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Background Isolated sulfite oxidase deficiency (ISOD) caused by sulfite oxidase gene (SUOX) mutations is a rare neurometabolic disease associated with ectopia lentis (EL). However, few genotype–phenotype correlations have been established yet. Methods Potentially pathogenic SUOX mutations were screened from a Chinese cohort of congenital EL using panel-based next-generation sequencing and analyzed with multiple bioinformatics tools. The genotype–phenotype correlations were evaluated via a systematic review of SUOX mutations within our data and from the literature. Results A novel paternal missense mutation, c.205G > C (p.A69P), and a recurrent maternal nonsense mutation, c.1200 C > G (p.Y400*), of SUOX were identified in a 4-year-old boy from 312 probands. The biochemical assays manifested elevated urine sulfite and S-sulfocysteine accompanied by decreased homocysteine in the blood. The patient had bilateral EL and normal fundus, yet minimal neurological involvement and normal brain structure. Molecular modeling simulation revealed the p.A69P mutant had an unstable structure but an unchanged affinity for sulfite, while the truncated p.Y400* mutant showed decreased binding capacity. Genotype–phenotype analysis demonstrated patients with biallelic missense mutations had milder symptoms (P = 0.023), later age of onset (P P = 0.017) than other genotypes. No correlations were found regarding EL and other neurological symptoms. Conclusion The data from this study not only enrich the known mutation spectrum of SUOX but also suggest that missense mutations are associated with mild and atypical symptoms.

The origin(s) of dissimilatory sulfate and/or (bi)sulfite reducing organisms (SRO) remains enigmatic despite their importance in global carbon and sulfur cycling since at least 3.4 Ga. Here, we describe novel, deep-branching archaeal SRO populations distantly related to other Diaforarchaea from two moderately acidic thermal springs. Dissimilatory (bi)sulfite reductase homologs, DsrABC, encoded in metagenome assembled genomes (MAGs) from spring sediments comprise one of the earliest evolving Dsr lineages. DsrA homologs were expressed in situ under moderately acidic conditions. MAGs lacked genes encoding proteins that activate sulfate prior to (bi)sulfite reduction. This is consistent with sulfide production in enrichment cultures provided sulfite but not sulfate. We suggest input of volcanic sulfur dioxide to anoxic spring-water yields (bi)sulfite and moderately acidic conditions that favor its stability and bioavailability. The presence of similar volcanic springs at the time SRO are thought to have originated (>3.4 Ga) may have supplied (bi)sulfite that supported ancestral SRO. These observations coincide with the lack of inferred SO42− reduction capacity in nearly all organisms with early-branching DsrAB and which are near universally found in hydrothermal environments.

1-Cyanocyclohexaneacetic acid (1-CHAA) is a critical intermediate for the synthesis of the antiepileptic agent gabapentin. Previously, our group has established a novel manufacturing route for 1-CHAA through bioconversion catalyzed by an Escherichia coli (E. coli) nitrilase whole cell catalyst. However, the nitrilase expressed in E. coli has several drawbacks such as a low level of reusability, which hampered its industrial application. Herein, we investigated the potential of using the methylotrophic yeast Pichia pastoris (P. pastoris) for producing the nitrilase whole cell catalyst. To achieve strains with high catalytic activities, we investigated the effects of the promoter choice, expressing cassette copy number, and co-expression of chaperone on the production of nitrilase. Our results demonstrated that the strain harboring the multicopy integrations of nitrilase gene under the control of the alcohol oxidase 1 (AOX1) promoter and co-expressing of ER oxidoreductin 1 (ERO1) exhibited an 18-fold enhancement in the nitrilase activity compared with the strain containing a single integration of nitrilase gene under the control of glyceraldehyde-3-phosphate (GAP) dehydrogenase promoter. This optimized P. pastoris strain, compared with the E. coli nitrilase whole cell catalyst, shows greatly improved levels of reusability and thermostability while has a similar high-substrate tolerance.

Mitochondria are the center of energy metabolism in the cell and the preferential target of various toxicants and ischemic injury. Renal ischemia–reperfusion (I/R) injury triggers proximal tubule injury and the mitochondria are believed to be the primary subcellular target of I/R injury. The promotion of mitochondrial biogenesis (MB) is critical for the prevention I/R injury. The results of our previous study showed that augmenter of liver regeneration (ALR) has anti-apoptotic and anti-oxidant functions. However, the modulatory mechanism of ALR remains unclear and warrants further investigation. To gain further insight into the role of ALR in MB, human kidney (HK)-2 cells were treated with lentiviruses carrying ALR short interfering RNA (siRNA) and a model of hypoxia reoxygenation (H/R) injury in vitro was created. We observed that knockdown of ALR promoted apoptosis of renal tubular cells and aggravated mitochondrial injury, as evidenced by the decrease in the mitochondrial respiratory proteins adenosine triphosphate (ATP) synthase subunit β, cytochrome c oxidase subunit 1, and nicotinamide adenine dinucleotide dehydrogenase (ubiquinone) beta subcomplex 8. Meanwhile, the production of reactive oxygen species was increased and ATP levels were decreased significantly in HK-2 cells, as compared with the siRNA/control group (p

Cysteine sulfinic acid or S-sulfinylation is an oxidative post-translational modification (OxiPTM) that is known to be involved in redox-dependent regulation of protein function but has been historically difficult to analyze biochemically. To facilitate the detection of S-sulfinylated proteins, we demonstrate that a clickable, electrophilic diazene probe (DiaAlk) enables capture and site-centric proteomic analysis of this OxiPTM. Using this workflow, we revealed a striking difference between sulfenic acid modification (S-sulfenylation) and the S-sulfinylation dynamic response to oxidative stress, which is indicative of different roles for these OxiPTMs in redox regulation. We also identified >55 heretofore-unknown protein substrates of the cysteine sulfinic acid reductase sulfiredoxin, extending its function well beyond those of 2-cysteine peroxiredoxins (2-Cys PRDX1-4) and offering new insights into the role of this unique oxidoreductase as a central mediator of reactive oxygen species-associated diseases, particularly cancer. DiaAlk therefore provides a novel tool to profile S-sulfinylated proteins and study their regulatory mechanisms in cells.

Postmenopausal osteoporosis is a major health problem with important genetic factors in postmenopausal women. We explored the relationship between SQRDL and osteoporosis in a cohort of 1006 patients and 2027 controls from Han Chinese postmenopausal women. Our evidence supported the significant role of SQRDL in the etiology of postmenopausal osteoporosis. Postmenopausal osteoporosis (PMOP) is a metabolic bone disease leading to progressive bone loss and the deterioration of the bone microarchitecture. The sulfide-quinone reductase-like protein is an important enzyme regulating the cellular hydrogen sulfide levels, and it can regulate bone metabolism balance in postmenopausal women. In this study, we aimed to investigate whether SQRDL is associated with susceptibility to PMOP in the Han Chinese population. A total of 3033 postmenopausal women, comprised of 1006 cases and 2027 controls, were recruited in the study. Twenty-two SNPs were selected for genotyping to evaluate the association of SQRDL gene with BMD and PMOP. Association analyses in both single marker and haplotype levels were performed for PMOP. Bone mineral density (BMD) was also utilized as a quantitative phenotype in further analyses. Bioinformatics tools were applied to predict the functional consequences of targeted polymorphisms in SQRDL. The SNP rs1044032 (P = 6.42 × 10−5, OR = 0.80) was identified as significantly associated with PMOP. Three SNPs (rs1044032, rs2028589, and rs12913151) were found to be significantly associated with BMD. Although limited functional significance can be obtained for these polymorphisms, significant hits for association with PMOP were found. Moreover, further association analyses with BMD identified three SNPs with significantly independent effects. Our evidence supported the significant role of SQRDL in the etiology of PMOP and suggest that it may be a genetic risk factor for BMD and osteoporosis in Han Chinese postmenopausal women.

Autophagy may have protective effects in renal ischemia-reperfusion (I/R) injury, although the underlying mechanisms remain unclear. Augmenter of liver regeneration (ALR), a widely distributed multifunctional protein that is originally identified as a hepatic growth factor, may participate in the process of autophagy. To investigate the role of ALR in autophagy, ALR expression is knocked-down in human kidney 2 (HK-2) cells with short hairpin RNA lentivirals. Then, the level of autophagy is measured in the shRNA/ALR group and the shRNA/control group in an in vitro model of ischemia-reperfusion (I/R). The results indicate that the level of autophagy in two groups increase, accompanied by increased reactive oxygen species production, especially in the shRNA/ALR group. The AMPK/mTOR signaling pathway is hyperactive in the shRNA/ALR group. Inhibition of autophagy with the AMPK inhibitor compound C induce apoptosis, especially in the shRNA/ALR group. These findings collectively indicate that ALR negatively regulates the autophagy process through an association with the AMPK/mTOR signaling pathway. Autophagy inhibit apoptosis and play a protective role under conditions of oxidative stress.

Acidithiobacillus ferrooxidans is a gram-negative, autotrophic and rod-shaped bacterium. It can gain energy through the oxidation of Fe(II) and reduced inorganic sulfur compounds for bacterial growth when oxygen is sufficient. It can be used for bio-leaching and bio-oxidation and contributes to the geobiochemical circulation of metal elements and nutrients in acid mine drainage environments. The iron and sulfur oxidation pathways of A. ferrooxidans play key roles in bacterial growth and survival under extreme circumstances. Here, the electrons transported through the thermodynamically favourable pathway for the reduction to H2O (downhill pathway) and against the redox potential gradient reduce to NAD(P)(H) (uphill pathway) during the oxidation of Fe(II) were reviewed, mainly including the electron transport carrier, relevant operon and regulation of its expression. Similar to the electron transfer pathway, the sulfur oxidation pathway of A. ferrooxidans, related genes and operons, sulfur oxidation mechanism and sulfur oxidase system are systematically discussed.

Chronic obstructive pulmonary disease (COPD) is a leading cause of death world-wide. Recently, we showed that COPD is associated with gene polymorphisms in SUMF1, a master regulator of sulfatases. Sulfatases are involved in extracellular matrix remodeling and activated by SUMF1, but their role in the lung is poorly described. We aimed to examine how sulfatases are affected in the airways of patients with COPD compared to ever smokers and never smokers. We observed that mRNA expression of the sulfatases GALNS, GNS and IDS was increased, while protein expression of many sulfatases was decreased in COPD fibroblasts. Several sulfatases, including GALNS, IDS, and SGSH, showed increased activity in COPD fibroblasts. Examination of different sulfatases by immunofluorescence showed that IDS, ARSB, GNS and SGSH in fibroblasts were localized to sites other than their reported destination. Using a master panel from different organs, RNA expression of all sulfatases could be observed in lung tissue. Additionally, immunohistochemistry on lung biopsies indicated differing expression of sulfatases in COPD patients. In conclusion, mRNA, protein expression, sulfatase activity levels, and localization of sulfatases are altered in lung fibroblasts and lung tissue from COPD patients and may be mechanistically important in COPD pathogenesis. This could contribute to the understanding of the disease mechanism in COPD and in the long run, to lead to more individualized therapies.

Background Non-alcoholic fatty liver disease (NAFLD) is a major health burden in need for new medication. To identify potential drug targets a genomic study was performed in lipid-laden primary human hepatocyte (PHH) and human hepatoma cell cultures. Methods PHH, HuH7 and HepG2 hepatoma cell cultures were treated with lipids and/or TNFα. Intracellular lipid load was quantified with the ORO assay. The Affymetrix HG-U133+ array system was employed to perform transcriptome analysis. The lipid droplet (LD) growth and fusion was determined by fluorescence microscopy. LD associated proteins were imaged by confocal immunofluorescence microscopy and confirmed by Western immunoblotting. Bioinformatics defined perturbed metabolic pathways. Results Whole genome expression profiling identified 227, 1031 and 571 significant regulated genes. Likewise, the combined lipid and TNFα treatment of PHH, HuH7 and HepG2 cell cultures revealed 154, 1238 and 278 differentially expressed genes. Although genomic responses differed among in-vitro systems, commonalities were ascertained by filtering the data for LD associated gene regulations. Among others the LD-growth and fusion associated cell death inducing DFFA like effector C (CIDEC), perilipins (PLIN2, PLIN3), the synaptosome-associated-protein 23 and the vesicle associated membrane protein 3 were strongly up-regulated. Likewise, the PPAR targets pyruvate-dehydrogenase-kinase-4 and angiopoietin-like-4 were up-regulated as was hypoxia-inducible lipid droplet-associated (HILPDA), flotilin and FGF21. Their inhibition ameliorates triglyceride and cholesterol accumulation. TNFα treatment elicited strong induction of the chemokine CXCL8, the kinases MAP3K8, MAP4K4 and negative regulators of cytokine signaling, i.e. SOCS2&SOCS3. Live cell imaging of DsRED calreticulin plasmid transfected HuH7 cells permitted an assessment of LD growth and fusion and confocal immunofluorescence microscopy evidenced induced LD-associated PLIN2, CIDEC, HIF1α, HILPDA, JAK1, PDK4 and ROCK2 expression. Notwithstanding, CPT1A protein was repressed to protect mitochondria from lipid overload. Pharmacological inhibition of the GTPase-dynamin and the fatty acid transporter-2 reduced lipid uptake by 28.5 and 35%, respectively. Finally, a comparisons of in-vitro/NAFLD patient biopsy findings confirmed common gene regulations thus demonstrating clinical relevance. Conclusion The genomics of fat-laden hepatocytes revealed LD-associated gene regulations and perturbed metabolic pathways. Immunofluorescence microscopy confirmed expression of coded proteins to provide a rationale for therapeutic intervention strategies. Collectively, the in-vitro system permits testing of drug candidates. Electronic supplementary material The online version of this article (10.1186/s12920-018-0438-7) contains supplementary material, which is available to authorized users.

Osm1 and Frd1 are soluble fumarate reductases from yeast that are critical for allowing survival under anaerobic conditions. Although they maintain redox balance during anaerobiosis, the underlying mechanism is not understood. Here, we report the crystal structure of a eukaryotic soluble fumarate reductase, which is unique among soluble fumarate reductases as it lacks a heme domain. Structural and enzymatic analyses indicate that Osm1 has a specific binding pocket for flavin molecules, including FAD, FMN, and riboflavin, catalyzing their oxidation while reducing fumarate to succinate. Moreover, ER-resident Osm1 can transfer electrons from the Ero1 FAD cofactor to fumarate either by free FAD or by a direct interaction, allowing de novo disulfide bond formation in the absence of oxygen. We conclude that soluble eukaryotic fumarate reductases can maintain an oxidizing environment under anaerobic conditions, either by oxidizing cellular flavin cofactors or by a direct interaction with flavoenzymes such as Ero1., Soluble fumarate reductases are essential for eukaryotic cell survival under anaerobic conditions but their mechanism is not fully understood. Here, the authors present structural and enzymatic analyses of yeast fumarate reductase Osm1, elucidating the molecular basis of maintaining redox balance during anaerobiosis.

Glutathione is a valuable tripeptide widely used in the pharmaceutical, food, and cosmetic industries. In industrial fermentation, glutathione is currently produced primarily using the yeast Saccharomyces cerevisiae. Intracellular glutathione exists in two forms; the majority is present as reduced glutathione (GSH) and a small amount is present as oxidized glutathione (GSSG). However, GSSG is more stable than GSH and is a more attractive form for the storage of glutathione extracted from yeast cells after fermentation. In this study, intracellular GSSG content was improved by engineering thiol oxidization metabolism in yeast. An engineered strain producing high amounts of glutathione from over-expression of glutathione synthases and lacking glutathione reductase was used as a platform strain. Additional over-expression of thiol oxidase (1.8.3.2) genes ERV1 or ERO1 increased the GSSG content by 2.9-fold and 2.0-fold, respectively, compared with the platform strain, without decreasing cell growth. However, over-expression of thiol oxidase gene ERV2 showed almost no effect on the GSSG content. Interestingly, ERO1 over-expression did not decrease the GSH content, raising the total glutathione content of the cell, but ERV1 over-expression decreased the GSH content, balancing the increase in the GSSG content. Furthermore, the increase in the GSSG content due to ERO1 over-expression was enhanced by additional over-expression of the gene encoding Pdi1, whose reduced form activates Ero1 in the endoplasmic reticulum. These results indicate that engineering the thiol redox metabolism of S. cerevisiae improves GSSG and is critical to increasing the total productivity and stability of glutathione.

Human serum albumin (HSA) and human growth hormone (hGH) fusion protein [HSA/GH] is a promising long-acting form of GH to treat GH deficiency. This study attempted to engineer a P. pastoris strain for high-level production of HSA/GH to be used in basic research and clinical application. Strains contained two, three, and seven copies of HSA/GH gene were screened by selecting against Zeocin resistance. The results revealed that introducing two to three copies of HSA/GH gene was sufficient to give a significant increase in secretion level, compared with one copy of HSA/GH gene. No significant differences were observed between two to three copies and seven copies. Co-expression with either one copy of exogenous ERO1 or PDI in a strain carrying multicopies of HSA/GH gene led to varying degrees of increase in HSA/GH secretion. The effect of introducing multicopies of PDI was similar to that of one copy of PDI, but introducing excess copies of ERO1 reduced HSA/GH secretion. Simultaneous co-expression with PDI and ERO1 was less effect than either PDI co-expression or ERO1 co-expression. A strain showing higher secretion level was successfully applied to large-scale fermentation with the productivity of 3-4 g/l.

Adenylylsulfate reductase is a heterodimeric complex of two subunits, AprB and AprA, and is a key enzyme in dissimilatory sulfate reduction and sulfur oxidation. Common use of aprA as a functional marker gene has revealed the diversity of sulfur-cycle prokaryotes in diverse environments. In this study, we established a comprehensive sequence set of apr genes and employed it to reanalyze apr phylogeny, evaluate the coverage of a widely used primer set (AprA-1-FW/AprA-5-RV), and categorize environmental aprA sequences. Phylogenetic tree construction revealed new members of Apr lineage II and several previously unrecognized lateral gene transfer events. Using the established phylogenetic tree, we classified all previously reported aprA sequences amplified from freshwater lakes with the primer pair AprA-1-FW/AprA-5-RV in addition to the aprA sequences newly retrieved from freshwater lakes; the obtained results were complemented by 16S rRNA clone library analysis. Apr-based classifications of some of operational taxonomic units were supported by 16S rRNA-based analysis. This study updates our knowledge on the phylogeny of aprBA and shows the identities of several sulfur-cycle bacteria, which could not be classified to a known taxa until now. The established apr sequence set is publicly available and can be applied to assign environmental sequences to known lineages.

Carboxypeptidase Y (CPY) is a yeast vacuolar protease with useful applications including C-terminal sequencing of peptides and terminal modification of target proteins. To overexpress CPY with the pro-sequence (proCPY) encoded by the Saccharomyces cerevisiae PRC1 gene in recombinant S. cerevisiae, the proCPY gene was combined with the gene coding for a signal sequence of S. cerevisiae mating factor α (MFα), invertase (SUC2), or Kluyveromyces marxianus inulinase (INU1). Among the three constructs, the MFα signal sequence gave the best specific activity of extracellular CPY. To enhance the CPY expression level, folding accessory proteins of Kar2p, Pdi1p and Ero1p located in the S. cerevisiae endoplasmic reticulum were expressed individually and combinatorially. A single expression of Kar2p led to a 28 % enhancement in extracellular CPY activity, relative to the control strain of S. cerevisiae CEN.PK2-1D/p426Gal1-MFαCPY. Coexpression of Kar2p, Pdi1p and Ero1p gave a synergistic effect on CPY expression, of which activity was 1.7 times higher than that of the control strain. This work showed that engineering of signal sequences and protein-folding proteins would be helpful to overexpress yeast proteins of interest.

Sulfur (S) is an essential macronutrient for plants, and deficiency in soil S availability limits plant growth. Adaptive strategies have been evolved by plants to respond to S deficiency by coordinating systemic regulatory mechanism. A split-root experiment using legume model plant Medicago truncatula Gaertn. was conducted to investigate the systemic response to S deficiency. Plant growth, root morphology and S contents under varying conditions of S supply were determined, and the expression of genes encoding sulfate transporter (MtSULTRs) and MtAPR1 encoding an enzyme involved in S assimilation was monitored. Our results demonstrated that there was an apparent systemic response of M. truncatula to heterogeneous S supply in terms of root length, S contents, and S uptake and assimilation at the transcriptional level. When exposed to heterogeneous S supply, M. truncatula plants showed proliferation of lateral roots in S-rich medium and reduction in investment to S-depleted roots. Growth was stimulated with half-part of roots exposed to S-deficient medium. There were different expression patterns of MtSULTRs and MtAPR1 in response to heterogeneous S supply both in roots and shoots of M. truncatula. Expression of MtSULTR1.1 and MtSULTR1.3 was systemically responsive to S deficiency, leading to an enhancement of S uptake in roots exposed to S-sufficient medium. In addition, the response of S-deprived seedlings to re-supply of sulfate and Cys was also analyzed. It was shown that sulfate, but not Cys, may serve as a systemic signal to regulate the expression of genes associated with S absorption and assimilation in M. truncatula. These findings provide a comprehensive picture of systemic responses to S deficiency in leguminous species.

Multiple sulfatase deficiency (MSD) is a rare inborn error of metabolism affecting posttranslational activation of sulfatases by the formylglycine generating enzyme (FGE). Due to mutations in the encoding SUMF1 gene, FGE's catalytic capacity is impaired resulting in reduced cellular sulfatase activities. Both, FGE protein stability and residual activity determine disease severity and have previously been correlated with the clinical MSD phenotype. Here, we report a patient with a late infantile severe course of disease. The patient is compound heterozygous for two so far undescribed SUMF1 mutations, c.156delC (p.C52fsX57) and c.390A>T (p.E130D). In patient fibroblasts, mRNA of the frameshift allele is undetectable. In contrast, the allele encoding FGE-E130D is expressed. FGE-E130D correctly localizes to the endoplasmic reticulum and has a very high residual molecular activity in vitro (55% of wildtype FGE); however, it is rapidly degraded. Thus, despite substantial residual enzyme activity, protein instability determines disease severity, which highlights that potential MSD treatment approaches should target protein folding and stabilization mechanisms.

Many bacteria reduce inorganic sulfate to sulfide to satisfy their need for sulfur, one of the most important elements for biological life. But little is known about the metabolic pathways involving hydrogen sulfide (H₂S) in mesophilic bacteria. By genomic sequence analysis, a complete set of genes for the assimilatory sulfate reduction pathway has been identified in the ethanologen Zymomonas mobilis. In this study, the first ATP sulfurylase- and final sulfite reductase-encoding genes cysND and cysIJ, respectively, in the putative pathway from sulfate to sulfite in Z. mobilis ZM4 was singly or doubly inactivated by homologous recombination and a site-specific FLP-FRT recombination. The resultant mutants, ∆cysND, ∆cysIJ and ∆cysND-cat∆cysIJ, were unable to produce detectable H₂S in glucose or sucrose-containing rich medium and sweet sorghum juice, in which the wild-type ZM4 produced detectable H₂S. While adding sulfite (SO₃²⁻) into media impaired the growth of the mutants and ZM4 to varying degrees, the sulfite restored the H₂S formation in the ∆cysND in the above media, but not in the ∆cysIJ and ∆cysND-cat∆cysIJ mutants. Although it seemed that the inactivation of cysND and cysIJ did not exert a significant negative effect on the cell growth at least in glucose or sucrose medium, the ethanol production of all mutants was inferior to that of ZM4 in sucrose medium and sweet sorghum juice. In addition, adding L-cysteine to glucose-containing rich media restored H₂S formation of all mutants, indicating the existence of another pathway for producing H₂S in Z. mobilis. All these results would help to further elucidate the metabolic pathways involving H₂S in Z. mobilis and exploit the biotechnological applications of this industrially important bacterium.

We describe a method for modifying proteins site-specifically using a chemoenzymatic bioconjugation approach. Formylglycine generating enzyme (FGE) recognizes a pentapeptide consensus sequence, CxPxR, and it specifically oxidizes the cysteine in this sequence to an unusual aldehyde-bearing formylglyine. The FGE recognition sequence, or aldehyde tag, can be inserted into heterologous recombinant proteins produced in either prokaryotic or eukaryotic expression systems. The conversion of cysteine to formylglycine is accomplished by co-overexpression of FGE, either transiently or as a stable cell line, and the resulting aldehyde can be selectively reacted with α-nucleophiles to generate a site-selectively modified bioconjugate. This protocol outlines both the generation and the analysis of proteins aldehyde-tagged at their termini and the methods for chemical conjugation to the formylglycine. The process of generating aldehyde-tagged protein followed by chemical conjugation and purification takes 20 d.

The ability to query enzyme molecules individually is transforming our view of catalytic mechanisms. Quiescin sulfhydryl oxidase (QSOX) is a multidomain catalyst of disulfide-bond formation that relays electrons from substrate cysteines through two redox-active sites to molecular oxygen. The chemical steps in electron transfer have been delineated, but the conformational changes accompanying these steps are poorly characterized. Here we use single-molecule Förster resonance energy transfer (smFRET) to probe QSOX conformation in resting and cycling enzyme populations. We report the discovery of unanticipated roles for conformational changes in QSOX beyond mediating electron transfer between redox-active sites. In particular, a state of the enzyme not previously postulated or experimentally detected is shown to gate, via a conformational transition, the entrance into a sub-cycle within an expanded QSOX kinetic scheme. By tightly constraining mechanistic models, smFRET data can reveal the coupling between conformational and chemical transitions in complex enzymatic cycles., A major challenge in following electron transfer through dithiol/disulfide exchange is the dearth of accompanying spectroscopic effects. Here, the authors use single-molecule Förster resonance energy transfer experiments to illuminate disulfide bond rearrangements within the enzyme quiescin sulfhydryl oxidase.

Quiescin sulfhydryl oxidases (QSOXs) catalyze the formation of disulfide bonds in peptides and proteins, and in vertebrates comprise two proteins: QSOX1 and QSOX2. QSOX1, the most extensively studied type, has been implicated in protein folding, production of extracellular matrix, redox regulation, protection from apoptosis, angiogenesis, and cell differentiation. Atherosclerosis is an immunopathological condition in which redox processes, apoptosis, cell differentiation, and matrix secretion/maturation have critical roles. Considering these data, we hypothesized that QSOX1 could be involved in this disease, possibly reducing apoptosis and angiogenesis inside the plaque. QSOX1 labeling in normal human carotid vessels showed predominant expression by endothelium, subendothelium, and adventitia. In atherosclerotic plaques, however, QSOX1 was highly expressed in macrophages at the lipid core. QSOX1 expression was also studied in terms of mRNA and protein in cell types present in plaques under apoptotic or activating stimuli, emulating conditions found in the atherosclerotic process. QSOX1 mRNA increased in endothelial cells and macrophages after the induction of apoptosis. At the protein level, the correlation between apoptosis and QSOX1 expression was not evident in all cell types, possibly because of a rapid secretion of QSOX1. Our results propose for the first time possible roles for QSOX1 in atherosclerosis, being upregulated in endothelial cells and macrophages by apoptosis and cell activation, and possibly controlling these processes, as well as angiogenesis. The quantitative differences in QSOX1 induction may depend on the cell type and also on local factors.

Multiple Sulfatase Deficiency (MSD) is caused by mutations in the sulfatase-modifying factor 1 gene encoding the formylglycine-generating enzyme (FGE). FGE post translationally activates all newly synthesized sulfatases by generating the catalytic residue formylglycine. Impaired FGE function leads to reduced sulfatase activities. Patients display combined clinical symptoms of single sulfatase deficiencies. For ten MSD patients, we determined the clinical phenotype, FGE expression, localization and stability, as well as residual FGE and sulfatase activities. A neonatal, very severe clinical phenotype resulted from a combination of two nonsense mutations leading to almost fully abrogated FGE activity, highly unstable FGE protein and nearly undetectable sulfatase activities. A late infantile mild phenotype resulted from FGE G263V leading to unstable protein but high residual FGE activity. Other missense mutations resulted in a late infantile severe phenotype because of unstable protein with low residual FGE activity. Patients with identical mutations displayed comparable clinical phenotypes. These data confirm the hypothesis that the phenotypic outcome in MSD depends on both residual FGE activity as well as protein stability. Predicting the clinical course in case of molecularly characterized mutations seems feasible, which will be helpful for genetic counseling and developing therapeutic strategies aiming at enhancement of FGE. European Journal of Human Genetics (2011) 19, 253-261; doi:10.1038/ejhg.2010.219; published online 12 January 2011

Augmenter of liver regeneration (ALR), which is critically important in liver regeneration and hepatocyte proliferation, is highly expressed in cirrhotic livers and hepatocellular carcinomas (HCC). In the current study, the functional role of ALR in hepatocancerogenesis was analyzed in more detail. HepG2 cells, in which the cytosolic 15 kDa ALR isoform was reexpressed stably, (HepG2-ALR) were used in migration and invasion assays using modified Boyden chambers. Epithelial-mesenchymal transition (EMT) markers were determined in HepG2-ALR cells in vitro and in HepG2-ALR tumors grown in nude mice. ALR protein was quantified in HCC and nontumorous tissues by immunohistochemistry. HepG2-ALR, compared with HepG2 cells, demonstrated reduced cell motility and increased expression of the epithelial cell markers E-cadherin and Zona occludens-1 (ZO-1), whereas SNAIL, a negative regulator of E-cadherin, was diminished. Matrix metalloproteinase MMP1 and MMP3 mRNA expression and activity were reduced. HepG2-ALR cell-derived subcutaneously grown tumors displayed fewer necrotic areas, more epithelial-like cell growth and fewer polymorphisms and atypical mitotic figures than tumors derived from HepG2 cells. Analysis of tumor tissues of 53 patients with HCC demonstrated an inverse correlation of ALR protein with histological angioinvasion and grading. The 15 kDa ALR isoform was found mainly in HCC tissues without histological angioinvasion 0. In summary the present data indicate that cytosolic ALR reduces hepatoma cell migration, augments epithelial growth and, therefore, may act as an antimetastatic and EMT reversing protein.

Sulfur is an essential nutrient, taken up as sulfate from soil, reduced and incorporated into bioorganic compounds in plant cells. The pathway of sulfate assimilation is highly regulated in a demand-driven manner in seed plants. To test the evolutionary conservation of the regulatory mechanisms, we analyzed regulation of the pathway in the model for basal plants, the moss Physcomitrella patens. While in Arabidopsis the key enzyme of sulfate assimilation, adenosine 5'-phosphosulfate reductase (APR), is feedback repressed by thiols and induced by reduced levels of glutathione, in P. patens such regulation does not occur. The control of the pathway was not moved to other components as these conditions affected neither mRNA accumulation of other genes of sulfate assimilation nor sulfate uptake. Other treatments known to regulate APR, O-acetylserine, cadmium and sulfur deficiency affected APR transcript levels, but not enzyme activity. It appears that the sulfate assimilation pathway in P. patens is much more robust than in seed plants. Thus, the regulatory networks controlling the pathway have probably evolved only later in the evolution of the seed plants after separation of the bryophytes.

Multiple Sulfatase Deficiency (MSD) is a rare autosomal recessive disease in which the activities of all sulfatases are reduced; its estimated prevalence is 1:1.4 million births. The disease is caused by mutations in SUMF1, which encodes an enzyme involved in the post-translational modification of sulfatases. The MSD phenotype is a combination of the clinical features found in diseases resulting from a deficiency of the individual sulfatases; i.e., mucopolysaccharidosis II, IIIA, IIID, IVA and VI, metachromatic leukodystrophy, X-linked ichthyosis, and the X-linked recessive form of chondrodysplasia punctata. We describe herein the first case of a Brazilian patient with MSD. The case was initially diagnosed as having mucopolysaccharidosis (MPS), due to skeletal alterations, coarse facial features, and urinary excretion of dermatan sulfate and heparan sulfate. Later, after a detailed biochemical investigation, the diagnosis of MSD was established. The analysis of the SUMF1 showed the patient was a compound heterozygote for two novel mutations (p.R349G and p.F244S). This case illustrates the challenges in the diagnosis of a disease considered rare, such as MSD. We point out that the availability of therapy for certain MPS disorders necessitates correct disease assignment, and the need to exclude the likelihood of MSD.

Adenosine 5'-phosphosulfate (APS) reductase is a key enzyme involved in the pathways of sulfate reduction and sulfide oxidation in the biological sulfur cycle. In this study, the gene of APS reductase from Acidithiobacillus ferrooxidans was cloned and expressed in Escherichia coli, the soluble protein was purified by one-step affinity chromatography to apparent homogeneity. The molecular mass of the recombinant APS reductase was determined to be 28 kDa using SDS-PAGE. According to optical and EPR spectra results of the recombinant protein confirmed that the iron-sulfur cluster inserted into the active site of the protein. Site-directed mutation for the enzyme revealed that Cys110, Cys111, Cys193, and Cys196 were in ligation with the iron-sulfur cluster. The [Fe4S4] cluster could be assembled in vitro, and exhibited electron transport and redox catalysis properties. As we know so far, this is the first report of expression in E. coli of APS reductase from A. ferrooxidans.

The X-ray crystal structure of peroxiredoxin bound to sulphiredoxin is solved. In this structure of the co-complex, the carboxy terminus of peroxiredoxin is completely unfolded, and it is 'packed' onto the backside of sulphiredoxin, away from the sulphiredoxin active site. Binding studies and activity analyses of site-directed mutants at this interface show that the interaction is required for repair to occur. Typical 2-Cys peroxiredoxins (Prxs) have an important role in regulating hydrogen peroxide-mediated cell signalling1. In this process, Prxs can become inactivated through the hyperoxidation of an active site Cys residue to Cys sulphinic acid. The unique repair of this moiety by sulphiredoxin (Srx) restores peroxidase activity and terminates the signal2. The hyperoxidized form of Prx exists as a stable decameric structure with each active site buried. Therefore, it is unclear how Srx can access the sulphinic acid moiety. Here we present the 2.6 A crystal structure of the human Srx–PrxI complex. This complex reveals the complete unfolding of the carboxy terminus of Prx, and its unexpected packing onto the backside of Srx away from the Srx active site. Binding studies and activity analyses of site-directed mutants at this interface show that the interaction is required for repair to occur. Moreover, rearrangements in the Prx active site lead to a juxtaposition of the Prx Gly-Gly-Leu-Gly and Srx ATP-binding motifs, providing a structural basis for the first step of the catalytic mechanism. The results also suggest that the observed interactions may represent a common mode for other proteins to bind to Prxs.

Sulfate assimilation provides reduced sulfur for the synthesis of the amino acids cysteine and methionine and for a range of other metabolites. The key step in control of plant sulfate assimilation is the reduction of adenosine 5'-phosphosulfate to sulfite. The enzyme catalyzing this reaction, adenosine 5'phosphosulfate reductase (APR), is found as an iron sulfur protein in plants, algae, and many bacteria. In the moss Physcomitrella patens, however, a novel isoform of the enzyme, APR-B, has recently been discovered lacking the co-factor. To assess the function of the novel APR-B we used homologous recombination to disrupt the corresponding gene in P. patens. The knock-out plants were able to grow on sulfate as a sole sulfur source and the content of low molecular weight thiols was not different from wild type plants or plants where APR was disrupted. However, when treated with low concentrations of cadmium the APR-B knockout plants were more sensitive than both wild type and APR knockouts. In wild type P. patens, the two APR isoforms were not affected by treatments that strongly regulate this enzyme in flowering plants. The data thus suggest that in P. patens APS reduction is not the major control step of sulfate assimilation.

Membrane-permeant oxidants have become a standard tool for studying eukaryotic organisms because they affect the redox state and the redox regulation of different compartments. The ero1-1 mutant is temperature sensitive (37 degrees C) and cannot grow under anaerobic conditions. Low micromolar concentrations of the specific thiol-oxidant dipyridyl disulfide (DPS) rescue both these growth defects. Furthermore, the unfolded protein response (UPR) is slightly induced by a DPS treatment. We infer that DPS may change the redox state of important ER-proteins by GSH-oxidation in the ER or, more likely, by directly oxidizing these targets. Therefore, DPS may be useful in genetic studies dealing with unknown factors of the Ero1p-driven pathway. The ero1-1 mutation and the overproduction of Ero1p confer DPS-sensitivity that could be partially related to a more oxidized cytosolic GSH redox potential and the presence of reactive oxidative species (ROS) in the cell.

Purpose: Identify the roles of sulfide oxidase and rhodanese in sulfide detoxification in rat colonic mucosa. Results: Gel filtration of colonic mucosa and purified bovine rhodanese showed that rhodanese and sulfide oxidizing activities resided in different proteins. In the presence of cyanide, rhodanese shifted the major mucosal metabolite of sulfide from thiosulfate to thiocyanate. The purported ability of purified rhodanese to metabolize sulfide reflects: (a) contamination with a sulfide oxidase, and (b) the spontaneous conversion of sulfide to thiosulfate during storage; rhodanese then catalyzes the conversion of this thiosulfate to thiocyanate. Conclusions: Rhodanese does not metabolize sulfide. The rate-limiting step in sulfide detoxification is oxidation by a sulfide oxidase to thiosulfate. Rhodanese then converts this thiosulfate to thiocyanate, but this reaction does not increase the rate of sulfide detoxification. The recent use of rhodanese activity as a surrogate for the rate that colonic mucosa detoxifies sulfide is inappropriate.

Real-time polymerase chain reaction (PCR) is considered a highly sensitive method for the quantification of microbial organisms in environmental samples. This study was conducted to evaluate real-time PCR with SybrGreen detection as a quantification method for sulfate-reducing bacteria (SRB) in industrial wastewater produced by several chemical industries. We designed four sets of primers and developed standard curves based on genomic DNA of Desulfovibrio vulgaris from pure culture and on plasmids containing dissimilatory sulfate reductase (dsrA) or adenosine-5'-phosphosulfate reductase (apsA) genes of SRB. All the standard curves, two for dsrA and two for apsA genes, had a linear range between 0.95 x 10(2) and 9.5 x 10(6) copies/microL and between 1.2 x 10(3) and 1.2 x 10(7) copies/microL, respectively. The theoretical copy numbers of the tenfold dilutions of D. vulgaris genomic DNA were best estimated (between 2.7 to 10.5 times higher than theoretical numbers) by the standard curve with DSR1F and RH3-dsr-R primers. To mimic the effect of foreign DNA in environmental samples, serial dilutions of D. vulgaris genomic DNA were mixed with Escherichia coli chromosomal DNA (40 ng per assay). This influenced neither PCR amplification nor the quantification of target DNA. Industrial wastewater was sampled during a 15-month period and analyzed for the presence of SRB, based on dsrA gene amplification. SRB displayed a higher abundance during the summer (about 10(7)-10(8) targets mL(-1)) and lower during the winter (about 10(4)-10(5) targets mL(-1)). The results indicate that our real-time PCR approach can be used for detection of uncultured SRB and will provide valuable information related to the abundance of SRB in durable environmental samples, such as complex and saline industrial wastewaters.

Background Medulloblastoma is a malignant, invasive embryonal tumor of the cerebellum and accounts for 20% of intracranial tumors in children. QSOX1, whose functions include formation of disulphide bridges, which are needed for correct protein folding and stability, formation of the extracellular matrix, regulation of the redox status and cell cycle control, appears to be involved in apoptosis in pathological states such as cancer. Thus, the aim of this study was to investigate the immunohistochemical expression of QSOX1 in medulloblastomas and nonneoplastic cerebellum. Methods Histology blocks of pediatric medulloblastomas were separated and two representative areas of the tumors and non-neoplastic cerebellum samples were used to construct tissue microarrays (TMAs) that were stained with an anti-QSOX1 antibody, and the slides were read using image analysis software. Results QSOX1 immunoexpression was observed in the non-neoplastic cerebellum samples and the medulloblastoma samples. There was no statistically significant relationship between QSOX1 immunopositivity in the medulloblastoma samples and the clinical and pathological variables. Conclusions Although QSOX1 did not prove useful for stratifying patients into risk groups, tumor cells and the fibrillar extracellular matrix were positive for this marker, indicating that this enzyme may be involved in the pathogenesis of medulloblastoma. Virtual Slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1822040654139436

Sulfatases are key enzymatic regulators of sulfate homeostasis with several biological functions including degradation of glycosaminoglycans (GAGs) and other macromolecules in lysosomes. In a severe lysosomal storage disorder, multiple sulfatase deficiency (MSD), global sulfatase activity is deficient due to mutations in the sulfatase-modifying factor 1 (SUMF1) gene, encoding the essential activator of all sulfatases. We identify a novel regulatory layer of sulfate metabolism mediated by a microRNA. miR-95 depletes SUMF1 protein levels and suppresses sulfatase activity, causing the disruption of proteoglycan catabolism and lysosomal function. This blocks autophagy-mediated degradation, causing cytoplasmic accumulation of autophagosomes and autophagic substrates. By targeting miR-95 in cells from MSD patients, we can effectively increase residual SUMF1 expression, allowing for reactivation of sulfatase activity and increased clearance of sulfated GAGs. The identification of this regulatory mechanism opens the opportunity for a unique therapeutic approach in MSD patients where the need for exogenous enzyme replacement is circumvented.

Gene transcription changes dramatically in response to various stresses. This event is an obligatory step for adaptation of cells to certain environments. Endoplasmic reticulum (ER) oxidoreductin encoded by the ERO1 gene of the yeast Saccharomyces cerevisiae is essential for the formation of protein disulfide bonds in the ER and for cell viability. We show that transcription of ERO1 is regulated by two transcriptional activators in response to different stresses. In the unfolded protein response induced by the reductant dithiothreitol, transcription factor Hac1 activates ERO1 transcription through a sequence that diverges from the consensus Hac1-binding sequence. Heat shock transcription factor Hsf1 activates ERO1 in response to heat, ethanol, and oxidative stresses. Using cells containing mutations in the Hac1- and Hsf1-binding sequences of the chromosomal ERO1 promoter, we demonstrate that Hac1-regulated transcription of ERO1 confers resistance to dithiothreitol. Although mutations in the Hsf1-binding sequences do not affect the sensitivity of cells to heat, ethanol, or oxidative stresses, both the Hac1- and Hsf1-regulated pathways are critical for normal growth under complex stress conditions.

Cysteine is the first organic product of sulfate assimilation and as such is the precursor of all molecules containing reduced sulfur including methionine, glutathione, and their many metabolites. In plants, 5'-adenylylsulfate (APS) reductase is hypothesized to be a key regulatory point in sulfate assimilation and reduction. APS reductase catalyzes the two-electron reduction of APS to sulfite using glutathione as an electron donor. This paper reviews the experimental basis for this hypothesis. In addition, the results of an experiment designed to test the hypothesis by bypassing the endogenous APS reductase and its regulatory mechanisms are described. Two different bacterial assimilatory reductases were expressed in transgenic Zea mays, the thioredoxin-dependent APS reductase from Pseudomonas aeruginosa and the thioredoxin-dependent 3'-phosphoadenylylsulfate reductase from Escherichia coli. Each of them was placed under transcriptional control of the ubiquitin promoter and the protein products were targeted to chloroplasts. The leaves of transgenic Z. mays lines showed significant accumulation of reduced organic thiol compounds including cysteine, gamma-glutamylcysteine, and glutathione; and reduced inorganic forms of sulfur including sulfite and thiosulfate. Both bacterial enzymes appeared to be equally capable of deregulating the assimilative sulfate reduction pathway. The reduced sulfur compounds accumulated to such high levels that the transgenic plants showed evidence of toxicity. The results provide additional evidence that APS reductase is a major control point for sulfate reduction in Z. mays.

Applied Microbiology and Biotechnology, 68 (2), ISSN:0175-7598

The phylogenetic diversity of dissimilatory sulfite reductase (DSR, EC 1.8.99.3) alpha-subunit genes from a deep-sea cold seep was analyzed. Bulk genomic DNA was extracted from the cold seep sediment and used for amplification by polymerase chain reaction (PCR) of DSR alpha-subunit gene. Two sizes of PCR products, 1.4 kb (expected) and 1.3 kb (unexpected), were amplified. Sixteen clones of the 1.4-kb amplicons and 16 clones of 1.3-kb amplicons, a total of 32 clones, were obtained and grouped into operational DSR units (ODUs) based on restriction fragment length polymorphism (RFLP) by digestion with HaeIII and MboI. A total of 14 ODUs, i.e., 5 ODUs from 1.4-kb amplicon clones and 9 ODUs from 1.3-kb amplicon clones, were recovered. About 400 bp of the 5' ends of all the clones was sequenced and validated the RFLP-based ODU grouping. All the 5'-end 400-bp sequences of ODUs, even from the 1.3-kb amplicons, showed the characteristic DSR amino acid sequence motifs. The ODUs from 1.4-kb amplicons were closely related to the delta-Proteobacterial lineage with the DSR genes from epsilon-Proteobacterial epibionts of the hot vent worm Alvinella pompejana. The ODUs from 1.3-kb amplicons were mostly related to the unknown but possibly archaeal lineage. The diversity of the DSR genes may indicate the diversity of sulfate reducers in the seep sediment as well as the complexity of electron donors including methane.

A benzothiophene (BT) and dibenzothiophene (DBT) monooxygenase (TdsC), which catalyzes the oxidation of the sulfur atoms in BT and DBT molecules, was purified from Paenibacillus sp. strain A11-2. The molecular mass of the purified enzyme and its subunit were determined to be 200 kDa and 43 kDa by gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis, respectively, indicating a tetrameric structure. The N-terminal amino acid sequence of the purified TdsC completely matched the amino acid sequence deduced from the nucleotide sequence of the tdsC gene reported previously [Ishii et al. (2000) Biophys Biochem Res Commun 270:81-88]. The optimal temperature and pH for the TdsC reaction were 65 degrees C and pH 9, respectively. TdsC required NADH, FMN and TdsD, a NADH-dependent FMN oxidoreductase, for its activity, as was observed for TdsA. FAD, lumiflavin and/or NADPH had some effect on the maintenance of TdsC activity. A comparison of the substrate specificity of TdsC and DszC, the homologous monooxygenase purified from Rhodococcus erythropolis strain KA2-5-1, demonstrated a contrasting pattern towards alkylated DBTs and BTs.

The titration of chicken liver sulfite oxidase (SO) with the one-electron reductant Ti(III) citrate, at pH 7.0, results in nearly quantitative selective reduction of the Mo(VI) center to Mo(V), while the b-type heme center remains in the fully oxidized Fe(III) state. The selective reduction of the Mo(VI/V) couple has been established from electronic and EPR spectra. The elec- tronic spectrum of the Fe(III) heme center is essentially unchanged during the titration, and the continuous wave (CW)-EPR spectrum shows the appearance of the well-known Mo(V) signal due to the low pH (lpH) form of SO. Further confirmation of the selective formation of the Mo(V)/Fe(III) form of SO is provided by the � 1:1 ratio of the integrated intensities of the Mo(V) and low- spin Fe(III) EPR signals after addition of one equivalent of Ti(III). The selective generation of the Mo(V)/Fe(III) form of SO is unexpected, considering that previous microcoulometry and flash photolysis investigations have indicated that the Mo(VI/V) and Fe(III/II) couples of SO have similar reduction potentials at pH 7. The nearly quantitative preparation of the one-electron re- duced Mo(V)/Fe(III) form of SO by reduction with Ti(III) has enabled the interaction between these two paramagnetic metalcenters, which are l inked by a flex- ible loop with no secondary structure, to be investigated for the first time by variable-frequency pulsed electron- electron double resonance (ELDOR) spectroscopy. The ELDOR kinetics were obtained from frozen solutions at 4.2 K at severalmicrowave frequencies by pumping on the narrow Mo(V) signaland observing the effect on the Fe(III) primary echo at both higher and lower fre- quencies within the microwave C-band region. The ELDOR data indicate that freezing the solution of one- electron reduced SO produces localized regions where the concentration of SO approaches that in the crystal structure, which results in the interpair interactions be- ing the dominant dipolar interaction. However, thor- ough analysis of the ELDOR decay curves and simulations suggests a distribution of intramolecular Mo ... Fe distances, consistent with the proposalof mul - tiple conformations in solution for the flexible loop that connects the Mo and heme domains of SO.

Efficient protein folding and quality control in the endoplasmic reticulum (ER) require that disulphide bonds are formed in nascent proteins, isomerised during assisted folding and reduced in terminally misfolded molecules. Recent findings in yeast and mammalian cells indicate that specific protein-protein interactions underlie redox control in the ER, allowing these competing reactions to occur simultaneously during protein quality control.

Background Efficient utilization of both glucose and xylose is necessary for a competitive ethanol production from lignocellulosic materials. Although many advances have been made in the development of xylose-fermenting strains of Saccharomyces cerevisiae, the productivity remains much lower compared to glucose. Previous transcriptional analyses of recombinant xylose-fermenting strains have mainly focused on central carbon metabolism. Very little attention has been given to other fundamental cellular processes such as the folding of proteins. Analysis of previously measured transcript levels in a recombinant XR/XDH-strain showed a wide down-regulation of genes targeted by the unfolded protein response during xylose fermentation. Under anaerobic conditions the folding of proteins is directly connected with fumarate metabolism and requires two essential enzymes: FADH2-dependent fumarate reductase (FR) and Ero1p. In this study we tested whether these enzymes impair the protein folding process causing the very slow growth of recombinant yeast strains on xylose under anaerobic conditions. Results Four strains over-expressing the cytosolic (FRD1) or mitochondrial (OSM1) FR genes and ERO1 in different combinations were constructed. The growth and fermentation performance was evaluated in defined medium as well as in a complex medium containing glucose and xylose. Over-expression of FRD1, alone or in combination with ERO1, did not have any significant effect on xylose fermentation in any medium used. Over-expression of OSM1, on the other hand, led to a diversion of carbon from glycerol to acetate and a decrease in growth rate by 39% in defined medium and by 25% in complex medium. Combined over-expression of OSM1 and ERO1 led to the same diversion of carbon from glycerol to acetate and had a stronger detrimental effect on the growth in complex medium. Conclusions Increasing the activities of the FR enzymes and Ero1p is not sufficient to increase the anaerobic growth on xylose. So additional components of the protein folding mechanism that were identified in transcription analysis of UPR related genes may also be limiting. This includes i) the transcription factor encoded by HAC1 ii) the activity of Pdi1p and iii) the requirement of free FAD during anaerobic growth.

The individual rate constants for intramolecular electron transfer (IET) between the Mo(VI)Fe(II) and Mo(V)Fe(III) forms of chicken liver sulfite oxidase (SO) have been determined at a variety of pH values, and at high and low anion concentrations. Large anions such as EDTA do not inhibit IET as dramatically as do small anions such as SO4(2-) and Cl-, which suggests that specific anion binding at the sterically constrained Mo active site is necessary for IET inhibition to occur.IET may require that SO adopt a conformation in which the Mo and Fe centers are held in close proximity by electrostatic interactions between the predominantly positively charged Mo active site, and the negatively charged heme edge. Thus, small anions which can fit into the Mo active site will weaken this electrostatic attraction and disfavor IET. The rate constant for IET from Fe(II) to Mo(VI) decreases with increasing pH, both in the presence and absence of 50 mM SO4(2-) . However, the rate constant for the reverse process exhibits no significant pH dependence in the absence of SO4(2-), and increases with pH in the presence of 50 mM S04(2-). This behavior is consistent with a mechanism in which IET from Mo(V) to Fe(III) is coupled to proton transfer from Mo(V)-OH to OH-, and the reverse IET process is coupled to proton transfer from H2O to Mo(VI) = O. At high concentrations of small anions, direct access of H2O or OH- to the Mo-OH will be blocked, which provides a second possible mechanism for inhibition of IET by such anions. Inhibition by anions is not strictly competitive, however, and Tyr322 may play an important intermediary role in transferring the proton when an anion blocks direct access of H2O or OH- to the Mo-OH. Competing H-bonding interactions of the Mo-OH moiety with Tyr322 and with the anion occupying the active site may also be responsible for the well-known equilibrium between two EPR-distinct forms of SO that is observed for the two-electron reduced enzyme.

The genes encoding the alpha- and beta-subunits of dissimilatory sulfite reductase, dsrAB, from the hyperthermophilic archaeon Archaeoglobus profundus and the thermophilic gram-positive bacterium Desulfotomaculum thermocisternum were cloned and sequenced. The dsrAB genes are contiguous, and most probably comprise an operon also including a dsrD homolog, a conserved gene of unknown function located downstream of dsrAB in all four sulfate reducers so far sequenced. Sequence comparison confirms that dissimilatory sulfite reductase, Dsr, is a highly conserved enzyme. A phylogenetic analysis using the available Dsr sequences, including Dsr-like proteins from nonsulfate reducers, suggests a paralogous origin of the alpha- and beta-subunits. Furthermore, the Dsr from sulfate reducers forms a separate cluster, with Dsr from the bacterial sulfate reducers Desulfotomaculum thermocisternum and Desulfovibrio vulgaris branching together, next to Dsr from Archaeoglobus profundus and Archaeoglobus fulgidus. Based on an alignment with the assimilatory sulfite reductase from Escherichia coli, the amino acid residues involved in binding of sulfite, siroheme, and [Fe4S4]-clusters have been tentatively identified, which is consistent with the binding of two sirohemes and four [Fe4S4]-clusters per alpha2beta2 structure. The evolution of Dsr and the structural basis for the binding of substrate and cofactors are discussed.

Expression of eukaryotic proteins in Escherichia coli is challenging, especially when they contain disulfide bonds. Since the discovery of the prion protein (PrP) and its role in transmissible spongiform encephalopathies, the need to obtain large quantities of the recombinant protein for research purposes has been essential. Currently, production of recombinant PrP is achieved by refolding protocols. Here, we show that the co-expression of two different PrP with the human Quiescin Sulfhydryl OXidase (QSOX), a human chaperone with thiol/disulfide oxidase activity, in the cytoplasm of E. coli produces soluble recombinant PrP. The structural integrity of the soluble PrP has been confirmed by nuclear magnetic resonance spectroscopy, demonstrating that properly folded PrP can be easily expressed in bacteria. Furthermore, the soluble recombinant PrP produced with this method can be used for functional and structural studies.

Five facultative sulfur chemolithotrophs were isolated from soils to study the diversity of sulfur lithotrophy. Phenotypic characteristics, including sulfur lithotrophic properties and chemotaxonomic features of the isolates, were similar to those of the members of the colorless sulfur bacteria. 16S rDNA sequence analyses rendered placing the isolates to three distinct phylogenetic clusters of alpha-proteobacteria. Three isolates, AS001, AS002, and KCT002, were identified as members of the genus Paracoccus. The strains AS001 and AS002, having identical 16S-rDNA sequence, showed significant 16S rDNA sequence similarity (99.1%) to Paracoccus versutus. The strain KCT002 showed highest (98%) 16S rDNA sequence similarity to P. alcaliphilus and 96% similarity to the pair AS001 and AS002. Isolate KCT001 appeared to be closely related to Pseudaminobacter salicylatoxidans, although sulfur lithotrophy of P. salicylotoxidans is not known. The other isolate, TCK, showed almost identical 16S rDNA (99.9%) sequence with two recently described unclassified chemolithoautotrophic arsenite oxidizing strains. Physiological and chemotaxonomic characteristics and phylogenetic analyses of the five new strains emphasize the need of polyphasic bacterial taxonomy of sulfur lithotrophs.

Background The sulfate assimilation pathway is present in photosynthetic organisms, fungi, and many bacteria, providing reduced sulfur for the synthesis of cysteine and methionine and a range of other metabolites. In photosynthetic eukaryotes sulfate is reduced in the plastids whereas in aplastidic eukaryotes the pathway is cytosolic. The only known exception is Euglena gracilis, where the pathway is localized in mitochondria. To obtain an insight into the evolution of the sulfate assimilation pathway in eukaryotes and relationships of the differently compartmentalized isoforms we determined the locations of the pathway in lineages for which this was unknown and performed detailed phylogenetic analyses of three enzymes involved in sulfate reduction: ATP sulfurylase (ATPS), adenosine 5'-phosphosulfate reductase (APR) and sulfite reductase (SiR). Results The inheritance of ATPS, APR and the related 3'-phosphoadenosine 5'-phosphosulfate reductase (PAPR) are remarkable, with multiple origins in the lineages that comprise the opisthokonts, different isoforms in chlorophytes and streptophytes, gene fusions with other enzymes of the pathway, evidence a eukaryote to prokaryote lateral gene transfer, changes in substrate specificity and two reversals of cellular location of host- and endosymbiont-originating enzymes. We also found that the ATPS and APR active in the mitochondria of Euglena were inherited from its secondary, green algal plastid. Conclusion Our results reveal a complex history for the enzymes of the sulfate assimilation pathway. Whilst they shed light on the origin of some characterised novelties, such as a recently described novel isoform of APR from Bryophytes and the origin of the pathway active in the mitochondria of Euglenids, the many distinct and novel isoforms identified here represent an excellent resource for detailed biochemical studies of the enzyme structure/function relationships.

A case of combined deficiency of sulphite-oxidase and xanthine-oxidase with a defect of the molybdenum cofactor, which is vital to the activity of sulphite-, xanthine- and aldehyde-oxidase, is reported here. Seven cases of combined deficiencies have been described with regard to both clinical and laboratory findings. The clinical, laboratory and anatomo-pathological features and, in particular, the central nervous system lesions of the present case correspond exactly to those in the case described Rosenblum in which an isolated deficiency in sulphite-oxidase was present. As the cerebral alterations in the present case are comparable to those described in Rosenblum's case, they probably result from the defect in sulphite-oxidase activity.