Two tissue-specific factors bind the erythroid promoter of the human porphobilinogen deaminase gene.

We have studied the erythroid-specific promoter of the human gene coding for Porphobilinogen Deaminase (PBGD) by DNaseI footprinting, gel retardation and methylation interference assays. We show that this promoter, which is inducible during MEL cell differentiation, contains three binding sites for the erythroid-specific factor NF-E1 and one site for a second erythroid-specific factor, which we name NF-E2. NF-E1 is a factor that also binds the promoter and the enhancer (present in the 3' flanking region) of the human beta-globin gene. NF-E2 has not yet been described and although it binds to a sequence containing the Ap1 consensus, it appears to be different from Ap1.     

Linkage disequilibrium between DNA polymorphisms within the porphobilinogen deaminase gene

Summary Three restriction fragment length polymorphisms (RFLPs) (MspI, PstI, ScrFI/BstNI) within the human porphobilinogen deaminase (PBG-D) gene have been studied in 47 unrelated patients with the autosomal dominant disorder, acute intermittent porphyria (AIP), and in 92 control subjects. Each enzyme identified a two-allele polymorphism with allele frequencies close to 0.50; however, marked linkage disequilibrium limited the number of observed haplotypes to four, of which one is uncommon. No association was detected between any haplotype and AIP.     

No association between RFLPs at the porphobilinogen deaminase gene and schizophrenia

Summary An association study of restriction fragment length polymorphisms (RFLPs) in the porphobilinogen deaminase (PBGD) gene and schizophrenia was conducted. RFLPs detected by MspI, PstI, ApaLI and BstNI in intron 1 of the gene were studied in 49 patients and 79 controls. There were no significant differences between the groups in allele frequencies, genotype counts or haplotype distribution.     

Mechanism of action of porphobilinogen deaminase. The participation of stable enzyme substrate covalent intermediates between porphobilinogen and the porphobilinogen deaminase from Rhodopseudomonas spheroides.

Highly stable labelled complexes are formed between porphobilinogen deaminase and stoicheiometric amounts of [14C]porphobilinogen. On completion of the catalytic cycle by the addition of excess of substrate, the complexes yield labelled product and display all the properties expected from covalently bound enzyme intermediates involved in the deaminase catalytic sequence.     

Cloning and functional characterization of LCR-F1: a bZIP transcription factor that activates erythroid-specific,human globin gene expression.

DNase I hypersensitive site 2 (HS 2) of the human beta-globin Locus Control Region (LCR) directs high level expression of the beta-globin gene located 50 kilobases downstream. Experiments in cultured cells and in transgenic mice demonstrate that duplicated AP1-like sites in HS 2 are required for this powerful enhancer activity. A cDNA clone encoding a basic, leucine-zipper protein that binds to these sites was isolated and designated Locus Control Region-Factor 1 (LCR-F1). This protein is a member of a new family of regulatory factors that contain a 63 amino acid ''CNC domain'' overlapping the basic region. This domain is approximately 70% identical in the Drosophila Cap N Collar (CNC) protein, NF-E2 and LCR-F1. LCR-F1 transactivates an HS 2/gamma-globin reporter gene over 170-fold in transient transfection experiments specifically in erythroid cells. These results suggest that LCR-F1 may be a critical factor involved in LCR-mediated, human globin gene expression.     

Two new polymorphisms in introns 2 and 3 of the human porphobilinogen deaminase gene

Sequencing of the polymerase chain reaction amplified exon 2–4 fragment of the human porphobilinogen deaminase gene revealed a G/T polymorphism (I2G and I2T) in intron 2, and a G/A polymorphism (I3G and I3A) in intron 3 of the gene. The frequencies of these alleles are presented.The nucleotide sequence data reported in this paper (the sequence of introns 2 and 3, and the polymorphic sites) will appear in the DDBJ, EMBL, and GenBank Nucleotide Sequence Database with accession nos. D10608 and D12722     

Kinetic and molecular parameters of human hepatic porphobilinogen deaminase

A basal level of human liver porphobilinogen deaminase of 3.66 units/g wet weight was found in adult tissue. Activity in neonatal liver was at least three fold higher. Physico-chemical studies revealed that the enzyme has the approximate form of a symmetrical molecule and exhibits hyperbolic kinetics with a Km value of 3.6 microM at pH 7.6. Two ionizable groups with pK values of 7.35 and 8.90 are prominent for catalysis. A set of pI (5.8-4.9) were observed under different conditions. Results demonstrate the existence of a single protein differentially charged with multiple molecular forms in adult liver.     

Haplotyping of the human porphobilinogen deaminase gene in acute intermittent porphyria by polymerase chain reaction

Summary Acute intermittent porphyria (AIP) is due to a defect in porphobilinogen deaminase (PBGD, E.C. 4.1.3.8) inherited as an autosomal dominant trait. Presymptomatic carrier detection is important in order to avoid exposure to factors inducing severe clinical symptoms. Carriers and noncarriers of the AIP gene can be distinguished by linkage analysis using three intragenic RFLPs in AIP families. In the present study, the polymerase chain reaction (PCR) was used to amplify 3.3-kb genomic sequences covering three polymorphic sites. Haplotypes were identified after cleavage of amplified products with three restriction enzymes, showing that the technique can be successfully used for linkage analysis in AIP families.     

Structural basis of pyrrole polymerization in human porphobilinogen deaminase

Human porphobilinogen deaminase (PBGD), the third enzyme in the heme pathway, catalyzes four times a single reaction to convert porphobilinogen into hydroxymethylbilane. Remarkably, PBGD employs a single active site during the process, with a distinct yet chemically equivalent bond formed each time. The four intermediate complexes of the enzyme have been biochemically validated and they can be isolated but they have never been structurally characterized other than the apo- and holo-enzyme bound to the cofactor. We present crystal structures for two human PBGD intermediates: PBGD loaded with the cofactor and with the reaction intermediate containing two additional substrate pyrrole rings. These results, combined with SAXS and NMR experiments, allow us to propose a mechanism for the reaction progression that requires less structural rearrangements than previously suggested: the enzyme slides a flexible loop over the growing-product active site cavity. The structures and the mechanism proposed for this essential reaction explain how a set of missense mutations result in acute intermittent porphyria.