Human delta-aminolevulinate dehydratase: nucleotide sequence of a full-length cDNA clone.
Two cDNAs encoding human delta-aminolevulinate dehydratase (ALA-D; porphobilinogen synthase; EC 4.2.1.24), the second enzyme in the heme biosynthetic pathway, were identified, recloned into bacteriophage M13, and sequenced by primer extension. The first clone with an 827-base-pair (bp) pEX-ALA-D cDNA insert, shown to contain DNA sequences that were colinear with four bovine ALA-D peptide sequences, was used to screen a pKT218 human liver library. A second clone containing a 1200-bp insert was identified that contained an open reading frame of 990 bp as well as 5' (66 bp)- and 3' (94 bp)-untranslated regions, the latter terminating in poly(dA). The predicted N-terminal amino acid sequence was colinear with the first 13 residues of microsequenced ALA-D purified from human erythrocytes. The ATG initiation codon was preceded by ACGCC, a functional initiation sequence, while an upstream (position -32), in-phase AACTG ATG sequence was entirely nonhomologous with the initiation consensus sequence and, therefore, presumed to be nonfunctional. The unusual polyadenylylation signal, AGTAAA, has been reported only in the human HRAS1 gene. The nucleotide sequences of the two cDNA clones differed at position 730 or 733 and encoded two differently charged amino acids. This nucleotide difference may be the basis for the polymorphic charge isozymes of human ALA-D. The sequence encoding this zinc metalloenzyme contained a cysteine- and histidine-rich binding site for zinc and an unusual region of charge complementarity surrounding the active lysine residue in the catalytic site.
Wetmur
J G
JG
Bishop
D F
DF
Cantelmo
C
C
Desnick
R J
RJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis*; D006801:Humans; D000623:Porphobilinogen Synthase / Q000032:analysis / Q000235:genetics*; D011487:Protein Conformation; D015032:Zinc / Q000378:metabolism
D004247:DNA; D000623:Porphobilinogen Synthase; D015032:Zinc