Co-synthesis of Human δ-Aminolevulinate Dehydratase (ALAD) Mutants with the Wild-type Enzyme in Cell-free System—Critical Importance of Conformation on Enzyme Activity—

Properties of mutant δ-aminolevulinate dehydratase (ALAD) found in patients with ALAD porphyria were studied by enzymological and immunological analyses after the synthesis of enzyme complexes using a cell-free system. Enzyme activities of homozygous G133R, K59N/G133R, V153M, and E89K mutants were 11%, 22%, 67%, and 75% of the wild-type ALAD, respectively, whereas that of K59N, a normal variant, was 112%. Enzyme activities of L273R, C132R and F12L were undetectable. Co-synthesis of F12L, L273R, G133R, K59N/G133R, or C132R mutants with the wild-type at various ratios showed that ALAD activity was proportionally decreased in the amount of the wild-type in the complex. In contrast, co-synthesis of V153M, K59N, and E89K with the wild-type did not influence enzyme activity of the wild-type. Surface charge changes in K59N, E89K, C132R and G133R predicted by mutations were also confirmed by native polyacrylamide gel electrophoresis. A compound E89K and C132R complex showed ALAD activity similar to that was found in erythrocytes of the patient. These findings indicate that cell-free synthesis of ALAD proteins reflects enzymatic activities found in patients, and suggest that, in addition to the direct effect of mutations on the catalytic activity, conformational effects play an important role in determining enzyme activity

A precursor‐inducible zebrafish model of acute protoporphyria with hepatic protein aggregation and multiorganelle stress

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154358/1/fsb2030005012.pd

Porfirias agudas: aspectos laboratoriais

Porphyrias are disorders characterized by identical neurological disfunctions arising from an enzymatic defect in the heme biosynthetic pathway. The acute porphyrias, namely 5-aminolevulinic acid dehydratase porphyria, acute intermittent porphyria, hereditary coproporphyria and variegate porphyria are caused respectively by deficiencies in the enzymes 5aminolevulinic acid dehydratase, porphobilinogen deaminase, coproporphyrinogen decarboxilase and protoporphyrinogen oxidase. Acute porphyrias are characterized by an increase of plasma and liquor 5aminolevulinic acid levels, followed by increased urinary excretion of this compound. Increases in the urinary excretion of porphyrins or other porphyrin precursors may be observed depending on the type of acute porphyria. Diagnosis of acute porphyria cases rests on the measurement of porphyrins or porphyrin precursors in urine, blood and faeces. Since the clinical manifestations are generally triggered by ambiental or acquired factors, screenings for gene carriers among relatives of AIP are important to identify latent cases. The diagnosis of gene carriers was usually based on the determination of the activity of blood heme biosynthetic enzymes and more recently on DNA analysis.As porfirias são causadas por deficiência parcial de uma das enzimas da via de biossíntese do heme, caracterizando-se por disfunções neuroviscerais bastante semelhantes. As porfirias agudas são decorrentes da deficiência das enzimas delta-aminolevulinato desidratase (ALAD), porfobilinogênio desaminase, coproporfirinogênio oxidase ou protoporfirinogênio oxidase, que provocam, respectivamente, porfiria por deficiência da ALAD, porfiria aguda intermitente, coproporfiria hereditária e porfiria variegada. Todas as porfirias agudas caracterizam-se por um aumento na concentração de ácido 5-aminolevulínico no plasma e no líquor, acompanhado de um aumento na excreção urinária deste composto. Dependendo do tipo de porfiria aguda que acomete o paciente, podem ser observados ainda aumentos na excreção urinária de outros precursores porfirínicos e mesmo de porfirinas. Os casos de porfirias agudas podem ser detectados pela dosagem de porfirinas e seus precursores na urina, sangue e fezes. A triagem das famílias portadoras é importante para detectar casos latentes, pois a expressão clínica da doença geralmente está ligada a fatores ambientais ou adquiridos que provocam os ataques agudos. A identificação de portadores pode ser realizada através da determinação da atividade das enzimas da via de biossíntese do heme no sangue e mais recentemente através da análise do DNA

Environmental chemical exposures and disturbances of heme synthesis.

Porphyrias are relatively uncommon inherited or acquired disorders in which clinical manifestations are attributable to a disturbance of heme synthesis (porphyrin metabolism), usually in association with endogenous or exogenous stressors. Porphyrias are characterized by elevations of heme precursors in blood, urine, and/or stool. A number of chemicals, particularly metals and halogenated hydrocarbons, induce disturbances of heme synthesis in experimental animals. Certain chemicals have also been linked to porphyria or porphyrinuria in humans, generally involving chronic industrial exposures or environmental exposures much higher than those usually encountered. A noteworthy example is the Turkish epidemic of porphyria cutanea tarda produced by accidental ingestion of wheat treated with the fungicide hexachlorobenzene. Measurements of excreted heme precursors have the potential to serve as biological markers for harmful but preclinical effects of certain chemical exposures; this potential warrants further research and applied field studies. It has been hypothesized that several otherwise unexplained chemical-associated illnesses, such as multiple chemical sensitivity syndrome, may represent mild chronic cases of porphyria or other acquired abnormalities in heme synthesis. This review concludes that, although it is reasonable to consider such hypotheses, there is currently no convincing evidence that these illnesses are mediated by a disturbance of heme synthesis; it is premature or unfounded to base clinical management on such explanations unless laboratory data are diagnostic for porphyria. This review discusses the limitations of laboratory measures of heme synthesis, and diagnostic guidelines are provided to assist in evaluating the symptomatic individual suspected of having a porphyria

Expression, purification and characterisation of protoporphyrinogen oxidases from diverse species

Bibliography: leaves 163-203.This work involved the characterisation of protoporphyrinogen oxidase (PPO), the penultimate enzyme in haem biosynthesis, from Bacillus subtilis, Myxococcus xanthus, and human. A defect in human PPO causes variegate porphyria, an autosomal dominant disorder characterised by skin photosensitivity and propensity towards acute neurovisceral crises. At the beginning of this project little information was available on the kinetic and biophysical properties of isolated PPOs due largely to difficulties associated with their purification from natural sources