Comparative studies on substrate specificity of succinic semialdehyde reductase from Gluconobacter oxydans and glyoxylate reductase from Acetobacter aceti.

Gluconobacter oxydans succinic semialdehyde reductase (GoxSSAR) and Acetobacter aceti glyoxylate reductase (AacGR) represent a novel class in the ß-HAD superfamily. Kinetic analyses revealed GoxSSAR's activity with both glyoxylate and succinic semialdehyde, while AacGR is glyoxylate-specific. GoxSSAR K167A lost activity with succinic semialdehyde but retained some with glyoxylate, whereas AacGR K175A lost activity. These findings elucidate differences between these homologous enzymes.

Apolipoprotein E-mediated regulation of selenoprotein P transportation via exosomes.

Selenoprotein P (SELENOP), secreted from the liver, functions as a selenium (Se) supplier to other tissues. In the brain, Se homeostasis is critical for physiological function. Previous studies have reported that SELENOP co-localizes with the apolipoprotein E receptor 2 (ApoER2) along the blood-brain barrier (BBB). However, the mechanism underlying SELENOP transportation from hepatocytes to neuronal cells remains unclear. Here, we found that SELENOP was secreted from hepatocytes as an exosomal component protected from plasma kallikrein-mediated cleavage. SELENOP was interacted with apolipoprotein E (ApoE) through heparin-binding sites of SELENOP, and the interaction regulated the secretion of exosomal SELENOP. Using in vitro BBB model of transwell cell culture, exosomal SELENOP was found to supply Se to brain endothelial cells and neuronal cells, which synthesized selenoproteins by a process regulated by ApoE and ApoER2. The regulatory role of ApoE in SELENOP transport was also observed in vivo using ApoE-/- mice. Exosomal SELENOP transport protected neuronal cells from amyloid ß (Aß)-induced cell death. Taken together, our results suggest a new delivery mechanism for Se to neuronal cells by exosomal SELENOP.

Initial Step of Selenite Reduction via Thioredoxin for Bacterial Selenoprotein Biosynthesis.

Many organisms reductively assimilate selenite to synthesize selenoprotein. Although the thioredoxin system, consisting of thioredoxin 1 (TrxA) and thioredoxin reductase with NADPH, can reduce selenite and is considered to facilitate selenite assimilation, the detailed mechanism remains obscure. Here, we show that selenite was reduced by the thioredoxin system from Pseudomonas stutzeri only in the presence of the TrxA (PsTrxA), and this system was specific to selenite among the oxyanions examined. Mutational analysis revealed that Cys33 and Cys36 residues in PsTrxA are important for selenite reduction. Free thiol-labeling assays suggested that Cys33 is more reactive than Cys36. Mass spectrometry analysis suggested that PsTrxA reduces selenite via PsTrxA-SeO intermediate formation. Furthermore, an in vivo formate dehydrogenase activity assay in Escherichia coli with a gene disruption suggested that TrxA is important for selenoprotein biosynthesis. The introduction of PsTrxA complemented the effects of TrxA disruption in E. coli cells, only when PsTrxA contained Cys33 and Cys36. Based on these results, we proposed the early steps of the link between selenite and selenoprotein biosynthesis via the formation of TrxA-selenium complexes.

Characterization of a novel class of glyoxylate reductase belonging to the ß-hydroxyacid dehydrogenase family in Acetobacter aceti.

Enzymes related to ß-hydroxyacid dehydrogenases/3-hydroxyisobutyrate dehydrogenases are ubiquitous, but most of them have not been characterized. An uncharacterized protein with moderate sequence similarities to Gluconobacter oxydans succinic semialdehyde reductase and plant glyoxylate reductases/succinic semialdehyde reductases was found in the genome of Acetobacter aceti JCM20276. The corresponding gene was cloned and expressed in Escherichia coli. The gene product was purified and identified as a glyoxylate reductase that exclusively catalyzed the NAD(P)H-dependent reduction of glyoxylate to glycolate. The strict substrate specificity of this enzyme to glyoxylate, the diverged sequence motifs for its binding sites with cofactors and substrates, and its phylogenetic relationship to homologous enzymes suggested that this enzyme represents a novel class of enzymes in the ß-hydroxyacid dehydrogenase family. This study may provide an important clue to clarify the metabolism of glyoxylate in bacteria. Abbreviations: GR: glyoxylate reductase; GRHPR: glyoxylate reductase/hydroxypyruvate reductase; HIBADH: 3-hydroxyisobutyrate dehydrogenase; SSA: succinic semialdehyde; SSAR: succinic semialdehyde reductase.

Selenite uptake by outer membrane porin ExtI and its involvement in the subcellular localization of rhodanese-like lipoprotein ExtH in Geobacter sulfurreducens.

Selenite reduction is a key step in the biogeochemical cycle of selenium-an essential trace element for life. A variety of bacteria can transform selenite into elemental selenium nanoparticles on the cell surface via anaerobic respiration or detoxification processes. However, the proteins associated with the uptake of selenite for these processes are poorly understood. In this study, we investigated the role of an outer membrane porin-like protein, ExtI, in selenite permeation in Geobacter sulfurreducens. We demonstrated that selenite uptake and selenium nanoparticle formation were impaired in an extI-deficient strain. A putative rhodanese-like lipoprotein is encoded by an extH gene located immediately upstream of extI in the genome. We showed that ExtH is translocated into inner and outer membranes and that extI deficiency exclusively affects the localization of ExtH in the outer membrane. Coelution of ExtI and ExtH during gel filtration analysis of the outer membrane fraction of wild-type cells suggests a direct protein-protein interaction between them. Taken together, these results lead us to propose a physiological role for ExtI as a selenite channel associated with ExtH in the outer membrane.

Characterization of a Novel Porin-Like Protein, ExtI, from Geobacter sulfurreducens and Its Implication in the Reduction of Selenite and Tellurite.

The extI gene in Geobacter sulfurreducens encodes a putative outer membrane channel porin, which resides within a cluster of extHIJKLMNOPQS genes. This cluster is highly conserved across the Geobacteraceae and includes multiple putative c-type cytochromes. In silico analyses of the ExtI sequence, together with Western blot analysis and proteinase protection assays, showed that it is an outer membrane protein. The expression level of ExtI did not respond to changes in osmolality and phosphate starvation. An extI-deficient mutant did not show any significant impact on fumarate or Fe(III) citrate reduction or sensitivity to ß-lactam antibiotics, as compared with those of the wild-type strain. However, extI deficiency resulted in a decreased ability to reduce selenite and tellurite. Heme staining analysis revealed that extI deficiency affects certain heme-containing proteins in the outer and inner membranes, which may cause a decrease in the ability to reduce selenite and tellurite. Based on these observations, we discuss possible roles for ExtI in selenite and tellurite reduction in G. sulfurreducens.

Selective fluorescence detection method for selenide and selenol using monochlorobimane.

The low redox potential of selenide and selenol is physiologically important, as it confers efficient catalytic abilities to selenoproteins. Quantitative determination of selenol and selenide provide important clues for understanding the metabolism and physiological function of selenium. However, selective detection of selenol and selenide is extremely difficult because of their chemical similarity to thiol and sulfide. In this study, we established a highly sensitive, selective, quantitative, and simple method for detection of selenol and selenide, using a reaction with monochlorobimane (MCB), followed by ethyl acetate extraction of the product syn-(methyl,methyl)bimane. We analyzed selenide production from selenite, catalyzed by human glutathione reductase, and also determined selenide and selenol concentrations in Hepa1-6 cells using the MCB method, to demonstrate its practical applications. This study provides a new tool for selenium detection in biology.

Purification and properties of 4-methyl-5-hydroxyethylthiazole kinase from Escherichia coli.

4-Methyl-5-hydroxyethylthiazole kinase (ThiM) participates in thiamin biosynthesis as the key enzyme in its salvage pathway. We purified and characterized ThiM from Escherichia coli. It has broad substrate specificity toward various nucleotides and shows a preference for dATP as a phosphate donor over ATP. It is activated by divalent cations, and responds more strongly to Co(2+) than to Mg(2+).

A non-radioactive assay for selenophosphate synthetase activity using recombinant pyruvate pyrophosphate dikinase from Thermus thermophilus HB8.

Biosynthesis of selenocysteine-containing proteins requires monoselenophosphate, a selenium-donor intermediate generated by selenophosphate synthetase (Sephs). A non-radioactive assay was developed as an alternative to the standard [8-(14)C] AMP-quantifying assay. The product, AMP, was measured using a recombinant pyruvate pyrophosphate dikinase from Thermus thermophilus HB8. The KM and kcat for Sephs2-Sec60Cys were determined to be 26 µM and 0.352 min(-1), respectively.

Functional expression of L-lysine α-oxidase from Scomber japonicus in Escherichia coli for one-pot synthesis of L-pipecolic acid from DL-lysine.

L-Pipecolic acid is a key component of biologically active molecules and a pharmaceutically important chiral building block. It can be stereoselectively produced from L-lysine by a two-step bioconversion involving L-lysine α-oxidase and ∆(1)-piperideine-2-carboxylae (Pip2C) reductase. In this study, we focused on an L-lysine α-oxidase from Scomber japonicus that was originally identified as an apoptosis-inducing protein (AIP) and applied the enzyme to one-pot fermentation of L-pipecolic acid in Escherichia coli. A synthetic gene coding for an AIP was expressed in E. coli, and the recombinant enzyme was purified and characterized. The purified enzyme was determined to be a homodimer with a molecular mass of 133.9 kDa. The enzyme essentially exhibited the same substrate specificity as the native enzyme. Optimal temperature and pH for the enzymatic reaction were 70 °C and 7.4, respectively. The enzyme was stable below 60 °C and at a pH range of 5.5-7.5 but was markedly inhibited by Co(2+). To establish a one-pot fermentation system for the synthesis of optically pure L-pipecolic acid from DL-lysine, an E. coli strain carrying a plasmid encoding AIP, Pip2C reductase from Pseudomonas putida, lysine racemase from P. putida, and glucose dehydrogenase from Bacillus subtilis was constructed. The one-pot process produced 45.1 g/L of L-pipecolic acid (87.4 % yield from DL-lysine) after a 46-h reaction with high optical purity (>99.9 % enantiomeric excess).

Global identification of genes affecting iron-sulfur cluster biogenesis and iron homeostasis.

Iron-sulfur (Fe-S) clusters are ubiquitous cofactors that are crucial for many physiological processes in all organisms. In Escherichia coli, assembly of Fe-S clusters depends on the activity of the iron-sulfur cluster (ISC) assembly and sulfur mobilization (SUF) apparatus. However, the underlying molecular mechanisms and the mechanisms that control Fe-S cluster biogenesis and iron homeostasis are still poorly defined. In this study, we performed a global screen to identify the factors affecting Fe-S cluster biogenesis and iron homeostasis using the Keio collection, which is a library of 3,815 single-gene E. coli knockout mutants. The approach was based on radiolabeling of the cells with [2-(14)C]dihydrouracil, which entirely depends on the activity of an Fe-S enzyme, dihydropyrimidine dehydrogenase. We identified 49 genes affecting Fe-S cluster biogenesis and/or iron homeostasis, including 23 genes important only under microaerobic/anaerobic conditions. This study defines key proteins associated with Fe-S cluster biogenesis and iron homeostasis, which will aid further understanding of the cellular mechanisms that coordinate the processes. In addition, we applied the [2-(14)C]dihydrouracil-labeling method to analyze the role of amino acid residues of an Fe-S cluster assembly scaffold (IscU) as a model of the Fe-S cluster assembly apparatus. The analysis showed that Cys37, Cys63, His105, and Cys106 are essential for the function of IscU in vivo, demonstrating the potential of the method to investigate in vivo function of proteins involved in Fe-S cluster assembly.

Glutathione contributes to the efflux of selenium from hepatoma cells.

Selenite is a selenium source for selenoprotein biosynthesis in mammalian cells. Although previous studies have suggested the involvement of glutathione (GSH) and/or thioredoxin reductase in selenite metabolism, intracellular selenite metabolism remains largely unknown. Here, we report that GSH depletion did not affect the amount of selenoprotein in Hepa 1-6 cells, suggesting that GSH does not play a central role in the reduction of selenite in selenoprotein biosynthesis. On the other hand, we found that GSH is involved in the efflux of low-molecular-weight selenium compounds from cells, presumably via the formation of selenodiglutathione. Moreover, selenite inhibited the efflux of a fluorescent bimane-GS conjugate that is mediated by ATP-dependent multidrug-resistant proteins, implying the existence of an active transporter for selenodiglutathione. This is the first report demonstrating that GSH plays a role in selenium excretion from cells by forming a GSH-conjugate, which may contribute to the distribution, detoxification, and homeostasis of selenium in the body.

Novel neogala-series glycosphingolipids with a terminal glucose residue from the fungus Mariannaea elegans.

Glycosphingolipids (GSLs) are essential membrane components of eukaryotic cells. Recently, a new type of fungal neogala-series GSL was identified in aureobasidin A-resistant fungi. In this study, we analyzed GSLs from four pathogenic fungal strains belonging to the order Hypocreales, and found that Mariannaea elegans contained both acidic GSLs and neutral GSLs with mono- and di-saccharides. The structures of the neutral GSLs of M. elegans were determined by compositional sugar, fatty acid, and sphingoid analyses by GC/MS, MALDI time-of-flight/MS, and 1H NMR. The ceramide moiety of Glcß1-Cer consisted mainly of the 2-hydroxylated C18:0-fatty acid 9-methyl-octadeca-4-sphinganine or 9-methyl-octadeca-4,8-sphingadienine. In contrast, the ceramides of Galß1-6Galß1-Cer and Glc1-6Galß1-Cer consisted mainly of saturated 2-hydroxylated C24:0-fatty acids and C18:0-phytosphingosine. To our knowledge, Glc1-6Galß1-Cer is a novel GSL in fungi, and M. elegans is the first example of an aureobasidin A-sensitive fungus that possesses fungal neogala series GSLs.

Comparison of Nutritional Availability of Biogenic Selenium Nanoparticles and Chemically Synthesized Selenium Nanoparticles.

Selenium (Se) is an essential micronutrient, and animals biosynthesize selenoproteins from various selenocompounds such as inorganic salts and organic selenocompounds as a Se source. In addition to the inorganic and organic forms of Se, it is also known that elemental Se is biologically synthesized at the nanoscale in nature. Biologically synthesized Se nanoparticles (Se-NPs), i.e., biogenic Se-NPs (Se-BgNPs), have not been fully investigated as a Se source compared with the other forms of Se. In this study, we evaluated the nutritional availability of Se-BgNPs biosynthesized in E. coli and revealed that Se-BgNPs were less assimilated into selenoproteins in rats as a Se source than inorganic Se salt or chemically synthesized Se-NPs. Se-BgNPs showed tolerance toward digestion and low absorbability in gut, which resulted in the low nutritional availability. Se-BgNPs seem to be coated with a biomaterial that functions to reduce their toxicity toward E. coli and at the same time lowers their availability to animals.

Group II truncated haemoglobin YjbI prevents reactive oxygen species-induced protein aggregation in Bacillus subtilis.

Oxidative stress-mediated formation of protein hydroperoxides can induce irreversible fragmentation of the peptide backbone and accumulation of cross-linked protein aggregates, leading to cellular toxicity, dysfunction, and death. However, how bacteria protect themselves from damages caused by protein hydroperoxidation is unknown. Here, we show that YjbI, a group II truncated haemoglobin from Bacillus subtilis, prevents oxidative aggregation of cell-surface proteins by its protein hydroperoxide peroxidase-like activity, which removes hydroperoxide groups from oxidised proteins. Disruption of the yjbI gene in B. subtilis lowered biofilm water repellence, which associated with the cross-linked aggregation of the biofilm matrix protein TasA. YjbI was localised to the cell surface or the biofilm matrix, and the sensitivity of planktonically grown cells to generators of reactive oxygen species was significantly increased upon yjbI disruption, suggesting that YjbI pleiotropically protects labile cell-surface proteins from oxidative damage. YjbI removed hydroperoxide residues from the model oxidised protein substrate bovine serum albumin and biofilm component TasA, preventing oxidative aggregation in vitro. Furthermore, the replacement of Tyr63 near the haem of YjbI with phenylalanine resulted in the loss of its protein peroxidase-like activity, and the mutant gene failed to rescue biofilm water repellency and resistance to oxidative stress induced by hypochlorous acid in the yjbI-deficient strain. These findings provide new insights into the role of truncated haemoglobin and the importance of hydroperoxide removal from proteins in the survival of aerobic bacteria.

Escherichia coli dihydropyrimidine dehydrogenase is a novel NAD-dependent heterotetramer essential for the production of 5,6-dihydrouracil.

The reductive pyrimidine catabolic pathway is absent in Escherichia coli. However, the bacterium contains an enzyme homologous to mammalian dihydropyrimidine dehydrogenase. Here, we show that E. coli dihydropyrimidine dehydrogenase is the first member of a novel NADH-dependent subclass of iron-sulfur flavoenzymes catalyzing the conversion of uracil to 5,6-dihydrouracil in vivo.

IscS functions as a primary sulfur-donating enzyme by interacting specifically with MoeB and MoaD in the biosynthesis of molybdopterin in Escherichia coli.

The persulfide sulfur formed on an active site cysteine residue of pyridoxal 5'-phosphate-dependent cysteine desulfurases is subsequently incorporated into the biosynthetic pathways of a variety of sulfur-containing cofactors and thionucleosides. In molybdenum cofactor biosynthesis, MoeB activates the C terminus of the MoaD subunit of molybdopterin (MPT) synthase to form MoaD-adenylate, which is subsequently converted to a thiocarboxylate for the generation of the dithiolene group of MPT. It has been shown that three cysteine desulfurases (CsdA, SufS, and IscS) of Escherichia coli can transfer sulfur from l-cysteine to the thiocarboxylate of MoaD in vitro. Here, we demonstrate by surface plasmon resonance analyses that IscS, but not CsdA or SufS, interacts with MoeB and MoaD. MoeB and MoaD can stimulate the IscS activity up to 1.6-fold. Analysis of the sulfuration level of MoaD isolated from strains defective in cysteine desulfurases shows a largely decreased sulfuration level of the protein in an iscS deletion strain but not in a csdA/sufS deletion strain. We also show that another iscS deletion strain of E. coli accumulates compound Z, a direct oxidation product of the immediate precursor of MPT, to the same extent as an MPT synthase-deficient strain. In contrast, analysis of the content of compound Z in DeltacsdA and DeltasufS strains revealed no such accumulation. These findings indicate that IscS is the primary physiological sulfur-donating enzyme for the generation of the thiocarboxylate of MPT synthase in MPT biosynthesis.

Reaction mechanism and molecular basis for selenium/sulfur discrimination of selenocysteine lyase.

Selenocysteine lyase (SCL) catalyzes the pyridoxal 5'-phosphate-dependent removal of selenium from l-selenocysteine to yield l-alanine. The enzyme is proposed to function in the recycling of the micronutrient selenium from degraded selenoproteins containing selenocysteine residue as an essential component. The enzyme exhibits strict substrate specificity toward l-selenocysteine and no activity to its cognate l-cysteine. However, it remains unclear how the enzyme distinguishes between selenocysteine and cysteine. Here, we present mechanistic studies of selenocysteine lyase from rat. ESI-MS analysis of wild-type and C375A mutant SCL revealed that the catalytic reaction proceeds via the formation of an enzyme-bound selenopersulfide intermediate on the catalytically essential Cys-375 residue. UV-visible spectrum analysis and the crystal structure of SCL complexed with l-cysteine demonstrated that the enzyme reversibly forms a nonproductive adduct with l-cysteine. Cys-375 on the flexible loop directed l-selenocysteine, but not l-cysteine, to the correct position and orientation in the active site to initiate the catalytic reaction. These findings provide, for the first time, the basis for understanding how trace amounts of a selenium-containing substrate is distinguished from excessive amounts of its cognate sulfur-containing compound in a biological system.

Favourable effects of eicosapentaenoic acid on the late step of the cell division in a piezophilic bacterium, Shewanella violacea DSS12, at high-hydrostatic pressures.

Shewanella violacea DSS12, a deep-sea bacterium, produces eicosapentaenoic acid (EPA) as a component of membrane phospholipids. Although various isolates from the deep sea, such as Photobacterium profundum SS9, Colwellia psychrerythraea 34H and various Shewanella strains, produce EPA- or docosahexaenoic acid-containing phospholipids, the physiological role of these polyunsaturated fatty acids remains unclear. In this article, we illustrate the physiological importance of EPA for high-pressure adaptation in strain DSS12 with the help of an EPA-deficient mutant (DSS12(pfaA)). DSS12(pfaA) showed significant growth retardation at 30 MPa, but not at 0.1 MPa. We also found that DSS12(pfaA) grown at 30 MPa forms filamentous cells. When an EPA-containing phospholipid (sn-1-oleoly-sn-2-eicosapentaenoyl phosphatidylethanolamine) was supplemented, the growth retardation and the morphological defect of DSS12(pfaA) were suppressed, indicating that the externally added EPA-containing phospholipid compensated for the loss of endogenous EPA. In contrast, the addition of an oleic acid-containing phospholipid (sn-1,2-dioleoyl phosphatidylethanolamine) did not affect the growth and the morphology of the cells. Immunofluorescent microscopic analysis with anti-FtsZ antibody revealed a number of Z-rings and separated nucleoids in DSS12(pfaA) grown at 30 MPa. These results demonstrate the physiological importance of EPA for the later step of Z-ring formation of S. violacea DSS12 under high-pressure conditions.

Bacterial cysteine desulfurases: versatile key players in biosynthetic pathways of sulfur-containing biofactors.

Cysteine desulfurases are pyridoxal 5'-phosphate-dependent homodimeric enzymes that catalyze the conversion of L-cysteine to L-alanine and sulfane sulfur via the formation of a protein-bound cysteine persulfide intermediate on a conserved cysteine residue. The enzymes are capable of donating the persulfide sulfur atoms to a variety of biosynthetic pathways for sulfur-containing biofactors, such as iron-sulfur clusters, thiamin, transfer RNA thionucleosides, biotin, and lipoic acid. The enormous advances in biochemical and structural studies of these biosynthetic pathways over the past decades provide an opportunity for detailed understanding of the nature of the excellent sulfur transfer mechanism of cysteine desulfurases.