Bernhard Grimm

Structural genes of glutamate 1-semialdehyde aminotransferase for porphyrin synthesis in a cyanobacterium and Escherichia coli.

SummaryIn bacteria 5-aminolevulinate, the universal precursor in the biosynthesis of the porphyrin nucleus of hemes, chlorophylls and bilins is synthesised by two different pathways: in non-sulphur purple bacteria (Rhodobacter) or Rhizobium 5-aminolevulinate synthase condenses glycine and succinyl-CoA into 5-aminolevulinate as is the case in mammalian cells and yeast. In cyanobacteria, green and purple sulphur bacteria, as in chloroplasts of higher plants and algae a three step pathway converts glutamate into 5-aminolevulinate. The last step is the conversion of glutamate 1-semialdehyde into 5-aminolevulinate. Using a cDNA clone encoding glutamate 1-semialdehyde aminotransferase from barley, genes for this enzyme were cloned from Synechococcus PCC6301 and Escherichia coli and sequenced. The popC gene of E. coli, previously considered to encode 5-aminolevulinate synthase, appears to be a structural gene for glutamate 1-semialdehyde aminotransferase. Domains with identical amino acid sequences comprise 48% of the primary structure of the barley, cyanobacterial and putative E. coli glutamate 1-semialdehyde aminotransferases. The cyanobacterial and barley enzymes share 72% identical residues. The peptide containing a likely pyridoxamine phosphate binding lysine is conserved in all three protein sequences.

Purification and partial amino acid sequence of the glutamate 1-semialdehyde aminotransferase of barley and Synechococcus

Glutamate-1-semialdehyde aminotransferase (E.C. 5.4.3.8) was purified from barley and the cyanobacteria Synechococcus PCC 6301. The purification procedure involved serial affinity chromatography and preparative polyacrylamide gel electrophoresis under non-denaturing conditions. The aminotransferase of these two organisms showed different mobilities in non-denaturing gels. In SDS-PAGE the enzyme from both organisms migrated as a single protein with an apparent molecular weight of 46.000 Da. An antibody against the barley enzyme cross-reacted with the cyanobacterial aminotransferase. This antibody also recognized a 17 kDa peptide cleaved from the barley protein with cyanogen bromide. Amino acid sequences of the NH2-termini revealed significant homology between the eucaryotic and cyanobacterial enzyme.

The Glutamate 1-Semialdehyde Aminotransferase of Barley: Purification of the Protein, c-DNA Cloning, Sequencing and Characterisation of the mRNA

Chlorophyll synthesis in plants is regulated at steps converting glutamate into δ-aminolevulinate (ALA). The stromal protein glutamate 1-semialdehyde (GSA) aminotransferase catalyzes the last reaction in the synthesis of ALA. We identified and sequenced a c-DNA clone encoding the GSA-aminotransferase in order to study the expression of this enzyme and its catalytic mechanism of transamination.