Structure and regulation of vertebrate δ‐aminolevulinate synthases

Abstract

Two distinct patterns of regulating heme biosynthesis have been observed in animals: while heme negatively regulates the synthesis of δ-aminolevulinate (ALA) synthase in non-erythroid cells, the expression of the enzyme is regulated developmentally in red blood cells. This observation eventually led to the cloning of both a tissue-specific ALA synthase isozyme (ALAS-E) that is expressed in erythroid-lineage cells and is distinct from the housekeeping isozyme (ALAS-N). We originally isolated cDNA clones encoding chicken ALAS-E by the combined use of an anti-chicken ALAS-N antibody, which was partially cross-reactive to chicken ALAS-E, and a λgt11 expression library. ALAS-E was also purified to homogeneity from rat reticulocyte lysate using a papain digestion method. The papain-resistant core catalytic domain overlaps with the evolutionarily conserved segment that had been described by sequence alignment of ALA synthases from a variety of species, suggesting that the papain-resistant domain represents the ancestral core of the enzyme. Blot hybridization analysis of RNA isolated from various developmental stage rat livers and from chicken and mouse erythroleukemia cells demonstrated that ALAS-E is the key enzyme which supplies large quantities of heme for hemoglobin synthesis. We are currently investigating the mechanisms which confer erythroid-specific transcriptional activation of the ALAS-E gene through transient transfection assays in erythroid cells using human and chicken ALAS-E genes. These experiments have identified promoter elements which are required for high level, erythroid-specific transcription of the genes.