All reported mutations in ALAS2, which encodes the rate-regulating enzyme of erythroid heme biosynthesis, cause X-linked sideroblastic anemia. We describe eight families with ALAS2 deletions, either c.1706-1709 delAGTG (p.E569GfsX24) or c.1699-1700 delAT (p.M567EfsX2), resulting in frameshifts that lead to replacement or deletion of the 19-20 C-terminal residues of the enzyme. Prokaryotic expression studies show that both mutations markedly increase ALAS2 activity. These gain-of-function mutations cause a previously unrecognized form of porphyria, X-linked dominant protoporphyria, characterized biochemically by a high proportion of zinc-protoporphyrin in erythrocytes, in which a mismatch between protoporphyrin production and the heme requirement of differentiating erythroid cells leads to overproduction of protoporphyrin in amounts sufficient to cause photosensitivity and liver disease.

Variegate porphyria (VP), a low-penetrant autosomal dominant inherited disorder of haem metabolism, is characterised by photosensitivity (Fig. 1) and a propensity to develop acute neuropsychiatric attacks with abdominal pain, vomiting, constipation, tachycardia, hypertension, psychiatric symptoms and, in the worst cases, quadriplegia. Acute attacks, often precipitated by inappropriate drug therapy, are potentially fatal. While earlier workers thought the distal haem biosynthetic enzyme ferrochelatase may be involved in the genesis of VP, it was shown in the early 1980's, and is now accepted, that VP is associated with decreased protoporphyrinogen oxidase activity (PPO) (E.C.1.3.3.4). VP prevalence is much higher in South Africa than elsewhere; probably due to a founder effect with patients descending from a 17th century Dutch immigrant. PPO cDNAs from Bacillus subtilis, Myxococcus xanthus, human placenta and mouse liver have been cloned, sequenced and expressed. Human and mouse cDNAs consist of open reading frames 1431 nucleotides long, encoding a 477 amino acid protein. The human PPO gene contains thirteen exons, spanning approximately 4.5 kb. We have identified a C to T transition in codon 59 (in exon 3) resulting in an arginine to tryptophan substitution (R59W). A protein expressed from an in vitro-mutagenized PPO construct exhibits substantially less activity than the wild type. The R59W mutation was present in 43 of 45 patients with VP from 26 of 27 South African families investigated, but not in 34 unaffected relatives or 9 unrelated British patients with PPO deficiency. Since at least one of these families is descended from the founder of South African VP, this defect may represent the founder gene defect associated causally with VP in South Africa.

Protoporphyrinogen IX oxidase, a monotopic membrane protein, which catalyzes the oxidation of protoporphyrinogen IX to protoporphyrin IX in the heme/chlorophyll biosynthetic pathway, is distributed widely throughout nature. Here we present the structure of protoporphyrinogen IX oxidase from Myxococcus xanthus, an enzyme with similar catalytic properties to human protoporphyrinogen IX oxidase that also binds the common plant herbicide, acifluorfen. In the native structure, the planar porphyrinogen substrate is mimicked by a Tween 20 molecule, tracing three sides of the macrocycle. In contrast, acifluorfen does not mimic the planarity of the substrate but is accommodated by the shape of the binding pocket and held in place by electrostatic and aromatic interactions. A hydrophobic patch surrounded by positively charged residues suggests the position of the membrane anchor, differing from the one proposed for the tobacco mitochondrial protoporphyrinogen oxidase. Interestingly, there is a discrepancy between the dimerization state of the protein in solution and in the crystal. Conserved structural features are discussed in relation to a number of South African variegate porphyria-causing mutations in the human enzyme.Protoporphyrinogen oxidase (PPOX) 5 (EC 1.3.3.4), the penultimate enzyme in the heme biosynthetic pathway, is the last common enzyme in heme and chlorophyll biosynthesis. It catalyzes the six-electron oxidation of protoporphyrinogen IX to protoporphyrin IX (1, 2). In humans, defects in the PPOX gene result in the dominantly inherited disorder variegate porphyria (VP) (3, 4), characterized by cutaneous photosensitivity and the propensity to develop acute neurovisceral crisis (5). This disease is particularly prevalent in South Africa because of a founder gene effect (an R59W mutation) (6, 7). In plants, PPOX is a target of light-dependent peroxidizing herbicides, which act as competitive inhibitors (8) resulting in desiccation and photobleaching of green plant tissue. Of the herbicides known to inhibit PPOX, the diphenylethers, of which acifluorfen (AF) is the most well studied, are of great interest. Such inhibition has also been documented in mammals and bacteria (9, 10). Inhibition of the enzyme leads to the accumulation and export of protoporphyrinogen to the cytoplasm, where it undergoes non-enzymatic oxidation to porphyrin. In the presence of light, accumulation of protoporphyrin can lead to reactive oxygen species that may induce cellular damage, primarily via peroxidation of membrane lipids, and ultimately cell death. A similar photodestructive mechanism, because of accumulating porphyrin intermediates in the skin of patients with VP, is probably responsible for the photocutaneous manifestations in this condition (11).In eukaryotes, PPOX is functionally conserved despite a low sequence identity. It is an intrinsic protein of the inner mitochondrial membrane and requires molecular oxygen and a flavin cofactor (in most cases, flavin adenine dinucleotide (FAD)) for this conversion. However, it is p...

Human alpha, pi, and mu class glutathione S-transferases (GSH S-T) have been localized immunohistologically in a variety of organs. Alpha GSH S-T are found principally in hepatocytes, proximal convoluted tubules of kidney, the deep reticular layer of the adrenal gland, interstitial cells of the testis, and oxyntic cells of the stomach. The pi GSH S-T are present in relative abundance in ductular, as opposed to parenchymal cells in the liver, pancreas, salivary glands, and kidney. The presence of mu GSH S-T in the tissues of certain patients and its absence in the same tissues from other patients has been demonstrated. The pi GSH S-T seems to be most persistently and strongly expressed in tumors but alpha GSH S-T are also found in some neoplasms whereas the mu GSH S-T are occasionally present when the other two transferases are weak or absent.

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