δ-Aminolevulinate dehydratase deficiency porphyria (ADP, also known as Doss porphyria, MIM 125270.) is a very rare autosomal recessive porphyric disorder due to a deficiency of cytosolic enzyme delta-aminolevulinate dehydratase (ALAD, also known as porphobilinogen synthase or PBGS.).
This is the rarest form of porphyria and only few cases have been reported to date, which include two young men from Germany reported by Doss and coworkers, one child from Sweden studied by Thunell and colleagues and one elderly patient from Belgium reported by Hassoun and associated.
The autosomal recessive disease is very rare, but it has been estimated that the prevalence of individuals with 50% of normal ALAD activity is about 2% in the normal asymptomatic population (see below).
Patients with this condition have clinical symptoms of acute hepatic porphyria such as abdominal pain, vomiting, pain in the arms and legs, hypochromic anemia and neuropathy.
Characteristic laboratory findings are markedly increased urinary excretion of ALA due to the enzyme deficiency. Urinary coproporphyrinogen III and erythrocyte zinc protoporphyrin are also increased, although the reason for these findings are not clear.
ALAD porphyria is a "conformational disease" of ALAD. To date a total of nine point mutations of the alad gene have been identified, which resulted in different amino acid changes (K59N, A274T, V153M, G133R, K59N, G133R, F12L, R240W and V275M); all these point mutations prevent the folding and/or the zinc binding site, thus the catalytic activity of the enzyme. All known porphyria-associated PBGS mutations increase the propensity of the human protein to exist in the low activity hexameric assembly, thus establishing the physiologic relevance of the quaternary structure equilibrium to human health.
Although aboveall mutation are not at the enzyme active site, the quaternary structure change can be seen todestabilize the active-site lid and to account for the low activity of the hexamer.
Spatial location of the porphyria-associated mutations of human ALAD. The crystal structure contains an asymmetric hugging dimer in the unit cell; the two subunits differ predominantly in their regions of disorder. The more highly ordered monomer is illustrated by use of a light-gray strand to illustrate the backbone. Product is seen bound at the enzyme active site and known human variations are shown using space-filling representation. Lys59, which has some disorder, is green, Phe12 is magenta, Ala274 is red, Gly133 is blue, Arg240 is yellow, Cys132 is cyan, Glu89 is orange, Val153 is pink, and Val275 is purple.
ALAD deficiency porphyria is an autosomal recessive disorder caused by homozygous ALAD deficiency and mutations are highly heterogeneous.
Although homozygous or compound heterozygous deficiency of ALAD is extremely rare, enzyme activities below 50% of normal level are not exceptional in the population, as found in 16 of 880 (2%) normal individuals examined in a Swedish study.
These individuals with low ALAD activity may be more vulnerable to toxic effects of chemical or compounds inhibiting ALAD activity such as lead, trichloroethylene, styrene oxide or bromobenzene
Physiologically, ALAD inhibitors can derive from environmental contaminants, natural products, or drugs such as amebicidal agents. A drug that functions as a strong inhibitor could provide a structural explanation for the drug’s side effects. One discovered inhibiting molecule, which was called ML- 3H2 (5-chloro-7-diethylaminomethyl-quinolin-8-ol), is chemically similar to two amebicidal agents currently used in surgery, Clioquinol and Iodoquinol which have porphirya-like side effects. It is possible that exposure to inhibiting compounds could contribute to the sporadic nature of the symptoms, which is a common and poorly understood aspect of porphyria. It is also possible that allosteric ligands of human ALAD could raise metabolic ALA levels and cause deleterious side effects on the nervous system because of the structural similarity between ALA and the neurotransmitter 4-aminobutyric acid.
Hormonal fluctuations in women contribute to cyclical attacks and are usually treated with oral contraceptives and luteinizing hormones to shut down menstrual cycles. Androgens and fertility hormones also trigger attacks.
A case of ALAD porphyria with syndrome of inappropriate secretion of antidiuretic hormone (SIADH) in a 69-year-old woman was reported (Muraoka et al, 1995).
Six diabetic, azotemic patients with no prior history of porphyria, who developed a syndrome similar to acute intermittent porphyria after initiation of treatment with erythropoietin were described by Hedger and colleagues in 2006. The porphyria resolved when treatment with erythropoietin was stopped.
TISSUE SPECIFIC RISK FACTORS
anatomical (due its structure)
vascular (due to the local circulation)
physiopathological (due to tissue function and activity)
Tissue distribution of ALAD, depending on alternative splicing, drives clinical manifestations (i.e.
hepatic or erythripoietic).
Whilst haem, the final product of the biosynthetic pathway, is biologically important, porphyrins
and their precursors are not only useless, but also toxic. In fact, the structural similarity between
ALA and the neurotransmitter 4-aminobutyric acid is related to the neurologic sequelae of porphyrias and lead poisoning.
Therapy with heme arginate in combination with glucose infusion are effective in the management of acute crises in all ADP patients. Avoidance of drugs that are harmful in other acute porphyrias should be recommended.
ALAD is an allosteric enzyme that catalyzes the Knorr-type (asymmetric) condensation of two molecules of δ-aminolevulinic acid (ALA) to form one molecule of the monopyrrole ring porphobilinogen.
CHEMICAL STRUCTURE AND IMAGES
CHEMICAL STRUCTURE AND IMAGES
Protein Aminoacids Percentage
The Protein Aminoacids Percentage gives useful information on the local environment and the metabolic status of the cell (starvation, lack of essential AA, hypoxia). For example, in ALAD, alanine, leucine and valine are more abundant and all these aminoacids are hydrophobic.
SYNTHESIS AND TURNOVER
The gene contains two first exons, named 1A and 1B, wich are alternatively spliced to exon 2, where the coding region begins. Each exon has its own promoter. The promoter driving exon 1A expression is TATA less and contains many GC boxes. In contrast, the exon 1B promoter bears regulatory sequences similar to those found in beta-globin and other erythroid specific genes, such as GATA-1 and NF-E2 sites. Tissue distribution studies reveal that ALAD mRNA containing exon 1A is ubiquitous, whereas mRNA containing exon 1B is found only in erythroid tissues. In fact, exon 1B expression increases 3-fold during maturation of murine erythroid progenitor cells simultaneously to GATA-1 mRNA levels.
Tahara et al described a decrease of hepatic delta-aminolevulinate dehydratase activity in an animal model of fatigue. The ALAD activity, as well as its gene expression, was restored after as the treatment stopped.
ALAD is a cytosolic enzyme and is committed in the heme biosynthetic pathway.
Heme biosynthesis in humans is an essential metabolic function controlled predominantly at the first committed step, which is the mitochondrial synthesis of 5- aminolevulinic acid from succinyl–coenzyme A (CoA) and glycine. ALAD activity is present in large excess compared to other enzymes in the heme biosynthetic pathway.
- Alternative functions
Mammals contain as much as 100-fold more enzyme activity than is necessary for required heme formation. Alternative possible moonlighting functions for ALAD include roles in proteosome-directed protein degradation and in chaperone-assisted protein folding.
- Structural protein
The human ALAD protein is a oligomer of 330 amino acids composed of eight identical subunits of about 36 kDa. Each of the subunits has a binding site for an atom of zinc or of magnesium (in plants). The sequence of the region in the vicinity of the active site residue is conserved in ALAD from various prokaryotic and eukaryotic species.
Human δ-aminolevulinate dehydratase or ALAD exists as a quaternary structure equilibrium consisting of a high activity octamer, a low activity hexamer and a dimer that can take on two distinct conformations, each of which dictates assembly to either the octamer or the hexamer, that suggest a dynamic flexibility of this homo oligomeric protein. For the wild type protein at neutral pH, the octamer is the dominant assembly in the equilibrium. Addition of magnesium favors the examer-octamer transition suggesting an iondependent allosteric regulation of protein function. In the last years emerged the concept of protein quaternary structure dynamics of ALAD, viewed as an ensemble of conformations, which is the consequence of an allosteric regulation. In this new model one functional homo-oligomer can dissociate, change conformation, and reassociate into a different oligomer, that is the concept of morpheeins. ALAD porphyria is the first identified morpheein-based conformational disease as each porphyria associated mutation shifts the human ALAD morpheein equilibrium toward the exameric conformation.
The substrate ALA stabilizes the octameric assembly of ALAD, promoting a closed conformation of the active site lid, which in turn stabilizes a subunit interaction that is found in the octamer but not the hexamer. The arrangement of the subunits in the ALAD hexamer creates a surface cavity that is not present in the octamer or the dimers. Ligand binding to this cavity is posited to stabilize the hexamer, draw the quaternary structure equilibrium toward the hexamer, and thus inhibit function. Small molecules that bind selectively to the hexamer, presumably by binding in this surface cavity, can act as inhibitors whose mechanism of action is stabilization of a low activity oligomer. ALAD inhibitors, discovered to stabilize the wild type human PBGS hexamer, are predicted to have increased potency against the naturally occurring ALAD porphyria associated variants. These data support the hypothesis that inhibition of human ALAD, for example by Clioquinol, is an off-target side effect of these drugs and may contribute to clinically observed side effects. It is possible that
other known drugs, toxins or environmental contaminants could have a similar hexamer-stabilizing inhibitory effect on human ALAD.
Equilibrium of human ALAD morpheeins. The ALAD quaternary structure isoforms are the high-activity octamer (wild-type protein, shades of red), the transient hugging dimer, the transient detached dimer, and the low-activity hexamer (F12L variant, shades of blue).
Molecular analysis could identify heterozygous carriers of ALAD deficiency among normal population (about 2%) wich are more vulnerable to toxic effects of chemical or compounds inhibiting ALAD activity.