Highly heterogeneous nature of delta-aminolevulinate dehydratase (ALAD) deficiencies in ALAD porphyria

The properties of 9 delta -aminolevulinate dehydratase (ALAD) mutants from patients with ALAD porphyria (ADP) were examined by bacterial expression of their complementary DNAs and by enzymologic and immunologic assays. ALADs were expressed as glutathione-S-transferase (GST) fusion proteins in Escher ichia coli and purified by glutathione-affinity column chromatography. The GST-ALAD fusion proteins were recognized by anti-ALAD antibodies and were e nzymatically active as ALAD. The enzymatic activities of 3 ALAD mutants, K5 9N, A274T, and V153M, were 69.9%, 19.3%, and 41.0% of that of the wild-type ALAD, respectively, whereas 6 mutants, G133R, K59N/G133R, F12L, R240W, V27 5M, and deITC, showed little activity (< 8%). These variations generally re flect the phenotype of ALAD in vivo in patients with ADP and indicate that GST-ALAD fusion protein is indeed useful for predicting of the phenotype of ALAD mutants. The location of F12L mutation in the enzyme's molecular stru cture indicates that its disturbance of the quaternary contact of the ALAD dimer appears to have a significant influence on the enzymatic activity. Mo use monoclonal antibodies to human ALAD were developed that specifically re cognized a carboxy terminal portion of ALAD, or other regions In the enzyme . This study represents the first complete analysis of 9 mutants of ALAD id entified In ADP and indicates the highly heterogeneous nature of mutations in this disorder. (Blood. 2001;97:2972-2978) (C) 2001 by The American Socie ty of Hematology.