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Histone post‐translational modifications (PTMs) are key players in chromatin regulation. The identification of novel histone acylations raises important questions regarding their role in transcription. In this study, we characterize the role of an acylation on the lateral surface of the histone octamer, H3K122 succinylation (H3K122succ), in chromatin function and transcription. Using chromatin succinylated at H3K122 in in vitro transcription assays, we show that the presence of H3K122succ is sufficient to stimulate transcription. In line with this, we found in our ChIP assays H3K122succ enriched on promoters of active genes and H3K122succ enrichment scaling with gene expression levels. Furthermore, we show that the co‐activators p300/CBP can succinylate H3K122 and identify sirtuin 5 (SIRT5) as a new desuccinylase. By applying single molecule FRET assays, we demonstrate a direct effect of H3K122succ on nucleosome stability, indicating an important role for histone succinylation in modulating chromatin dynamics. Together, these data provide the first insights into the mechanisms underlying transcriptional regulation by H3K122succ., Histone post‐translational modifications are key players in chromatin regulation. This study reveals the role of H3K122 succinylation in chromatin function and transcription.

Hepatitis B virus (HBV) persists by depositing a covalently closed circular DNA (cccDNA) in the nucleus of infected cells that cannot be targeted by available antivirals. Interferons can diminish HBV cccDNA via APOBEC3‐mediated deamination. Here, we show that overexpression of APOBEC3A alone is not sufficient to reduce HBV cccDNA that requires additional treatment of cells with interferon indicating involvement of an interferon‐stimulated gene (ISG) in cccDNA degradation. Transcriptome analyses identify ISG20 as the only type I and II interferon‐induced, nuclear protein with annotated nuclease activity. ISG20 localizes to nucleoli of interferon‐stimulated hepatocytes and is enriched on deoxyuridine‐containing single‐stranded DNA that mimics transcriptionally active, APOBEC3A‐deaminated HBV DNA. ISG20 expression is detected in human livers in acute, self‐limiting but not in chronic hepatitis B. ISG20 depletion mitigates the interferon‐induced loss of cccDNA, and co‐expression with APOBEC3A is sufficient to diminish cccDNA. In conclusion, non‐cytolytic HBV cccDNA decline requires the concerted action of a deaminase and a nuclease. Our findings highlight that ISGs may cooperate in their antiviral activity that may be explored for therapeutic targeting., Hepatitis B virus deposits a covalently closed circular (ccc)DNA in the nucleus of infected cells to persist. Interferons can purge cccDNA from the nucleus by inducing deaminases and the nuclease ISG20 that modify and subsequently degrade cccDNA.

Identification of interactors is a major goal in cell biology. Not only protein-protein but also protein-carbohydrate interactions are of high relevance for signal transduction in biological systems. Here we aim to identify novel interacting binding partners for the β-galactoside-binding proteins Galectin-1 (Gal-1) and Galectin-3 (Gal-3) relevant in the context of the eye disease proliferative vitreoretinopathy (PVR). PVR is one of the most common failures after retinal detachment surgeries and is characterized by the migration, adhesion and epithelial-to-mesenchymal transition (EMT) of retinal pigment epithelial cells (RPE) and the subsequent formation of sub- and epiretinal fibrocellular membranes. Gal-1 and Gal-3 bind in a dose- and carbohydrate-dependent manner to mesenchymal RPE cells and inhibit cellular processes like attachment and spreading. Yet knowledge about glycan-dependent interactors of Gal-1 and Gal-3 on RPE cells is very limited, although this is a prerequisite for unravelling the influence of galectins on distinct cellular processes in RPE cells. We identify here 131 Gal-3 and 15 Gal-1 interactors by galectin pull-down experiments combined with quantitative proteomics. They mainly play a role in multiple binding processes and are mostly membrane proteins. We focused on two novel identified interactors of Gal-1 and Gal-3 in the context of PVR: the low-density lipoprotein receptor LRP1 and the platelet-derived growth factor receptor beta PDGFRB. Addition of exogenous Gal-1 and Gal-3 induced crosslinking with LRP1/PDGFRB and Integrin-β1 (ITGB1) on the cell surface of human RPE cells and induced ERK/MAPK and Akt signaling. Treatment with Kifunensine, an inhibitor of complex-type-N-glycosylation, weakened the binding of Gal-1 and Gal-3 to these interactors and prevented lattice formation. In conclusion, the identified specific glycoprotein ligands shed light into the highly specific binding of galectins to dedifferentiated RPE cells and the resulting prevention of PVR-associated cellular events.

Summary: The neural crest (NC) gives rise to a multitude of fetal tissues, and its misregulation is implicated in congenital malformations. Here, we investigated molecular mechanisms pertaining to NC-related symptoms in Bohring-Opitz syndrome (BOS), a developmental disorder linked to mutations in the Polycomb group factor Additional sex combs-like 1 (ASXL1). Genetically edited human pluripotent stem cell lines that were differentiated to NC progenitors and then xenotransplanted into chicken embryos demonstrated an impairment of NC delamination and emigration. Molecular analysis showed that ASXL1 mutations correlated with reduced activation of the transcription factor ZIC1 and the NC gene regulatory network. These findings were supported by differentiation experiments using BOS patient-derived induced pluripotent stem cell lines. Expression of truncated ASXL1 isoforms (amino acids 1–900) recapitulated the NC phenotypes in vitro and in ovo, raising the possibility that truncated ASXL1 variants contribute to BOS pathology. Collectively, we expand the understanding of truncated ASXL1 in BOS and in the human NC. : In this study, Drukker and colleagues developed human pluripotent stem cell models for the rare congenital disorder Bohring-Opitz syndrome, which is caused by mutations in the Polycomb factor ASXL1. In these lines, they found impaired neural crest emigration in vitro and in vivo and link this phenotype to impaired activation of ZIC1. Keywords: ASXL1, neural crest, Bohring-Opitz syndrome, Polycomb, ZIC1

Proteasome dysfunction is emerging as a novel pathomechanism for the development of chronic obstructive pulmonary disease (COPD), a major leading cause of death in the world. Cigarette smoke, one of the main risk factors for COPD, impairs proteasome function in vitro and in vivo. In the present study, we dissected the molecular changes induced by cigarette smoke on the proteasome in lung epithelial cells and mouse lungs. 26S proteasome pull-down, MS interactome, and stoichiometry analyses indicated that 26S proteasome complexes become instable in cigarette smoke-treated lung epithelial cells as well as in lungs of mice after three day smoke exposure. The interactome of the 26S was clearly altered in mouse lungs upon smoke exposure but not in cells after 24 h of smoke exposure. Using native MS analysis of purified 20S proteasomes, we observed some destabilization of 20S complexes purified from cigarette smoke-exposed cells in the absence of any dominant and inhibitory modification of proteasomal proteins. Taken together, our results suggest that cigarette smoke induces minor but detectable changes in the stability of 20S and 26S proteasome complexes which might contribute to imbalanced proteostasis in a chronic setting as observed in chronic lung diseases associated with cigarette smoking.

Summary Superoxide dismutases (SODs) are differentially inhibited by peroxynitrite-mediated tyrosine nitration. Tyr63 is the main target responsible for inactivation of MnSOD1. This mechanism seems to be evolutionarily conserved in multicellular organisms., Despite the importance of superoxide dismutases (SODs) in the plant antioxidant defence system little is known about their regulation by post-translational modifications. Here, we investigated the in vitro effects of nitric oxide derivatives on the seven SOD isoforms of Arabidopsis thaliana. S-nitrosoglutathione, which causes S-nitrosylation of cysteine residues, did not influence SOD activities. By contrast, peroxynitrite inhibited the mitochondrial manganese SOD1 (MSD1), peroxisomal copper/zinc SOD3 (CSD3), and chloroplastic iron SOD3 (FSD3), but no other SODs. MSD1 was inhibited by up to 90% but CSD3 and FSD3 only by a maximum of 30%. Down-regulation of these SOD isoforms correlated with tyrosine (Tyr) nitration and both could be prevented by the peroxynitrite scavenger urate. Site-directed mutagenesis revealed that—amongst the 10 Tyr residues present in MSD1—Tyr63 was the main target responsible for nitration and inactivation of the enzyme. Tyr63 is located nearby the active centre at a distance of only 5.26 Å indicating that nitration could affect accessibility of the substrate binding pocket. The corresponding Tyr34 of human manganese SOD is also nitrated, suggesting that this might be an evolutionarily conserved mechanism for regulation of manganese SODs.

Nitric oxide (NO) has emerged as a signaling molecule in plants being involved in diverse physiological processes like germination, root growth, stomata closing and response to biotic and abiotic stress. S-nitrosoglutathione (GSNO) as a biological NO donor has a very important function in NO signaling since it can transfer its NO moiety to other proteins (trans-nitrosylation). Such trans-nitrosylation reactions are equilibrium reactions and depend on GSNO level. The breakdown of GSNO and thus the level of S-nitrosylated proteins are regulated by GSNO-reductase (GSNOR). In this way, this enzyme controls S-nitrosothiol levels and regulates NO signaling. Here we report that Arabidopsis thaliana GSNOR activity is reversibly inhibited by H2O2 in vitro and by paraquat-induced oxidative stress in vivo. Light scattering analyses of reduced and oxidized recombinant GSNOR demonstrated that GSNOR proteins form dimers under both reducing and oxidizing conditions. Moreover, mass spectrometric analyses revealed that H2O2-treatment increased the amount of oxidative modifications on Zn2+-coordinating Cys47 and Cys177. Inhibition of GSNOR results in enhanced levels of S-nitrosothiols followed by accumulation of glutathione. Moreover, transcript levels of redox-regulated genes and activities of glutathione-dependent enzymes are increased in gsnor-ko plants, which may contribute to the enhanced resistance against oxidative stress. In sum, our results demonstrate that reactive oxygen species (ROS)-dependent inhibition of GSNOR is playing an important role in activation of anti-oxidative mechanisms to damping oxidative damage and imply a direct crosstalk between ROS- and NO-signaling.

The seven-transmembrane receptor Smoothened (Smo) activates all Hedgehog (Hh) signaling by translocation into the primary cilia (PC), but how this is regulated is not well understood. Here we show that Pitchfork (Pifo) and the G protein-coupled receptor associated sorting protein 2 (Gprasp2) are essential components of an Hh induced ciliary targeting complex able to regulate Smo translocation to the PC. Depletion of Pifo or Gprasp2 leads to failure of Smo translocation to the PC and lack of Hh target gene activation. Together, our results identify a novel protein complex that is regulated by Hh signaling and required for Smo ciliary trafficking and Hh pathway activation.

The IκB kinase (IKK) complex acts as the gatekeeper of canonical NF-κB signaling, thereby regulating immunity, inflammation and cancer. It consists of the catalytic subunits IKKα and IKKβ and the regulatory subunit NEMO/IKKγ. Here, we show that the ubiquitin binding domain (UBAN) in NEMO is essential for IKK/NF-κB activation in response to TNFα, but not IL-1β stimulation. By screening a natural compound library we identified an anthraquinone derivative that acts as an inhibitor of NEMO-ubiquitin binding (iNUB). Using biochemical and NMR experiments we demonstrate that iNUB binds to NEMOUBAN and competes for interaction with methionine-1-linked linear ubiquitin chains. iNUB inhibited NF-κB activation upon UBAN-dependent TNFα and TCR/CD28, but not UBAN-independent IL-1β stimulation. Moreover, iNUB was selectively killing lymphoma cells that are addicted to chronic B-cell receptor triggered IKK/NF-κB activation. Thus, iNUB disrupts the NEMO-ubiquitin protein-protein interaction interface and thereby inhibits physiological and pathological NF-κB signaling.

Epstein-Barr nuclear antigen 1 (EBNA1) is the essential Epstein-Barr virus (EBV) protein at the interface between the EBV genome and the host chromatin. It is EBNA1's task to guarantee replication and segregation of the multicopy closed circular viral genome in infected cells. While EBNA1's functions are relatively well understood, little is known about the molecular mechanisms of EBNA1 mediating chromatin tethering and DNA replication. To characterize those, purified EBNA1 would be a very useful tool in many different biochemical assays. For long, it was not possible to overexpress sufficient quantities of EBNA1 in Escherichia coli (E. coli) due to its rare codon usage, especially in the N-terminal part of the protein. Recently, some groups succeeded in purifying EBNA1 from bacteria using advanced inducible E. coli cells [1-3]. However, all purification procedures ended in a His-tagged version of EBNA1, which might influence EBNA1's function in biological assays. Therefore, we inserted a tobacco etch virus (TEV)-cleavage site between the N-terminal His-tag and the following open reading frame of EBNA1. Using sequential Ni-NTA and gel filtration columns and TEV protease-mediated cleavage upon autoinduction, we were able to purify functional EBNA1 protein featuring just a single additional, artificial N-terminal glycine residue. Following our simple and fast purification scheme we were able to synthesize 2mg of highly pure EBNA1 protein per liter culture.

In prokaryotes, operon encoded proteins often form protein–protein complexes. Here, we show that the native structure of operons can be used to efficiently overexpress protein complexes. This study focuses on operons from mycobacteria and the use of Mycobacterium smegmatis as an expression host. We demonstrate robust and correct stoichiometric expression of dimers to higher oligomers. The expression efficacy was found to be largely independent of the intergenic distances. The strategy was successfully extended to express mycobacterial protein complexes in Escherichia coli, showing that the operon structure of gram-positive bacteria is also functional in gram-negative bacteria. The presented strategy could become a general tool for the expression of large quantities of pure prokaryotic protein complexes for biochemical and structural studies.Structured summaryMINT-7542207: ESAT-6 (uniprotkb:Q50206) and CFP-10 (uniprotkb:O33084) bind (MI:0407) by blue native page (MI:0276)MINT-7542534: ESAT-6 (uniprotkb:P0A564) and CFP-10 (uniprotkb:P0A566) bind (MI:0407) by X-ray crystallography (MI:0114)MINT-7542187: CFP-10 (uniprotkb:P0A566) and ESAT-6 (uniprotkb:P0A564) bind (MI:0407) by blue native page (MI:0276)MINT-7542652: CFP-10 (uniprotkb:P0A566) and ESAT-6 (uniprotkb:P0A564) bind (MI:0407) by molecular sieving (MI:0071)MINT-7542474, MINT-7542303: CFP-10 (uniprotkb:P0A566) physically interacts (MI:0915) with ESAT-6 (uniprotkb:P0A564) by pull down (MI:0096)

Two C-terminally truncated variants of the small subunit of Mycobacterium tuberculosis isopropylmalate isomerase (Rv2987c; LeuD), LeuD_1-156 and LeuD_1-168, have been cloned, heterologously expressed in Escherichia coli, purified using standard chromatographic techniques and crystallized. The crystals of LeuD_1-156 belonged to the hexagonal system (space group P6(1)22 or P6(5)22) with up to four subunits in the asymmetric unit, whereas the crystals of LeuD_1-168 belonged to the monoclinic system (space group P2(1)) with two subunits in the asymmetric unit. Both crystals diffracted X-rays to beyond 2.0 A resolution and were suitable for further crystallographic analysis.

The vacuolar (H+)-ATPase (or V-ATPase) is a membrane protein complex that is structurally related to F1 and F0 ATP synthases. The V-ATPase is composed of an integral domain (V0) and a peripheral domain (V1) connected by a central stalk and up to three peripheral stalks. The number of peripheral stalks and the proteins that comprise them remain controversial. We have expressed subunits E and G in Escherichia coli as maltose binding protein fusion proteins and detected a specific interaction between these two subunits. This interaction was specific for subunits E and G and was confirmed by co-expression of the subunits from a bicistronic vector. The EG complex was characterized using size exclusion chromatography, cross-linking with short length chemical cross-linkers, circular dichroism spectroscopy, and electron microscopy. The results indicate a tight interaction between subunits E and G and revealed interacting helices in the EG complex with a length of about 220 A. We propose that the V-ATPase EG complex forms one of the peripheral stators similar to the one formed by the two copies of subunit b in F-ATPase.

Telethonin interacts specifically with the two Z-disk IG-like domains (Z1Z2) at the N terminus of titin, the largest presently known protein. Analytical ultracentrifugation and synchrotron radiation x-ray scattering were employed to study the solution structures of Z1Z2 and its complexes with telethonin, and low resolution models were constructed ab initio from the scattering data. A seven residues-long polyhistidine tag was localized at the tip of the Z1 domain by comparison of independent models of native and His-tagged versions of Z1Z2. The stoichiometry and shape of the complex between the telethonin construct lacking the C terminus and Z1Z2 indicate antiparallel association of two Z1Z2 molecules with telethonin acting as a central linker. The complex of full-length telethonin with Z1Z2 appears to also have a 1:2 stoichiometry at concentrations below 1 mg/ml but dimerizes at higher concentrations. These results suggest a possible role of telethonin in linking titin filaments at the Z-disk periphery.

In human medicine, procalcitonin (PCT) is a very common and well-established biomarker for sepsis. Even though sepsis is also a leading cause of death in foals and adult horses, up to now, no data about the role of equine PCT in septic horses has been available. Based on monoclonal antibodies targeted against human PCT, we report here the development of a sandwich ELISA for the quantification of equine PCT in equine plasma samples. The ELISA was characterized for intra- and interassay variance and a working range from 25 to 1,000 ng mL(-1) was defined as within this range; both intra- and interassay variances were below 15 %. The target recovery ranged between 73 and 106 %. The ELISA was used to determine the equine PCT concentration in 24 healthy and 5 septic horses to show the potential for clinical evaluation of equine PCT. Significantly different (P = 0.0006) mean equine PCT concentrations were found for the healthy control group and the sepsis group (47 and 8,450 ng mL(-1)).

Quinohaemoprotein alcohol dehydrogenases, QH-ADHs, show appreciable enantioselectivity in the oxidation of certain chiral primary and secondary alcohols. We determined the effect of temperature on the enantiomeric ratio of structurally related enzymes, QH-ADH, Type I, from Comamonas testosteroni and QH-ADH, Type II, from Acetobacrer pasteurianus, in the kinetic resolution of racemic 2, 2-dimethyl-4- (hydroxymethyl)-1, 3-dioxolane (solketal) and 2-butanol, respectively. It appears that entropic contributions to the stabilization of the enantioselectivity-determining transition state play an important role in the enantiopreference. Consequences of these findings for the formulation of models that can be used to summarise the observed enan-tioselectivities are discussed.

Proliferative vitreoretinopathy (PVR) is a blinding disease frequently occurring after retinal detachment surgery. Adhesion, migration and matrix remodeling of dedifferentiated retinal pigment epithelial (RPE) cells characterize the onset of the disease. Treatment options are still restrained and identification of factors responsible for the abnormal behavior of the RPE cells will facilitate the development of novel therapeutics. Galectin-3, a carbohydrate-binding protein, was previously found to inhibit attachment and spreading of retinal pigment epithelial cells, and thus bares the potential to counteract PVR-associated cellular events. However, the identities of the corresponding cell surface glycoprotein receptor proteins on RPE cells are not known. Here we characterize RPE-specific Gal-3 containing glycoprotein complexes using a proteomic approach. Integrin-β1, integrin-α3 and CD147/EMMPRIN, a transmembrane glycoprotein implicated in regulating matrix metalloproteinase induction, were identified as potential Gal-3 interactors on RPE cell surfaces. In reciprocal immunoprecipitation experiments we confirmed that Gal-3 associated with CD147 and integrin-β1, but not with integrin-α3. Additionally, association of Gal-3 with CD147 and integrin-β1 was observed in co-localization analyses, while integrin-α3 only partially co-localized with Gal-3. Blocking of CD147 and integrin-β1 on RPE cell surfaces inhibited binding of Gal-3, whereas blocking of integrin-α3 failed to do so, suggesting that integrin-α3 is rather an indirect interactor. Importantly, Gal-3 binding promoted pronounced clustering and co-localization of CD147 and integrin-β1, with only partial association of integrin-α3. Finally, we show that RPE derived CD147 and integrin-β1, but not integrin-α3, carry predominantly β-1,6-N-actyl-D-glucosamine-branched glycans, which are high-affinity ligands for Gal-3. We conclude from these data that extracellular Gal-3 triggers clustering of CD147 and integrin-β1 via interaction with β1,6-branched N-glycans on RPE cells and hypothesize that Gal-3 acts as a positive regulator for CD147/integrin-β1 clustering and therefore modifies RPE cell behavior contributing to the pathogenesis of PVR. Further investigations at this pathway may aid in the development of specific therapies for PVR.

Epithelial-to-mesenchymal transition (EMT) of retinal pigment epithelial cells is a crucial event in the onset of proliferative vitreoretinopathy (PVR), the most common reason for treatment failure in retinal detachment surgery. We studied alterations in the cell surface glycan expression profile upon EMT of RPE cells and focused on its relevance for the interaction with galectin-3 (Gal-3), a carbohydrate binding protein, which can inhibit attachment and spreading of human RPE cells in a dose- and carbohydrate-dependent manner, and thus bares the potential to counteract PVR-associated cellular events. Lectin blot analysis revealed that EMT of RPE cells in vitro confers a glycomic shift towards an abundance of Thomsen-Friedenreich antigen, poly-N-acetyllactosamine chains, and complex-type branched N-glycans. Using inhibitors of glycosylation we found that both, binding of Gal-3 to the RPE cell surface and Gal-3-mediated inhibition of RPE attachment and spreading, strongly depend on the interaction of Gal-3 with tri- or tetra-antennary complex type N-glycans and sialylation of glycans but not on complex-type O-glycans. Importantly, we found that β1,6 N-acetylglucosaminyltransferase V (Mgat5), the key enzyme catalyzing the synthesis of tetra- or tri-antennary complex type N-glycans, is increased upon EMT of RPE cells. Silencing of Mgat5 by siRNA and CRISPR-Cas9 genome editing resulted in reduced Gal-3 binding. We conclude from these data that binding of recombinant Gal-3 to the RPE cell surface and inhibitory effects on RPE attachment and spreading largely dependent on interaction with Mgat5 modified N-glycans, which are more abundant on dedifferentiated than on the healthy, native RPE cells. Based on these findings we hypothesize that EMT of RPE cells in vitro confers glycomic changes, which account for high affinity binding of recombinant Gal-3, particularly to the cell surface of myofibroblastic RPE. From a future perspective recombinant Gal-3 may disclose a therapeutic option allowing for selectively targeting RPE cells with pathogenic relevance for development of PVR.

The stability and homogeneity of a protein sample is strongly influenced by the composition of the buffer that the protein is in. A quick and easy approach to identify a buffer composition which increases the stability and possibly the conformational homogeneity of a protein sample is the fluorescence-based thermal-shift assay (Thermofluor). Here, a novel 96-condition screen for Thermofluor experiments is presented which consists of buffer and additive parts. The buffer screen comprises 23 different buffers and the additive screen includes small-molecule additives such as salts and nucleotide analogues. The utilization of small-molecule components which increase the thermal stability of a protein sample frequently results in a protein preparation of higher quality and quantity and ultimately also increases the chances of the protein crystallizing.

Hydroxyacid dehydrogenases, responsible for the stereospecific conversion of 2-keto acids to 2-hydroxyacids in lactic acid producing bacteria, have a range of biotechnology applications including antibiotic synthesis, flavor development in dairy products and the production of valuable synthons. The genome of Lactobacillus delbrueckii ssp. bulgaricus, a member of the heterogeneous group of lactic acid bacteria, encodes multiple hydroxyacid dehydrogenases whose structural and functional properties remain poorly characterized. Here, we report the apo and coenzyme NAD⁺ complexed crystal structures of the L. bulgaricusD-isomer specific 2-hydroxyacid dehydrogenase, D2-HDH. Comparison with closely related members of the NAD-dependent dehydrogenase family reveals that whilst the D2-HDH core fold is structurally conserved, the substrate-binding site has a number of non-canonical features that may influence substrate selection and thus dictate the physiological function of the enzyme.

Acetobacter pasteurianus oxidizes glycidol with high activity, comparable to the oxidation of ethanol. The organism has a preference for the S -enantiomer, and the kinetic resolution process obeys a simple relationship, indicating an enantiomeric ratio (E) of 19. The compound is converted into glycidic acid, although a transient accumulation of glycidaldehyde occurs initially. Determination of other parameters revealed a temperature optimum of 50°C, long-term stability (cells in the resting state), and a pH optimum compatible with the chemical stability of glycidol. However, it was also noted that respiration rates decrease at concentrations of glycidol above 1 m . This is most likely caused by substrate inhibition of the glycidol-oxidizing enzyme, the quinohemoprotein ethanol dehydrogenase. Comparison with existing methods for enantiomerically pure glycidol production indicated a number of attractive points for the method described here, although definitive evaluation must await further studies on the long-term stability under process conditions, reusability of the cells, and the mechanism of glycidol inhibition.

Several purified or commercially available alcohol oxidoreductases of different kinds were tested for their ability to convert the racemic, glycerol-based C3-synthons glycidol (2,3-epoxy-1-propanol) and solketal (2,2-dimethyl-4-(hydroxymethyl)-1,3-dioxolane), with adequate activity and enantioselectivity. Quinohaemo-protein alcohol dehydrogenases (enzymes containing haem c as well as pyrroloquinoline quinone (PQQ) as cofactors) appeared to be excellently suited for such use. The bacteria from which the enzymes were purified had the same enantiomer preference and had an efficient respiratory chain for reoxidation of these dehydrogenases. In some cases, however, whole cells gave a lower enantiomer ratio (E) than the pure enzyme. NAD-dependent alcohol dehydrogenases also are present in these bacteria, but their presence may not explain the lower ratio because they oxidized the C3-synthons little if at all. It seems, therefore, that different kinetic mechanisms are responsible for the discrepancy between the ...

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, to obtain milligrams of soluble proteins is still challenging since many proteins are expressed in an insoluble form without optimization. Therefore when working with tens of proteins or protein domains it is recommended that high-throughput expression screening at a small scale (1-4ml of culture) is carried out to identify the optimal conditions for soluble protein production. Once determined, these culture conditions can be applied at a large scale to produce sufficient protein for structural or functional studies. We describe a procedure that has enabled the systematic screening of culture conditions or fusion-tags on hundreds of cultures per week. The analysis of the optimal conditions for the soluble production of these proteins helped us to design a simple and efficient protocol for soluble protein expression screening. This protocol has since been used on hundreds of proteins and is illustrated with the genome wide scale production of proteins containing the DNA binding domains of Ciona intestinalis.

Accurate and reliable methods are presented for the determination of enantiomeric excess values of glycidol (2,3-epoxy-1-propanol), glycidyl esters, solketal [2,2-dimethyl-4-(hydroxymethyl)-1,3-dioxolane], homologous 1,3-dioxolane alcohols and substituted primary propanols in biological samples. One method consists of derivatization of the samples with 2,3,4,6-tetra-O-acetyl-β-glucopyranosyl isothiocyanate, followed by separation of the resulting diastereomers on a non-chiral reversed-phase (C18) HPLC column. The second method uses direct injection of (aqueous) samples on to capillary GC columns coated with chiral stationary phases (2,3,6-tri-O-methyl-β- or a 2,3,6-tri-O-trifluoroacetyl-λ-cyclodextrin). The advantages and disadvantages of both methods are discussed.

Bacteria contain alcohol dehydrogenases (ADH's) which either occur in the cytosol and which interact with the NAD(P) pool or are directly coupled to the respiratory chain and can be assayed i n v i t ro with artificial electron acceptors, i.e. by decoloration o f dyes. During ihe past ten years, several of these dye-linked ADH's have been characterized and shown to be quinoproteins, i.e. enzymes with a quinone cofactor (in most cases this is pyrroloquinoline quinone, PQQ). These enzymes were investigated here for kinetic resolution of the C -synthons solketal ( 2,2-dimethyl-4-( hydroxymethyl) -1,J-dicxslane) and glycidol (2,3-epoxy-l-propanol). It appeared that quinohaemoprotein ADH's are excellently suited for this 4ince this type of enzymes has adequate substrate diversity, catalytic activity and resolving power, i n v i t r o as well as i n vivo.

The absence of the leucine biosynthesis pathway in humans makes the enzymes of this pathway in pathogenic bacteria such as Mycobacterium tuberculosis potential candidates for developing novel antibacterial drugs. One of these enzymes is isopropylmalate isomerase (IPMI). IPMI exists as a complex of two subunits: the large (LeuC) and the small (LeuD) subunit. The functional LeuCD complex catalyzes the stereospecific conversion reaction of α-isopropylmalate to β-isopropylmalate. Three C-terminally truncated variants of LeuD have been analyzed by X-ray crystallography to resolutions of 2.0 A (LeuD_1–156), 1.2 A (LeuD_1–168), and 2.5 A (LeuD_1–186), respectively. The two most flexible parts of the structure are the regions of residues 30–37, the substrate discriminating loop, and of residues 70–74, the substrate binding loop. The three determined structures were also compared with the structures of other bacterial LeuDs. This comparison suggests the presence of two LeuD subfamilies. A model for the structure of the inactive enzyme complex has been obtained from solution X-ray scattering experiments. The crystal structure of LeuD was shown to be compatible with the solution X-ray scattering data from the small subunit. In contrast, the solution scattering results suggest that the large subunit LeuC and the LeuCD complex have overall shapes, which are radically different from the ones observed in the crystals of the functional homolog mitochondrial aconitase. Proteins 2010. © 2010 Wiley-Liss, Inc.

This study focuses on two representatives of experimentally uncharacterized haloalkane dehalogenases from the subfamily HLD-III. We report biochemical characterization of the expression products of haloalkane dehalogenase genes drbA from Rhodopirellula baltica SH1 and dmbC from Mycobacterium bovis 5033/66. The DrbA and DmbC enzymes show highly oligomeric structures and very low activities with typical substrates of haloalkane dehalogenases.

Summary: Cytoplasmic membranes of Acinetobacter calcoaceticus cells, cultured under acetate-limiting conditions, contain cytochrome b554 and a cytochrome o-containing oxidase. Both have been purified to homogeneity and characterized. Cytochrome b554 is a monomeric protein (molecular mass 70 kDa) with a midpoint potential of +100 mV (in the membrane it has most probably a value of +50 mV). The cytochrome o-containing oxidase seems to be an αβγδ protein since the molecular mass of the native protein was estimated to be 150 kDa and the molecular masses of the subunits, determined by SDS-polyacrylamide-gel electrophoresis, are 55, 41, 33 and 17 kDa. Redox spectroscopy of the purified complex shows the presence of a cytochrome b555/563 having a midpoint potential of approximately +160 mV (both in purified form and in the membrane). CO difference spectroscopy shows the presence of a second b-type cytochrome, viz. cytochrome, o. Cytoplasmic membranes of A. calcoaceticus cells grown under oxygen-limiting conditions also show the presence of the cytochrome b554 and the cytochrome o-containing oxidase. In addition a protein has been solubilized with the spectral characteristics of a cytochrome d-containing oxidase. The cytochrome o- and d-containing oxidases appear to be similar to those reported for Escherichia coli and Proteus mirabilis. On the other hand, cytochrome b554 has no counterpart in these organisms since the cytochrome b556 described for E. coli is quite dissimilar.

The resuscitation-promoting factor RpfB, the most complex of the five resuscitation-promoting factors produced by M. tuberculosis, is devoted to bacterial reactivation from the dormant state. RpfB consists of 362 residues predicted to form five domains. An RpfB fragment containing the protein catalytic domain and a G5 domain has been successfully crystallized using vapour-diffusion methods. This is the first crystallographic study of a resuscitation-promoting factor. Crystals of this protein belong to space group I422, with unit-cell parameters a = 97.63, b = 97.63, c = 114.87 A. Diffraction data have also been collected from a selenomethionine derivative at 2.9 A resolution. Model building using the phases derived from the multiwavelength anomalous dispersion experiment is in progress.

Lipoic acid is essential for the activation of a number of protein complexes involved in key metabolic processes. Growth of Mycobacterium tuberculosis relies on a pathway in which the lipoate attachment group is synthesized from an endogenously produced octanoic acid moiety. In patients with multiple-drug-resistant M. tuberculosis , expression of one gene from this pathway, lipB , encoding for octanoyl-[acyl carrier protein]-protein acyltransferase is considerably up-regulated, thus making it a potential target in the search for novel antiinfectives against tuberculosis. Here we present the crystal structure of the M. tuberculosis LipB protein at atomic resolution, showing an unexpected thioether-linked active-site complex with decanoic acid. We provide evidence that the transferase functions as a cysteine/lysine dyad acyltransferase, in which two invariant residues (Lys-142 and Cys-176) are likely to function as acid/base catalysts. Analysis by MS reveals that the LipB catalytic reaction proceeds by means of an internal thioesteracyl intermediate. Structural comparison of LipB with lipoate protein ligase A indicates that, despite conserved structural and sequence active-site features in the two enzymes, 4′-phosphopantetheine-bound octanoic acid recognition is a specific property of LipB.

3-Phosphoglycerate kinase (PGK) is the first ATP-producing enzyme in glycolysis: ADP + 1,3-bisphosphoglycerate (bPG)--ATP + 3-phosphoglycerate (PG). Whereas extensive studies have been carried out on its structure, there is less information about its reaction pathway, which is usually studied in the reverse direction because of the instability of bPG. We studied the transients of the PGK reaction by chemical sampling in a rapid quench flow apparatus, using [gamma-(32)P]ATP, in 30% methanol at 4 degrees C to decrease k(cat). There were two types of experiment, both at low PG concentrations to prevent bPG release. In the first, reaction mixtures were quenched in acid at different times (from 4 ms) and the bPG concentrations were determined. This type gave information about the ATP binding and phospho-transfer steps. In the second, PGK reaction mixtures at equilibrium were perturbed by the injection of ADP, the new mixtures aged for different times and quenched in acid, and the bPG concentrations were determined. This gave information about the kinetics of the binding of ADP to a PGK intermediate. The data from the two types of experiments were fitted to simple schemes and then treated together by a global fitting procedure using a five-step pathway, deduced from previous structural studies. Under our conditions, it appears that (1) a binary PGK.bPG complex is an important intermediate on the reaction pathway, i.e., that ADP is released before bPG, (2) ADP binds to a "closed" conformation in the PGK.bPG complex, and (3) the PGK reaction can be studied in the physiologically important direction without having to handle bPG.

We developed a protocol for the fast purification of small proteins and peptides using heat incubation as the first purification step. The proteins are expressed from a new bacterial expression vector (pETM-90) fused to the C-terminus of thermostable Ftr from Methanopyrus kandleri. The vector further contains a 6xHis-tag to allow immobilised metal ion affinity purification and a TEV protease cleavage site to enable the removal of the His-tag and fusion partner. Heat incubation induces the specific denaturation and precipitation of the Escherichia coli proteins but not of the thermostable fusion protein. Using the fusion construct and the heat incubation protocol a number of fusion proteins were purified to near homogeneity. The thermostability was ensured when Ftr had a molecular weight higher than twice the target protein. The obtained purification yields were similar and, in some cases, even higher than the ones obtained by affinity purification with the same Ftr-fusion proteins or the same target proteins fused to other often used partners such as NusA, GST, or DsbA. The protocol does not depend on a specific thermostable protein as was shown by the exchange of Ftr for M. kandleri Mtd. Purification by heat incubation is a fast and inexpensive alternative to chromatographic techniques, particularly suitable for the production of antigenic sequences for which the loss of native structure is not detrimental. We proved that it can be easily automated.

Phosphopantetheine adenylyltransferase (PPAT) is an essential enzyme in bacteria that catalyses a rate-limiting step in coenzyme A (CoA) biosynthesis, by transferring an adenylyl group from ATP to 4'-phosphopantetheine, yielding dephospho-CoA (dPCoA). Each phosphopantetheine adenylyltransferase (PPAT) subunit displays a dinucleotide-binding fold that is structurally similar to that in class I aminoacyl-tRNA synthetases. Superposition of bound adenylyl moieties from dPCoA in PPAT and ATP in aminoacyl-tRNA synthetases suggests nucleophilic attack by the 4'-phosphopantetheine on the alpha-phosphate of ATP. The proposed catalytic mechanism implicates transition state stabilization by PPAT without involving functional groups of the enzyme in a chemical sense in the reaction. The crystal structure of the enzyme from Escherichia coli in complex with dPCoA shows that binding at one site causes a vice-like movement of active site residues lining the active site surface. The mode of enzyme product formation is highly concerted, with only one trimer of the PPAT hexamer showing evidence of dPCoA binding. The homologous active site attachment of ATP and the structural distribution of predicted sequence-binding motifs in PPAT classify the enzyme as belonging to the nucleotidyltransferase superfamily.

Pyrroloquinoline-quinone(PQQ)-free quinohaemoprotein ethanol dehydrogenase (QH-EDH) apoenzyme was isolated from ethanol-grown Comamonas testosteroni. The purified apoenzyme, showing a single band of 71 kDa on native gel electrophoresis, could be only partially converted into active holoenzyme by addition of PQQ in the presence of calcium ions. In addition to a band with a molecular mass of 71 kDa, additional bands of 51 kDa and 25 kDa were observed with SDS/PAGE. Analysis of the N-terminal sequences of the bands and comparison with the DNA sequence of the gene, suggested that the latter two originate from the former one, due to scission occurring at a specific site between two vicinal residues in the protein chain. The extent of scission appeared to increase during growth of the organism. After addition of PQQ to apoenzyme, holoenzyme and nicked, inactive enzyme could be separated. Holoenzyme prepared in this way was found to contain equimolar amounts of PQQ, Ca2+ and covalently bound haem. EPR spectra of fully oxidized apo-QH-EDH and holo-QH-EDH showed g values typical for low-spin haem c proteins. In partially oxidized holo-QH-EDH an organic radical signal attributed to the semiquinone form of PQQ was observed. Binding of PQQ leads to conformational changes, as reflected by changes of spectral and chromatographic properties. Reconstitution of apoenzyme with PQQ analogues resulted in a decreased activity and enantioselectivity for the oxidation of chiral alcohols. Compared with PQQ, analogues with a large substituent had a lower affinity for the apoenzyme. Results with other analogues indicated that possession of the o-quinone/o-quinol moiety is not essential for binding but it is for activity.

Some strains of Acinetobacter calcoaceticus convert glucose with oxygen into gluconolactone and further to gluconic acid as end product. This incomplete oxidation can provide the cell with useful energy (De Bont et al., 1984; Van Schie et al., 1985, 1987; Kitagawa et al., 1986; Muller and Babel, 1986).

International audience; The interaction between the Shiga toxin B-subunit (STxB) and its globotriaosylceramide receptor (Gb3) has a high potential for being exploited for targeted cancer therapy. The primary goal of this study was to evaluate the capacity of STxB to carry small molecules and proteins as cargo into cells. For this purpose, an assay was designed to provide real-time information about the StxB-Gb3 interaction as well as the dynamics and mechanism of the internalization process. The assay revealed the ability to distinguish the process of binding to the cell surface from internalization and presented the importance of receptor and STxB clustering for internalization. The overall setup demonstrated that the binding mechanism is complex, and the concept of affinity is difficult to apply. Hence, time-resolved methods, providing detailed information about the interaction of STxB with cells, are critical for the optimization of intracellular delivery.