Reaction mechanisms of flavins and flavoproteins from an electronic-structure perspective / Mecanismos de reação de flavinas e flavoproteínas por uma perspectiva de estrutura eletrônica

Proteins equipped with flavin adenine dinucleotides (FAD) or flavin mononucleotides (FMN) are named flavoproteins and constitute about 1% of all existing proteins. They catalyze redox, acid-base and photochemical reactions in a variety of biochemical phenomena that goes from energy metabolism to DNA repair and light sensing. The versatility observed in flavoproteins is ultimately a balance of flavin intrinsic properties modulated by a protein environment. This thesis aims to investigate how flavoproteins work by systematic evaluating flavin properties and reactivity. In particular, the mechanism of fumarate reduction by the flavoenzyme fumarate reductase Fcc3 was determined. Electronic-structure calculations were used for this task based on rigorous calibration with experimental data and error assessment. Flavin properties at chemical accuracy were obtained with single reference coupled-cluster CCSD(T) calculations at the complete basis set limit. Density functional theory was demonstrated an excellent alternative with lower computational costs and slightly less accuracy. Flavin protonation and tautomerism were shown to be important modulators of flavin properties and reactivity, with the possibility of various tautomers existing at neutral pH. Regarding flavin redox properties, an analysis based on multiconfigurational wave function weights was proposed for categorizing flavin redox reactions as hydride or hydrogen-atom transfers. This analysis is an upgrade over traditional partial charges methods and can be applied not only to flavin reactions but to any protoncoupled electron transfer. In the investigation of the enzymatic mechanism of fumarate reduction, the reaction was determined as a nucleophilic addition by hydride transfer with carbanion formation. Fumarate reductase employs electrostatic catalysis in contrast to previous proposals of substrate straining and general-acid catalysis. Also, hydride transfer was shown to be vibronically adiabatic with low tunneling contribution. These findings give new insights into the mechanisms of fumarate reductases and provide a framework for future computational studies of flavoproteins in general. The analyses and benchmark studies presented can be used to build better models of properties and reactivity of flavins and flavoproteins

Proteínas equipadas com dinucleotídeos de flavina-adenina (FAD) e mononucleotídeos de flavina (FMN) são chamadas flavoproteínas e constituem cerca de 1% de todas as proteínas existentes. Elas catalisam reações redox, ácido-base e fotoquímicas numa variedade de fenômenos bioquímicos que vão desde o metabolismo energético até reparo de DNA e captação de luz. A versatilidade observada em flavoproteínas é em última instância um balanço das propriedades intrínsecas de flavinas moduladas por um ambiente proteico. Esta tese busca investigar como flavoproteínas funcionam através de avaliações sistemáticas de propriedades e reatividade de flavinas. Em particular, o mecanismo de redução de fumarato pela flavoenzima fumarato redutase Fcc3 foi determinado. Cálculos de estrutura eletrônica foram usados para esta tarefa com base em rigorosa calibração com dados experimentais e avaliação de erros. As propriedades de flavinas foram determinadas com acurácia química com cálculos monoconfiguracionais de coupled-cluster CCSD(T) no limite de conjunto base completo. A teoria do funcional da densidade mostrou-se uma alternativa excelente com menor custo computacional e um pouco menos de acurácia. Protonação e tautomerismo de flavinas mostraram-se moduladores importantes de suas propriedades e reatividade, com a possibilidade de vários tautômeros existirem em pH neutro. Em relação às propriedades redox de flavinas, uma análise baseada nos pesos de funções de onda multiconfiguracionais foi proposta para categorizar as reações redox de flavinas como transferências de hidreto ou hidrogênio. Esta análise é uma melhoria em relação aos métodos tradicionais de cargas parciais e pode ser aplicada não apenas para reações de flavinas mas para qualquer transferência de próton acoplada a elétrons. Na investigação do mecanismo enzimático de redução de fumarato, a reação foi designada como uma adição nucleofílica por transferência de hidreto e formação de carbânion. A fumarato redutase usa catálise eletrostática diferentemente de prospostas anteriores envolvendo distorção do substrato e catálise ácida geral. Além disso, a transferência de hidreto mostrou-se vibronicamente adiabática com pouca contribuição de tunelamento. Estas descobertas abrem novas perspectivas sobre os mecanismos de fumarato redutases e fornecem uma base para estudos computacionais futuros sobre flavoproteínas em geral. As análises e estudos comparativos apresentados podem ser usados para construir melhores modelos para propriedades e reatividade de flavinas e flavoproteínas

orsai, the Drosophila homolog of human ETFRF1, links lipid catabolism to growth control.

BACKGROUND: Lipid homeostasis is an evolutionarily conserved process that is crucial for energy production, storage and consumption. Drosophila larvae feed continuously to achieve the roughly 200-fold increase in size and accumulate sufficient reserves to provide all energy and nutrients necessary for the development of the adult fly. The mechanisms controlling this metabolic program are poorly understood. RESULTS: Herein we identified a highly conserved gene, orsai (osi), as a key player in lipid metabolism in Drosophila. Lack of osi function in the larval fat body, the regulatory hub of lipid homeostasis, reduces lipid reserves and energy output, evidenced by decreased ATP production and increased ROS levels. Metabolic defects due to reduced Orsai (Osi) in time trigger defective food-seeking behavior and lethality. Further, we demonstrate that downregulation of Lipase 3, a fat body-specific lipase involved in lipid catabolism in response to starvation, rescues the reduced lipid droplet size associated with defective orsai. Finally, we show that osi-related phenotypes are rescued through the expression of its human ortholog ETFRF1/LYRm5, known to modulate the entry of ß-oxidation products into the electron transport chain; moreover, knocking down electron transport flavoproteins EtfQ0 and walrus/ETFA rescues osi-related phenotypes, further supporting this mode of action. CONCLUSIONS: These findings suggest that Osi may act in concert with the ETF complex to coordinate lipid homeostasis in the fat body in response to stage-specific demands, supporting cellular functions that in turn result in an adaptive behavioral response.

Photolyase Production and Current Applications: A Review.

The photolyase family consists of flavoproteins with enzyme activity able to repair ultraviolet light radiation damage by photoreactivation. DNA damage by the formation of a cyclobutane pyrimidine dimer (CPD) and a pyrimidine-pyrimidone (6-4) photoproduct can lead to multiple affections such as cellular apoptosis and mutagenesis that can evolve into skin cancer. The development of integrated applications to prevent the negative effects of prolonged sunlight exposure, usually during outdoor activities, is imperative. This study presents the functions, characteristics, and types of photolyases, their therapeutic and cosmetic applications, and additionally explores some photolyase-producing microorganisms and drug delivery systems.

Odd Loop Regions of XenA and XenB Enzymes Modulate Their Interaction with Nitro-explosives Compounds and Provide Structural Support for Their Regioselectivity.

The nitro-explosive compounds 2,4,6-trinitrotoluene, 2,4,6-trinitrophenol, and 1,2,3-trinitroglycerol are persistent environmental contaminants. The presence of different functional groups in these molecules represents a great challenge to enzymatic catalysis. The chemical variety of these three substrates is such that they do not bind and interact with catalytic residues within an enzyme with the same affinity. In this context, two Xenobiotic Reductase enzymes produced by the bacteria Pseudomonas putida can catalyze the reduction of these compounds with different affinities and regioselectivity. The structural bases that support this substrate promiscuity and catalytic preferences are unknown. Therefore, through molecular dynamics simulations and free energy calculations, we explored the structural properties driving the specific interactions of these enzymes with their substrates and cofactor. Models of Xenobiotic Reductase A and B enzymes in complex with 2,4,6-trinitrotoluene, 2,4,6-trinitrophenol, or 1,2,3-trinitroglycerol were built, and the ligand enzyme interaction was simulated by molecular dynamics. The structural analysis of the molecular dynamics simulations shows that loops 3, 5, 7, 9, 11, and 13 of Xenobiotic Reductase B, and loops 4, 5, 7, 11, 13, and 15 Xenobiotic Reductase A, are in contact with the ligands during the first stages of the molecular recognition. These loops are the most flexible regions for both enzymes; however, Xenobiotic Reductase B presents a greater range of movement and a higher number of residues interacting with the ligands. Finally, the distance between the cofactor and the different reactive groups in the substrate reflects the regioselectivity of the enzymes, and the free energy calculations are consistent with the substrate specificity of both enzymes studied. The simulation shows a stable interaction between the aromatic ring of the substrates and Xenobiotic Reductase B. In contrast, a less stable interaction with the different nitro groups of the aromatic ligands was observed. In the case of 1,2,3-trinitroglycerol, Xenobiotic Reductase B interacts more closely with the nitro groups of carbon 1 or 3, while Xenobiotic Reductase A is more selective by nitro groups of carbon 2. The obtained results suggest that the flexibility of the loops in Xenobiotic Reductase B and the presence of polar and aromatic residues present in loops 5 and 7 are fundamental to determine the affinity of the enzyme with the different substrates, and they also contribute to the proper orientation of the ligands that directs the catalytic reaction.

Selection and validation of reliable housekeeping genes to evaluate Piscirickettsia salmonis gene expression.

Piscirickettsia salmonis is a highly aggressive facultative intracellular bacterium that challenges the sustainability of Chilean salmon production. Due to the limited knowledge of its biology, there is a need to identify key molecular markers that could help define the pathogenic potential of this bacterium. We think a model system should be implemented that efficiently evaluates the expression of putative bacterial markers by using validated, stable, and highly specific housekeeping genes to properly select target genes, which could lead to identifying those responsible for infection and disease induction in naturally infected fish. Here, we selected a set of validated reference or housekeeping genes for RT-qPCR expression analyses of P. salmonis under different growth and stress conditions, including an in vitro infection kinetic. After a thorough screening, we selected sdhA as the most reliable housekeeping gene able to represent stable and highly specific host reference genes for RT-qPCR-driven P. salmonis analysis.

Characterization of Francisella species isolated from the cooling water of an air conditioning system.

Strains of Francisella spp. were isolated from cooling water from an air conditioning system in Guangzhou, China. These strains are Gram negative, coccobacilli, non-motile, oxidase negative, catalase negative, esterase and lipid esterase positive. In addition, these bacteria grow on cysteine-supplemented media at 20 °C to 40 °C with an optimal growth temperature of 30 °C. Analysis of 16S rRNA gene sequences revealed that these strains belong to the genus Francisella. Biochemical tests and phylogenetic and BLAST analyses of 16S rRNA, rpoB and sdhA genes indicated that one strain was very similar to Francisella philomiragia and that the other strains were identical or highly similar to the Francisella guangzhouensis sp. nov. strain 08HL01032 we previously described. Biochemical and molecular characteristics of these strains demonstrated that multiple Francisella species exist in air conditioning systems.

Characterization of Francisella species isolated from the cooling water of an air conditioning system

Strains of Francisella spp. were isolated from cooling water from an air conditioning system in Guangzhou, China. These strains are Gram negative, coccobacilli, non-motile, oxidase negative, catalase negative, esterase and lipid esterase positive. In addition, these bacteria grow on cysteine-supplemented media at 20 °C to 40 °C with an optimal growth temperature of 30 °C. Analysis of 16S rRNA gene sequences revealed that these strains belong to the genus Francisella. Biochemical tests and phylogenetic and BLAST analyses of 16S rRNA, rpoB and sdhA genes indicated that one strain was very similar to Francisella philomiragia and that the other strains were identical or highly similar to the Francisella guangzhouensis sp. nov. strain 08HL01032 we previously described. Biochemical and molecular characteristics of these strains demonstrated that multiple Francisella species exist in air conditioning systems..(AU)

Characterization of Francisella species isolated from the cooling water of an air conditioning system

Strains of Francisella spp. were isolated from cooling water from an air conditioning system in Guangzhou, China. These strains are Gram negative, coccobacilli, non-motile, oxidase negative, catalase negative, esterase and lipid esterase positive. In addition, these bacteria grow on cysteine-supplemented media at 20 °C to 40 °C with an optimal growth temperature of 30 °C. Analysis of 16S rRNA gene sequences revealed that these strains belong to the genus Francisella. Biochemical tests and phylogenetic and BLAST analyses of 16S rRNA, rpoB and sdhA genes indicated that one strain was very similar to Francisella philomiragia and that the other strains were identical or highly similar to the Francisella guangzhouensis sp. nov. strain 08HL01032 we previously described. Biochemical and molecular characteristics of these strains demonstrated that multiple Francisella species exist in air conditioning systems.

Diabetes mellitus increases reactive oxygen species production in the thyroid of male rats.

Diabetes mellitus (DM) disrupts the pituitary-thyroid axis and leads to a higher prevalence of thyroid disease. However, the role of reactive oxygen species in DM thyroid disease pathogenesis is unknown. Dual oxidases (DUOX) is responsible for H(2)O(2) production, which is a cosubstrate for thyroperoxidase, but the accumulation of H(2)O(2) also causes cellular deleterious effects. Nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) is another member of the nicotinamide adenine dinucleotide phosphate oxidase family expressed in the thyroid. Therefore, we aimed to evaluate the thyroid DUOX activity and expression in DM rats in addition to NOX4 expression. In the thyroids of the DM rats, we found increased H(2)O(2) generation due to higher DUOX protein content and DUOX1, DUOX2, and NOX4 mRNA expressions. In rat thyroid PCCL3 cells, both TSH and insulin decreased DUOX activity and DUOX1 mRNA levels, an effect partially reversed by protein kinase A inhibition. Most antioxidant enzymes remained unchanged or decreased in the thyroid of DM rats, whereas only glutathione peroxidase 3 was increased. DUOX1 and NOX4 expression and H(2)O(2) production were significantly higher in cells cultivated with high glucose, which was reversed by protein kinase C inhibition. We conclude that thyroid reactive oxygen species is elevated in experimental rat DM, which is a consequence of low-serum TSH and insulin but is also related to hyperglycemia per se.

Sexual dimorphism of thyroid reactive oxygen species production due to higher NADPH oxidase 4 expression in female thyroid glands.

BACKGROUND: Dual oxidases (DUOX1 and DUOX2) are NADPH oxidases (NOX) involved in hydrogen peroxide production necessary for thyroid hormonogenesis, but recently, the NOX4 has also been described in the thyroid gland. The prevalence of thyroid disease is higher in women, and the basis for this difference might involve a higher oxidative stress level in the female thyroid gland. Hence, we aimed at evaluating whether the function and the expression of enzymes involved in the thyroid redox balance differ between females and males. METHODS: DUOX1, DUOX2, NOX4, glutathione peroxidase (GPx), and catalase activities and expression levels were evaluated in the thyroids of prepubertal and adult male and female rats. The mRNA levels of DUOXA1 and DUOXA2, the DUOX maturation factors, and of p22phox and Poldip2 (subunits of NOX4) were also determined. RESULTS: A higher calcium-independent H(2)O(2) production was detected in the adult female rat thyroid, being higher in the estrous phase of the cycle. Moreover, the expression of NOX4 and Poldip2 mRNA was higher in the thyroids of adult female rats, as well as in PCCL3 cells treated with 17ß-estradiol. The GPx1 mRNA expression was higher in adult female thyroids, while GPx2 and GPx3 mRNA and total GPx activity were not significantly different. Catalase mRNA expression and activity, together with thyroid thiol levels were significantly lower in the adult female rat thyroid. CONCLUSIONS: Taken together, our results show that the thyroid gland of female rats is exposed to higher oxidative stress levels due both to increased reactive oxygen species (ROS) production through NOX4, and decreased ROS degradation.

A possible subcellular mechanism underlying the "French paradox": the opening of mitochondrial K(ATP) channels.

A reduction in the risk of coronary heart disease has been associated to moderate red wine consumption. We tested whether a nonalcoholic red wine extract would open mitochondrial K(ATP) channels in guinea pig myocytes. The opening of mitochondrial K(ATP) channels was assessed by endogenous flavoprotein fluorescence. Red wine extract (100 µg·mL(-1)) increased flavoprotein oxidation (10.9% ± 1.2%, n = 20). This effect was prevented by the mitochondrial K(ATP) channel blocker, 5-hydroxydecanoate (500 µmol·L(-1); 0.3% ± 1.1%, n = 13), confirming the hypothesis that red wine extract opens mitochondrial K(ATP) channels.

A deficiency in the flavoprotein of Arabidopsis mitochondrial complex II results in elevated photosynthesis and better growth in nitrogen-limiting conditions.

Mitochondrial complex II (succinate dehydrogenase [SDH]) plays roles both in the tricarboxylic acid cycle and the respiratory electron transport chain. In Arabidopsis (Arabidopsis thaliana), its flavoprotein subunit is encoded by two nuclear genes, SDH1-1 and SDH1-2. Here, we characterize heterozygous SDH1-1/sdh1-1 mutant plants displaying a 30% reduction in SDH activity as well as partially silenced plants obtained by RNA interference. We found that these plants displayed significantly higher CO(2) assimilation rates and enhanced growth than wild-type plants. There was a strong correlation between CO(2) assimilation and stomatal conductance, and both mutant and silenced plants displayed increased stomatal aperture and density. By contrast, no significant differences were found for dark respiration, chloroplastic electron transport rate, CO(2) uptake at saturating concentrations of CO(2), or biochemical parameters such as the maximum rates of carboxylation by Rubisco and of photosynthetic electron transport. Thus, photosynthesis is enhanced in SDH-deficient plants by a mechanism involving a specific effect on stomatal function that results in improved CO(2) uptake. Metabolic and transcript profiling revealed that mild deficiency in SDH results in limited effects on metabolism and gene expression, and data suggest that decreases observed in the levels of some amino acids were due to a higher flux to proteins and other nitrogen-containing compounds to support increased growth. Strikingly, SDH1-1/sdh1-1 seedlings grew considerably better in nitrogen-limiting conditions. Thus, a subtle metabolic alteration may lead to changes in important functions such as stomatal function and nitrogen assimilation.

Heterologous expression of two FAD-dependent oxidases with (S)-tetrahydroprotoberberine oxidase activity from Arge mone mexicana and Berberis wilsoniae in insect cells.

Berberine, palmatine and dehydrocoreximine are end products of protoberberine biosynthesis. These quaternary protoberberines are elicitor inducible and, like other phytoalexins, are highly oxidized. The oxidative potential of these compounds is derived from a diverse array of biosynthetic steps involving hydroxylation, intra-molecular C-C coupling, methylenedioxy bridge formation and a dehydrogenation reaction as the final step in the biosynthesis. For the berberine biosynthetic pathway, the identification of the dehydrogenase gene is the last remaining uncharacterized step in the elucidation of the biosynthesis at the gene level. An enzyme able to catalyze these reactions, (S)-tetrahydroprotoberberine oxidase (STOX, EC 1.3.3.8), was originally purified in the 1980s from suspension cells of Berberis wilsoniae and identified as a flavoprotein (Amann et al. 1984). We report enzymatic activity from recombinant STOX expressed in Spodoptera frugiperda Sf9 insect cells. The coding sequence was derived successively from peptide sequences of purified STOX protein. Furthermore, a recombinant oxidase with protoberberine dehydrogenase activity was obtained from a cDNA library of Argemone mexicana, a traditional medicinal plant that contains protoberberine alkaloids. The relationship of the two enzymes is discussed regarding their enzymatic activity, phylogeny and the alkaloid occurrence in the plants. Potential substrate binding and STOX-specific amino acid residues were identified based on sequence analysis and homology modeling.

The Paracoccidioides brasiliensis gp70 antigen is encoded by a putative member of the flavoproteins monooxygenase family.

Glycoprotein gp70 is an important intracellular antigen from Paracoccidioides brasiliensis that elicits both humoral and cellular immune responses. Herein, the PbGP70 gene cloning from isolate Pb18 using internal peptide sequence information is reported. The deduced protein sequence bears two N-glycosylation sites, antigenic sites and two mouse T-cell epitopes. Anti-recombinant gp70 (rPbgp70) polyclonal antibodies reacted with a 70-kDa component in total cell extract of P. brasiliensis, while MAbC5F11 and paracoccidioidomycosis patients' sera recognized rPbgp70. Confocal microscopy with anti-rPbgp70 and MAbC5F11 showed intense staining and cytoplasmatic co-localization. The protein sequence belongs to the flavoprotein monooxygenase family which groups important anti-oxidative bioactive compounds. We found increased PbGP70 transcript accumulation under oxidative stress induced by H(2)O(2), during fungal growth and in macrophage phagocyted/bound yeasts. Therefore, gp70 might play a dual role in P. brasiliensis by both eliciting immune cellular and humoral responses in the host and protecting the fungus from oxidative stress generated by phagocytic cells.

Cryptochrome 2 expression level is critical for adrenocorticotropin stimulation of cortisol production in the capuchin monkey adrenal.

Timely production of glucocorticoid hormones in response to ACTH is essential for survival by coordinating energy intake and expenditure and acting as homeostatic regulators against stress. Adrenal cortisol response to ACTH is clock time dependent, suggesting that an intrinsic circadian oscillator in the adrenal cortex contributes to modulate the response to ACTH. Circadian clock gene expression has been reported in the adrenal cortex of several species. However, there are no reports accounting for potential involvement of adrenal clock proteins on cortisol response to ACTH. Here we explored whether the clock protein cryptochrome 2 (CRY2) knockdown modifies the adrenal response to ACTH in a primate. Adrenal gland explants from adult capuchin monkey (n = 5) were preincubated for 6 h with transfection vehicle (control) or with two different Cry2 antisense and sense probes followed by 48 h incubation in medium alone (no ACTH) or with 100 nm ACTH. Under control and sense conditions, ACTH increased cortisol production, whereas CRY2 suppression inhibited ACTH-stimulated cortisol production. Expression of the steroidogenic enzymes steroidogenic acute regulatory protein and 3beta-hydroxysteroid dehydrogenase at 48 h of incubation was increased by ACTH in control explants and suppressed by Cry2 knockdown. Additionally, we found that Cry2 knockdown decreased the expression of the clock gene brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein (Bmal1) at the mRNA and protein levels. Altogether these results strongly support that the clock protein CRY2 is involved in the mechanism by which ACTH increases the expression of steroidogenic acute regulatory protein and 3beta-hydroxysteroid dehydrogenase. Thus, adequate expression levels of components of the adrenal circadian clock are required for an appropriate cortisol response to ACTH.

Suppression of pleiotropic effects of functional cryptochrome genes by Terminal Flower 1.

TERMINAL FLOWER 1 (TFL1) encodes a protein with similarity to animal phosphatidylethanolamine-binding proteins and is required for normal trafficking to the protein storage vacuole. In Arabidopsis thaliana the tfl1 mutation produces severe developmental abnormalities. Here we show that most aspects of the tfl1 phenotype are lost in the cry1 cry2 double-mutant background lacking cryptochromes 1 and 2. The inhibition of hypocotyl growth by light is reduced in the tfl1 mutant but this effect is absent in the cry1 or cry2 mutant background. Although the promotion of flowering under long rather than short days is a key function of cryptochromes, in the tfl1 background, cryptochromes promoted flowering under short days. Thus, normal CRY control of photoperiod-dependent flowering and hypocotyl growth inhibition requires a functional TFL1 gene.

Melatonin and the circadian entrainment of metabolic and hormonal activities in primary isolated adipocytes.

The aim of this work was to investigate the effect of the in vitro circadian-like exposure to melatonin [in the presence or absence of insulin (Ins)] on the metabolism and clock gene expression in adipocytes. To simulate the cyclic characteristics of the daily melatonin profile, isolated rat adipocytes were exposed in a circadian-like pattern to melatonin added to the incubating medium for 12 hr (mimicking the night), followed by an equal period without melatonin (mimicking the day) combined or not with Ins. This intermittent incubation was interrupted when four and a half 24-hr cycles were fulfilled. At the end, either during the induced night (melatonin present) or the induced day (melatonin absent), the rates of lipolysis and D-[U-(14)C]-glucose incorporation into lipids were estimated, in addition to the determination of lipogenic [glucose-6-phosphate dehydrogenase and fatty acid synthase (FAS)] and lipolytic (hormone sensitive lipase) enzymes and clock gene (Bmal-1b, Clock, Per-1 and Cry-1) mRNA expression. The leptin release was also measured. During the induced night, the following effects were observed: an increase in the mRNA expression of Clock, Per-1 and FAS; a rise in lipogenic response and leptin secretion; and a decrease in the lipolytic activity. The intermittent exposure of adipocytes to melatonin temporally and rhythmically synchronized their metabolic and hormonal function in a circadian fashion, mimicking what is observed in vivo in animals during the daily light-dark cycle. Therefore, this work helps to clarify the physiological relevance of the circadian pattern of melatonin secretion and its interactions with Ins, contributing to a better understanding of the adipocyte biology.

Genetic and biochemical studies in Argentinean patients with variegate porphyria.

BACKGROUND: A partial deficiency in Protoporphyrinogen oxidase (PPOX) produces the mixed disorder Variegate Porphyria (VP), the second acute porphyria more frequent in Argentina. Identification of patients with an overt VP is absolutely important because treatment depends on an accurate diagnosis but more critical is the identification of asymptomatic relatives to avoid acute attacks which may progress to death. METHODS: We have studied at molecular level 18 new Argentinean patients biochemically diagnosed as VP. PPOX gene was amplified in one or in twelve PCR reactions. All coding exons, flanking intronic and promoter regions were manual or automatically sequenced. For RT-PCR studies RNA was retrotranscripted, amplified and sequenced. PPOX activity in those families carrying a new and uncharacterized mutation was performed. RESULTS: All affected individuals harboured mutations in heterozygous state. Nine novel mutations and 3 already reported mutations were identified. Six of the novel mutations were single nucleotide substitutions, 2 were small deletions and one a small insertion. Three single nucleotide substitutions and the insertion were at exon-intron boundaries. Two of the single nucleotide substitutions, c.471G>A and c.807G>A and the insertion (c.388+3insT) were close to the splice donor sites in exons 5, 7 and intron 4 respectively. The other single nucleotide substitution was a transversion in the last base of intron 7, g.3912G>C (c.808-1G>C) so altering the consensus acceptor splice site. However, only in the first case the abnormal band showing the skipping of exon 5 was detected. The other single nucleotide substitutions were transversions: c.101A>T, c.995G>C and c.670 T>G that result in p.E34V, p.G332A and W224G aminoacid substitutions in exons 3, 10 and 7 respectively. Activity measurements indicate that these mutations reduced about 50% PPOX activity and also that they co-segregate with this reduced activity value. Two frameshift mutations, c.133delT and c.925delA, were detected in exons 3 and 9 respectively. The first leads to an early termination signal 22 codons downstream (p.S45fsX67) and the second leads to a stop codon 5 codons downstream (p.I309fsX314). One reported mutation was a missense mutation (p.G232R) and 2 were frameshift mutations: c.1082insC and 1043insT. The last mutation was detected in six new apparently unrelated Argentinean families. CONCLUSION: Molecular analysis in available family members revealed 14 individuals who were silent carriers of VP. Molecular techniques represent the most accurate approach to identify unaffected carriers and to provide accurate genetic counselling for asymptomatic individuals. The initial screening includes the insertion search.