Phospholipid hydroperoxide glutathione peroxidase (GPx4) is a moonlighting selenoprotein, which has been implicated in anti-oxidative defense, sperm development, and cerebral embryogenesis. Among GPx-isoforms, GPx4 is unique because of its capability to reduce complex lipid hydroperoxides and its tendency toward polymerization, but the structural basis for these properties remained unclear. To address this, we solved the crystal structure of the catalytically active U46C mutant of human GPx4 to 1.55 A resolution. X-ray data indicated a monomeric protein consisting of four alpha-helices and seven beta-strands. GPx4 lacks a surface exposed loop domain, which appears to limit the accessibility of the active site of other GPx-isoforms, and these data may explain the broad substrate specificity of GPx4. The catalytic triad (C46, Q81, and W136) is localized at a flat impression of the protein surface extending into a surface exposed patch of basic amino acids (K48, K135, and R152) that also contains polar T139. Multiple mutations of the catalytic triad indicated its functional importance. Like the wild-type enzyme, the U46C mutant exhibits a strong tendency toward protein polymerization, which was prevented by reductants. Site-directed mutagenesis suggested involvement of the catalytic C46 and surface exposed C10 and C66 in polymer formation. In GPx4 crystals, these residues contact adjacent protein monomers.

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Mammalian lipoxygenases constitute a heterogeneous family of lipid-peroxidizing enzymes, and the various isoforms are categorized with respect to their positional specificity of arachidonic acid oxygenation into 5-, 8-, 12-, and 15-lipoxygenases. Structural modeling suggested that the substrate binding pocket of the human 5-lipoxygenase is 20% bigger than that of the reticulocyte-type 15-lipoxygenase; thus, reduction of the activesite volume was suggested to convert a 5-lipoxygenase to a 15-lipoxygenating enzyme species. To test this "spacebased" hypothesis of the positional specificity, the volume of the 5-lipoxygenase substrate binding pocket was reduced by introducing space-filling amino acids at critical positions, which have previously been identified as sequence determinants for the positional specificity of other lipoxygenase isoforms. We found that single point mutants of the recombinant human 5-lipoxygenase exhibited a similar specificity as the wild-type enzyme but double, triple, and quadruple mutations led to a gradual alteration of the positional specificity from 5S-via 8S-toward 15S-lipoxygenation. The quadruple mutant F359W/A424I/N425M/A603I exhibited a major 15S-lipoxygenase activity (85-95%), with (8S,5Z,9E,11Z,14Z)-8-hydroperoxyeicosa-5,9,11,14-tetraenoic acid being a minor side product. These data indicate the principle possibility of interconverting 5-and 15-lipoxygenases by sitedirected mutagenesis and appear to support the spacebased hypothesis of positional specificity. Lipoxygenases (LOXs)1 constitute a heterogeneous family of lipid-peroxidizing enzymes that catalyze the dioxygenation of free and/or esterified polyunsaturated fatty acids to their corresponding hydroperoxy derivatives. In mammals LOXs are categorized with respect to their positional specificity of arachidonic acid oxygenation into 5-, 8-, 12-, and 15-LOXs (1, 2). In contrast, plant physiologists prefer a linoleic acid-related enzyme nomenclature since arachidonic acid is only a minor fatty acid in plants. Mammalian 5-LOXs are key enzymes in the biosynthesis of leukotrienes, which are important mediators of inflammatory and anaphylactic disorders (3, 4). During the past 10 years, 5-LOX inhibitors and leukotriene receptor antagonists have been developed as anti-asthmatic drugs, and some of them are now available for prescription (5, 6). Mammalian 15-LOXs have been implicated in peroxisome proliferation activating receptor-␥-mediated cell signaling (7), in cell development and maturation (8, 9), as well as in the pathogenesis of atherosclerosis (10, 11). The intracellular activity of LOXs is regulated on pre-translational, translational, and posttranslational levels. Expression of the human 5-LOX is upregulated by transforming growth factor (12), and melatonin represses the 5-LOX pathway in B-lymphocytes (13). The interleukins-4 (14) and -13 (15) induce 15-LOX expression in monocyte/macrophages, and this regulatory process involves activation of the transcription factor STAT6 (16) as well as JAK2 and Tyk2 kinases (17). Transl...

We investigated the DNA of 29 unrelated pyruvate kinase (PK) deficiency (PKD) patients from Central Europe with hereditary nonspherocytic hemolytic anemia for mutations in the PK-L/R gene. Among 58 potentially affected alleles, 53 mutations were identified, of which 17 were different from each other. Of these 17 mutations, 13 were single-nucleotide (nt) substitutions resulting in amino acid exchanges, G787A (Gly263-Arg), G994A (Gly332-Ser), G1006T (Ala336-Ser), G1010A (Arg337-Gln), A1081G (Asn361-Asp), G1127T (Ser376-Ile), G1174A (Ala392-Thr), G1281T (Glu427-Asp), C1454T (Ser485-Phe), C1456T (Arg486-Trp), G1493A (Arg498-His), G1529A (Arg510-Gln), and C1594T (Arg532-Trp); 1 in-frame triplet deletion, 1060delAAG (delLys354); 1 in-frame triplet insertion, 1203insAGC (insSer after Cys401); 1 splicesite mutation, 101-1G-A; and 1 frameshift deletion, 628delGT. Six mutations, 628delGT, G787A, G1010A, G1127T, G1281T, and C1454T, are described for the first time. To test the hypothesis of a single origin of the most common PK mutation in the European population, G1529A, we investigated all patients at four polymorphic sites in the PK-L/R gene: C/A at nt 1705, C/T at nt 1992, the (ATT)n microsatellite in intron J, and a polymorphism (T)10/(T)19 in intron I. Nine patients homozygous for mutation G1529A were consistent in all four markers. In the group of patients homozygous for mutation G1529A, the hematologic parameters and clinical manifestations have been studied in detail. Although having an identical mutation in the PK-L/R gene, the patients are affected differently. Their appearance ranges from a very mild compensated hemolysis to a severe anemia. Possible molecular explanations are discussed.