities were bound to Blue Sepharose as well as Matrex gel red A. Chlorophyllin Sepharose retained the tRNAGlu, while glutamate 1-semialdehyde aminotransferase passed through the affinity columns and was collected in the run-off protein fraction. The run-off fraction also contained δ-aminolevulinate dehydratase and porphobilinogen deaminase. Glutamate was converted to glutamate 1-semialdehyde in the presence of tRNAGlu, ATP, NADPH and Mg2+, either by the Blue Sepharose-bound protein fraction or the Matrex gel Red A-bound protein fraction. In the presence of the aminotransferase-containing fraction, the glutamate 1-semialdehyde produced was converted into δ-aminolevulinate. Two compounds in addition to glutamate 1-semialdehyde and glutamyl tRNAGlu were produced from glutamate when the Blue Sepharose-bound protein fraction was incubated with tRNAGlu, ATP, NADPH and Mg2+. One of these was converted into δ-aminolevulinate upon incubation with the run-off protein fraction and was assumed to be an additional intermediate in the δ-aminolevulinate biosynthesis pathway. The tRNA preparation from Cyanidium caldarium gave a minor and a major peak of glutamate acceptor activity when analysed by High Pressure Liquid Chromatography on a C18 column containing trioctyl methylammonium chloride. The ability to convert glutamate to glutamate 1-semialdehyde was associated with the major peak of glutamate acceptor activity. Glutamate 1-semialdehyde aminotransferase of Cyanidium caldarium was stimulated by low concentrations (up to 100 μm) of pyridoxal 5′ phosphate and pyridoxamine 5′ phosphate. At higher concentrations (0.5 to 5mm), pyridoxamine 5′ phosphate was further stimulatory while pyridoxal 5′ phosphate was inhibitory. Gabaculine inhibited glutamate 1-semialdehyde aminotransferase and was more effective than the gabaculine-pyridoxal 5′ phosphate adduct, m-carboxyphenyl pyridoxamine 5′ phosphate.