Human uroporphyrinogen III synthase: molecular cloning, nucleotide sequence, and expression of a full-length cDNA.

Abstract

Uroporphyrinogen III synthase [URO-synthase; hydroxymethylbilane hydro-lyase (cyclizing), EC 4.2.1.75], the fourth enzyme in the heme biosynthetic pathway, is responsible for conversion of the linear tetrapyrrole, hydroxymethylbilane, to the cyclic tetrapyrrole, uroporphyrinogen III. The deficient activity of URO-synthase is the enzymatic defect in the autosomal recessive disorder congenital erythropoietic porphyria. To facilitate the isolation of a full-length cDNA for human URO-synthase, the human erythrocyte enzyme was purified to homogeneity and 81 nonoverlapping amino acids were determined by microsequencing the N terminus and four tryptic peptides. Two synthetic oligonucleotide mixtures were used to screen 1.2 x 106 recombinants from a human adult liver cDNA library. Eight clones were positive with both oligonucleotide mixtures. Of these, dideoxy sequencing of the 1.3 kilobase insert from clone pUROS-2 revealed 5' and 3' untranslated sequences of 196 and 284 base pairs, respectively, and an open reading frame of 798 base pairs encoding a protein of 265 amino acids with a predicted molecular mass of 28,607 Da. The authenticity of this clone was established by colinearity of the predicted amino acid sequence with 81 microsequenced residues from the purified enzyme. In addition, high levels of enzymatic activity and immunoreactive protein were expressed when a blunt-ended 971-base-pair Ava II cDNA fragment containing the entire coding region was inserted into vectors for expression in Escherichia coli. The isolation and expression of this full-length cDNA for human URO-synthase should facilitate studies of the structure, organization, and chromosomal localization of this heme biosynthetic gene as well as the characterization of the molecular lesions causing congenital erythropoietic porphyria. were required for the conversion of the monopyrrole, porphobilinogen, to the cyclic tetrapyrrole, uroporphyrinogen III (2). During the next two decades, the precise function of the two enzymes in uroporphyrinogen III biosynthesis was the subject of active investigation and debate, particularly with regard to their reaction mechanisms and intermediates and their potential interaction in a cytosolic enzyme complex (for reviews, see refs. 3 and 4). It is now recognized that HMB-synthase catalyzes the head to tail condensation offour molecules of porphobilinogen to form the linear tetrapyrrole HMB (4, 5). In the presence of URO-synthase, HMB is rapidly converted to uroporphyrinogen III by an intramolecular rearrangement of the D-pyrrole group and ring closure (5-7). In the absence of URO-synthase, HMB nonenzymatically cyclizes to form the nonphysiologic uroporphyrinogen I isomer.The deficient activity of URO-synthase is the enzymatic defect in congenital erythropoietic porphyria, an inborn error of heme biosynthesis that is inherited as an autosomal recessive trait (8). Enzymatic diagnosis of affected homozygotes and asymptomatic heterozygous carriers can be made by the demonstration of deficient or intermediate lev...

Two genes in Saccharomyces cerevisiae encode a membrane-bound form of casein kinase-1.

“…Although it is likely that the association of CKI1 with membranes results from a similar mechanism, we cannot rule out the participation of other factors. Indeed, mammalian uroporphyrinogen III synthase contains the CC motif yet is classically described as a soluble enzyme (Tsai et al, 1988).…”

Congenital erythropoietic porphyria: identification and expression of 10 mutations in the uroporphyrinogen III synthase gene.

“…Recently, the fulllength cDNA encoding the human URO-synthase polypeptide of 265 amino acids was isolated, sequenced, and expressed in Escherichia coli (16). Using the cDNA as a probe, a single URO-synthase gene was assigned to the narrow chromosomal region, lOq25.3 -+ q26.3 (17).…”

“…The normal and mutant URO-synthase alleles were expressed in E. coli using the pKK223-3 vector (Pharmacia LKB Biotechnology, Inc., Piscataway, NJ) as previously described (8,16). pKK-UROS-V99A, pKK-UROS-A104V, pKK-UROS-633insA, and pKK-UROS-G225S were constructed by cassette substitution of the cloned mutant cDNA into the pKK-UROS vector (8), while the pKK-UROS-L4F, pKK-UROS-Y19C, pKK-UROS-V82F, pKK-UROS-Q249X constructs were engineered by mega-primer PCR mutagenesis (27), and then were cassette-substituted into the pKK-UROS vector.…”

Congenital erythropoietic porphyria: a novel uroporphyrinogen III synthase branchpoint mutation reveals underlying wild-type alternatively spliced transcripts

“…18 The pure wild-type and mutant enzymes were incubated in 20mM N-2-hydroxyethylpiperazine-NЈ-2-ethanesulfonic acid, pH 7.4, 0.1M NaCl, and 1mM dithiothreitol, for various times at 37°C, transferred to an ice bath, and then assayed to determine thermostability. 17 …”

“…17 These constructs were also cloned into a high-level pET SUMO expression vector as a SUMO fusion protein, cleaved, and purified to homogeneity as previously described. 18 The pure wild-type and mutant enzymes were incubated in 20mM N-2-hydroxyethylpiperazine-NЈ-2-ethanesulfonic acid, pH 7.4, 0.1M NaCl, and 1mM dithiothreitol, for various times at 37°C, transferred to an ice bath, and then assayed to determine thermostability.…”