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The scientific literature contains a tremendous amount of kinetic data describing the dynamic behaviour of biochemical reactions over time. These data are needed for computational modelling to create models of biochemical reaction networks and to obtain a better understanding of the processes in living cells. To extract the knowledge from the literature, biocurators are required to understand a paper and interpret the data. For modellers, as well as experimentalists, this process is very time consuming because the information is distributed across the publication and, in most cases, is insufficiently structured and often described without standard terminology. In recent years, biological databases for different data types have been developed. The advantages of these databases lie in their unified structure, searchability and the potential for augmented analysis by software, which supports the modelling process. We have developed the SABIO- RK database for biochemical reaction kinetics. In the present review, we describe the challenges for database developers and curators, beginning with an analysis of relevant publications up to the export of database information in a standardized format. The aim of the present review is to draw the experimentalist's attention to the problem (from a data integration point of view) of incompletely and imprecisely written publications. We describe how to lower the barrier to curators and improve this situation. At the same time, we are aware that curating experimental data takes time. There is a community concerned with making the task of publishing data with the proper structure and annotation to ontologies much easier. In this respect, we highlight some useful initiatives and tools. [ABSTRACT FROM AUTHOR]

This is a series of papers (three reviews and five regular papers) focused on Mitochondria from authors contributing to two FEBS courses on Mitochondria held in 2012. These papers focus on: (i) mitochondrial biogenesis (regulation by phosphorylation, import of SOD1 and Mia40, mitophagy and tRNA variants) or (ii) mitochondrial pathology (ischaemia, obesity and Parkinson's disease). [ABSTRACT FROM AUTHOR]

Two divergent modelling methodologies have been adopted to increase our understanding of metabolism and its regulation. Constraint-based modelling highlights the optimal path through a stoichiometric network within certain physicochemical constraints. Such an approach requires minimal biological data to make quantitative inferences about network behaviour; however, constraint-based modelling is unable to give an insight into cellular substrate concentrations. In contrast, kinetic modelling aims to characterize fully the mechanics of each enzymatic reaction. This approach suffers because parameterizing mechanistic models is both costly and time-consuming. In this paper, we outline a method for developing a kinetic model for a metabolic network, based solely on the knowledge of reaction stoichiometries. Fluxes through the system, estimated by flux balance analysis, are allowed to vary dynamically according to linlog kinetics. Elasticities are estimated from stoichiometric considerations. When compared to a popular branched model of yeast glycolysis, we observe an excellent agreement between the real and approximate models, despite the absence of (and indeed the requirement for) experimental data for kinetic constants. Moreover, using this particular methodology affords us analytical forms for steady state determination, stability analyses and studies of dynamical behaviour. [ABSTRACT FROM AUTHOR]

Presents an abstracts of research papers related to biochemistry. "Hemolysis of Human Red Blood Cells by Riboflavin-Cu(II) System:Enhancement by Azide," by I. Ali and I. Naseem; "Effects of Nat5 Aminotermional Acetyltransferase Inhibition on HeLa Cells," by A. Ametzazurra and E. Larrea; "Plants As Alternative Systems for Recombinant Protein Production:Medicago Truncatula As an Emerging Production System," by R. Abranches and S. Marcel.

Peter Macheroux is Professor of Biochemistry and Head of the Institute of Biochemistry at Graz University of Technology in Austria. Peter's research spans a diverse selection of topics, and his work has contributed significantly towards advancing our understanding of bacterial enzymology, plant physiology and the molecular pathways that underlie human pathophysiology. Among Peter's many scientific achievements, he has led the team that recognised DPP3 as a biomarker for cardiovascular diseases, with the subsequent therapeutic implications of the development of DPP3 inhibitors. In this interview‐based article, Peter provides an overview of his research focus and goals, recalls some of his great scientific breakthroughs and describes what the key current challenges in his research field are. [ABSTRACT FROM AUTHOR]

Coenzyme Q (CoQ, ubiquinone) is the electron‐carrying lipid in the mitochondrial electron transport system (ETS). In mammals, it serves as the electron acceptor for nine mitochondrial inner membrane dehydrogenases. These include the NADH dehydrogenase (complex I, CI) and succinate dehydrogenase (complex II, CII) but also several others that are often omitted in the context of respiratory enzymes: dihydroorotate dehydrogenase, choline dehydrogenase, electron‐transferring flavoprotein dehydrogenase, mitochondrial glycerol‐3‐phosphate dehydrogenase, proline dehydrogenases 1 and 2, and sulfide:quinone oxidoreductase. The metabolic pathways these enzymes are involved in range from amino acid and fatty acid oxidation to nucleotide biosynthesis, methylation, and hydrogen sulfide detoxification, among many others. The CoQ‐linked metabolism depends on CoQ reoxidation by the mitochondrial complex III (cytochrome bc1 complex, CIII). However, the literature is surprisingly limited as for the role of the CoQ‐linked metabolism in the pathogenesis of human diseases of oxidative phosphorylation (OXPHOS), in which the CoQ homeostasis is directly or indirectly affected. In this review, we give an introduction to CIII function, and an overview of the pathological consequences of CIII dysfunction in humans and mice and of the CoQ‐dependent metabolic processes potentially affected in these pathological states. Finally, we discuss some experimental tools to dissect the various aspects of compromised CoQ oxidation. [ABSTRACT FROM AUTHOR]

Provides information on the "FEBS Journal," the new title for the European Journal of Biochemistry (EJB). Reflection of new broader aims and scope in the areas of bioinformatics, genomics and proteomics, and molecular cell biology; Retention of benefits to authors that have long been associated with EJB; Availability of the journal either free or at substantially reduced cost in developing countries.

Presents an abstracts of research papers related to biochemistry. "Cocrystals of Photosystem I With Its Soluble Natural Electron Acceptor Ferredoxin at 4 A Resolution," by R. Fromme, and H. Yu; "Breaking Biological Symmetry in Membrane Proteins:How PsaC Evolved to Orient Asymmetrically on the Photosystem I Core," by J. H. Golbeck and M. Antonkine; "The Primary Light Energy Conversion in Bacterial Reaction Centers:Coupling Electron Transfer and Nuclear Motions," by V. A. Shuvalov.

Presents an abstracts of research papers related to biochemistry. "Mechanism of the Multiprotein RNA Polymerase II," by P. Cramer; "Regulation of Transcription by RNA Polymerase II by the Multi-functional Ccr4-Not Complex," by H. M. Timmers and K. W. Mulder; "Functionally Distinct ADA2-containing Histone Acetylase Complexes in Drosophila," by I. M. Boros and T. Pankotai.

Presents an abstracts of research papers related to biochemistry. "Reference Measurement Procedures and External Quality Assessment Schemes," by H. Reinauer and . Kaiser; "International Standardization of Protein Measurement," by C. C. Heuck; "The HUPO Brain Proteome Project," by H. E. Meyer.

Presents an abstracts of research papers related to biochemistry. "Protein interactions in cell signaling and polarity," by T. Pawson; "Regulation and Function of the Proto-oncogene Protein Kinase B/Akt," by B. A. Hemmings; "Casein Kinase 2 Interacts With and Regulates Subcellular Localization of S6K," by G. Panasyuk, V. Filonenko and I. Gout; "Knockdown of BChE by Sequence-specific siRNAs Changes PKCb- and JNK Expression," by E. Bodur and P. G. Layer.

Presents an abstracts of research papers related to biochemistry. "Aquaporin Water Channels:From Atomic Structure to Clinical Medicine," by P. Agre; "The Cardiac Glycoside Binding Site of the Na,K-ATPase Plays a Role in Blood Pressure Regulation," by J. Lingrel, I. Dostanic and J, Neumann; "TRP Channels, Mediators of Sensory Signaling," by C. Montell.

Presents an abstracts of research papers related to biochemistry. "Tricking Cancer Cells to Die," by A. Levitzki; "IkB Kinases in Innate Immunity and Cancer," by M. Karin; "Structure-based Discovery of Non-peptidic Small Molecule Inhibitors of Caspase-3," by J. Sakai and A. Yoshimori; "Receptor Tyrosine Kinase Inhibition in Cancer Therapy:From Monospecific to Multi-targeted Drugs," by A. Ullrich.

Presents abstracts of research papers related to biochemistry. "Sox9 Controls Both Chondrocyte Differentiation and Proliferation," by B. de Crombrugghe, A. Haruhiko and A. Yuko; "The Role of Chondrocyte-matrix Attachment Complexes in Skeletal Development," by A. Aszodi, C. Noclae and A. Raducanu; "The Extracellular Matrix 1 Gene Is Essential for Early Mouse Development," by S. Sercu, J. Liekens and L. Umans.

Presents an abstracts of research papers related to biochemistry. "Stress and Misfolded Proteins:Modulators of Neurodegenerative Diseases and Longevity," by R. I. Morimoto, J. Morley, H. Brignull, S. Garcia, K. Richter and C. Kaiser; "Chaperone Machines of the Cytosol," by B. Bukau; "The Substrate Spectrum of the Eucaryotic Chaperonin TRIC/CCT Revelead by Genomic Approaches," by J. Frydman.

Research on enzyme mechanisms is advancing knowledge of the chemistry and biochemistry of catalytic mechanisms by biological systems. The structural‐dynamical properties of enzymes are of key importance. Advanced methodological approaches and new insights into enzyme functioning, and new emerging approaches for the redesign/design of enzymes or artificial biocatalysts, were discussed at the EMBO conference on Catalytic Mechanisms by Biological Systems. [ABSTRACT FROM AUTHOR]

Corrections to articles in previous issues including an article on the need of phosphorylation for DRS-DA4-induced chemotaxis by C. Auvynet and colleagues in one of the previous issues and an article on antimicrobial activities of DRS-DA4 and DRS-B2 in one another previous issues are presented.

Nonenzymatic acetylation of Lys side chains (Lys‐SCs) by various in vivo reactive molecules has been suggested to play novel regulatory roles. Ubiquitin (UB) has seven Lys residues that are utilized for synthesis of specific poly‐UB chains. To understand the nature of these Lys‐SC modifications, the chemical acetylation rate and pKa and Hill coefficient of each UB‐Lys‐SC were measured. Mutagenesis studies combined with the determination of activation energy indicated that specific neighboring residues of the Lys‐SCs have a potential catalytic activity during nonenzymatic acetylation. Based on the shared chemistry between nonenzymatic Lys acetylation and ubiquitylation, the characterized chemical properties of the UB‐Lys‐SCs could be a reference for deciphering both mechanisms. Our NMR approaches could be useful for studying general nonenzymatic Lys acylations of various proteins. [ABSTRACT FROM AUTHOR]

The human dimethylglycine dehydrogenase ( hDMGDH) is a flavin adenine dinucleotide ( FAD)- and tetrahydrofolate ( THF)-dependent, mitochondrial matrix enzyme taking part in choline degradation, one-carbon metabolism and electron transfer to the respiratory chain. The rare natural variant H109R causes dimethylglycine dehydrogenase deficiency leading to increased blood and urinary dimethylglycine concentrations. A detailed biochemical and structural characterization of hDMGDH was thus far hampered by insufficient heterologous expression of the protein. In the present study, we report the development of an intracellular, heterologous expression system in Komagataella phaffii (formerly known as Pichia pastoris) providing the opportunity to determine kinetic parameters, spectroscopic properties, thermostability, and the redox potential of hDMGDH. Moreover, we have successfully crystallized the wild-type enzyme and determined the structure to 3.1-Å resolution. The structure-based analysis of our biochemical data provided new insights into the kinetic properties of the enzyme in particular with respect to oxygen reactivity. A comparative study with the H109R variant demonstrated that the variant suffers from decreased protein stability, cofactor saturation, and substrate affinity. Database Structural data are available in the PDB database under the accession number . [ABSTRACT FROM AUTHOR]

Current models of the cell interior emphasise its crowded, chemically complex and dynamically organised structure. Although the chemical composition of cells is known, the cooperative intermolecular interactions that govern cell ultrastructure are poorly understood. A major goal of biochemistry is to capture these myriad interactions in vivo. We consider the landmark discoveries that have shaped this objective, starting from the vitalist framework established by early natural philosophers. Through this historical revisionism, we extract important lessons for the bioinspired chemists of today. Scientific specialisation tends to insulate seminal ideas and hamper the unification of paradigms across biology. Therefore, we call for interdisciplinary collaboration in grappling with the complex cell interior. Recent successes in integrative structural biology and chemical biology demonstrate the power of hybrid approaches. The future roles of the (bio)chemist and model systems are also discussed as starting points for in vivo explorations. [ABSTRACT FROM AUTHOR]

The first three enzymatic steps of the strigolactone biosynthetic pathway catalysed by β-carotene cis-trans isomerase Dwarf27 (D27) from Oryza sativa and carotenoid cleavage dioxygenases CCD7 and CCD8 from Arabidopsis thaliana have been reconstituted in vitro, and kinetic assays have been developed for each enzyme, in order to develop selective enzyme inhibitors. Recombinant OsD27 shows a UV-visible λmax at 422 nm and is inactivated by silver(I) acetate, consistent with the presence of an iron -sulfur cluster that is used in catalysis. OsD27 and At CCD7 are not inhibited by hydroxamic acids that cause shoot branching in planta, but OsD27 is partially inhibited by terpene-like hydroxamic acids. The reaction catalysed by At CCD8 is shown to be a two-step kinetic mechanism using pre-steady-state kinetic analysis. Kinetic evidence is presented for acid-base catalysis in the CCD8 catalytic cycle and the existence of an essential cysteine residue in the CCD8 active site. At CCD8 is inhibited in a time-dependent fashion by hydroxamic acids D2, D4, D5 and D6 (> 95% inhibition at 100 μ m) that cause a shoot branching phenotype in A. thaliana, and selective inhibition of CCD8 is observed using hydroxamic acids D13H and D15 (82%, 71% inhibition at 10 μ m). The enzyme inhibition data imply that the biochemical basis of the shoot branching phenotype is due to inhibition of CCD8. [ABSTRACT FROM AUTHOR]

During prophase of meiosis I, homologous chromosomes interact and undergo recombination. Successful completion of these processes is required in order for the homologous chromosomes to mount the meiotic spindle as a pair. The organization of the chromosomes into pairs ensures orderly segregation to opposite poles of the dividing cell, such that each gamete receives one copy of each chromosome. Chiasmata, the cytological manifestation of crossover products of recombination, physically connect the homologs in pairs, providing a linkage that facilitates their segregation. Consequently, mutations that reduce the level of recombination are invariably associated with increased errors in meiotic chromosome segregation. In this review, we focus on recent biochemical and genetic advances in elucidating the mechanisms of meiotic DNA strand exchange catalyzed by the Dmc1 protein. We also discuss the mode by which two recombination mediators, Hop2 and Mnd1, facilitate rate-limiting steps of DNA strand exchange catalyzed by Dmc1. [ABSTRACT FROM AUTHOR]

During the last decades antimicrobial resistance has become a global health problem. Metallo-β-lactamases ( MBLs) which are broad-spectrum β-lactamases that inactivate virtually all β-lactams including carbapenems, are contributing to this health problem. In this study a novel MBL variant, termed VIM-26, identified in a Klebsiella pneumoniae isolate was studied. VIM-26 belongs to the Verona integron-encoded metallo-β-lactamase ( VIM) family of MBLs and is a His224Leu variant of the well-characterized VIM-1 variant. In this study, we report the kinetic parameters, minimum inhibitory concentrations and crystal structures of a recombinant VIM-26 protein, and compare them to previously published data on VIM-1, VIM-2 and VIM-7. The kinetic parameters and minimum inhibitory concentration determinations show that VIM-26, like VIM-7, has higher penicillinase activity but lower cephalosporinase activity than VIM-1 and VIM-2. The four determined VIM-26 crystal structures revealed mono- and di-zinc forms, where the Zn1 ion has distorted tetrahedral coordination geometry with an additional water molecule (W2) at a distance of 2.6-3.7 Å, which could be important during catalysis. The R2 drug binding site in VIM-26 is more open compared to VIM-2 and VIM-7 and neutrally charged due to Leu224 and Ser228. Thus, the VIM-26 drug binding properties are different from the VIM-2 (Tyr224/Arg228) and VIM-7 (His224/Arg228) structures, indicating a role of these residues in the substrate specificity. [ABSTRACT FROM AUTHOR]

Information regarding squalene synthases (SQSs) from prokaryotes is scarce. We aimed to characterize the SQS from Methylococcus capsulatus. We studied its reaction mechanism by kinetic analysis and evaluated the structure of the substrate/inhibitor-binding sites via homology modeling. The cloned M. capsulatus SQS was expressed in Escherichia coli and purified by nickel-nitrilotriacetic acid column chromatography. Interestingly, M. capsulatus SQS was water-soluble and did not require any detergent for its higher activity, unlike other SQSs studied previously; supplementation of any type of detergent inhibited enzyme activity. The specific activity and the kinetic values (Km and kcat) for the substrate farnesyl diphosphate and NADPH are reported. The substrate analog farnesyl methylenediphosphonate showed potent inhibition toward the enzyme. We prepared the sitespecific mutants directed at potential active-site residues 58DXX61E62D (S1 site) and 213DXX216D217D (S2 site), which were assumed to be involved in the binding of the substrate farnesyl diphosphate through the Mg2+ion. We first demonstrated that the S1 site and the two basic residues (R55 and K212) were responsible for the binding of farnesyl diphosphate. Furthermore, we examined the catalytic roles of the highly conserved aromatic residues and demonstrated that the Y164 residue abstracts the proton of cation 5, which is produced during the first half-reaction (Scheme 1), to afford presqualene diphosphate, and that the W224 residue stabilizes the intermediary cation 5 via the cation-π interaction. Furthermore, we confirm for the first time that the F32 and the Y51 residues also stabilize the carbocation intermediate(s) generated during the second half-reaction. [ABSTRACT FROM AUTHOR]

Available structures of HAMP domains suggest rotation as one potential mechanism in intraprotein signal transduction. It has been proposed that in poly- HAMP modules the signal sign is inverted with each additional HAMP. We examined signal transduction through the HAMP tandem domain from the phototaxis transducer of the halophilic archaeon Natronomonas pharaonis in membrane-bound chimeras consisting of the Escherichia coli chemotaxis receptor for serine, Tsr, as an input and the mycobacterial adenylyl cyclase Rv3645 as an output domain, i.e. the basic chimera was ' Tsr- Np HAMP tandem- Rv3645 cyclase'. Neither of the Np HAMP units alone nor the Np HAMP tandem transduced a serine signal. After five targeted point mutations in the first α-helix of Np HAMP1, the non-functional Np HAMP modules combined into a functional HAMP tandem. 1 m m serine significantly inhibited cyclase activity (−35%; IC50 = 30 μ m) in disagreement with the structure-based predictions. Surprisingly, replacement of Np AS11 in the tandem by the respective AS1 from HAMPTsr resulted in signal inversion, i.e. serine activated cyclase (+129%; EC50 = 10 μ m). Examination of 48 mutants of AS11 in the HAMP tandem including two residues of a putative N-terminal control cable identified five residues in Np AS11 which probably define different ground states of the output domain and thus affect the sign of signal output. The data question the predicted HAMP rotation as the predominant mechanism of intraprotein signal transduction and point to as yet unrecognized conformational motions of HAMP domains in intraprotein signaling. [ABSTRACT FROM AUTHOR]

Serine hydroxymethyltransferase ( SHMT) catalyzes the transfer of a hydroxymethyl group from l-serine to tetrahydrofolate to yield glycine and 5,10-methylenetetrahydrofolate. Our previous investigations have shown that SHMTs from Plasmodium spp. ( P. falciparum, Pf; P. vivax, Pv) are different from the enzyme from rabbit liver in that Plasmodium SHMT can use d-serine as a substrate. In this report, the biochemical and biophysical properties of the Plasmodium and the human cytosolic form (hc SHMT) enzymes including ligand binding and kinetics were investigated. The data indicate that, similar to Plasmodium enzymes, hc SHMT can use d-serine as a substrate. However, hc SHMT displays many properties that are different from those of the Plasmodium enzymes. The molar absorption coefficient of hc SHMT-bound pyridoxal-5′-phosphate ( PLP) is much greater than Pv SHMT-bound or Pf SHMT-bound PLP. The binding interactions of hc SHMT and Plasmodium SHMT with d-serine are different, as only the Plasmodium enzyme undergoes formation of a quinonoid-like species upon binding to d-serine. Furthermore, it has been noted that hc SHMT displays strong substrate inhibition by tetrahydrofolate ( THF) (at THF > 40 μ m), compared with SHMTs from Plasmodium and other species. The pH-activity profile of hc SHMT shows higher activities at lower pH values corresponding to a p Ka value of 7.8 ± 0.1. Thiosemicarbazide reacts with hc SHMT following a one-step model [ k1 of 12 ± 0.6 m−1·s−1 and k−1 of (1.0 ± 0.6) × 10−3 s−1], while the same reaction with Pf SHMT involves at least three steps. All data indicated that the ligand binding environment of SHMT from human and Plasmodium are different, indicating that it should be possible to develop species-selective inhibitors in future studies. Database serine hydroxymethyltransferase, ; 5,10-methylenetetrahydrofolate dehydrogenase [ABSTRACT FROM AUTHOR]

Bacteroides fragilis causes the majority of anaerobic infections in humans. The presence of a pathogenicity island in the genome discriminates pathogenic and commensal B. fragilis strains. The island encodes metalloproteinase II ( MPII), a potential virulence protein, and one of three homologous fragilysin isozymes ( FRA; also termed B. fragilis toxin or BFT). Here, we report biochemical data on the structural-functional characteristics of the B. fragilis pathogenicity island proteases by reporting the crystal structure of MPII at 2.13 Å resolution, combined with detailed characterization of the cleavage preferences of MPII and FRA3 (as a representative of the FRA isoforms), identified using a high-throughput peptide cleavage assay with 18 583 substrate peptides. We suggest that the evolution of the MPII catalytic domain can be traced to human and archaebacterial proteinases, whereas the prodomain fold is a feature specific to MPII and FRA. We conclude that the catalytic domain of both MPII and FRA3 evolved differently relative to the prodomain, and that the prodomain evolved specifically to fit the B. fragilis pathogenicity. Overall, our data provide insights into the evolution of cleavage specificity and activation mechanisms in the virulent metalloproteinases. [ABSTRACT FROM AUTHOR]

Cyanobacterial fructose-1,6/sedoheptulose-1,7-bisphosphatase (cy- FBP/ SBPase) plays a vital role in gluconeogenesis and in the photosynthetic carbon reduction pathway, and is thus a potential enzymatic target for inhibition of harmful cyanobacterial blooms. Here, we describe the crystal structure of cy- FBP/ SBPase in complex with AMP and fructose-1,6-bisphosphate ( FBP). The allosteric inhibitor AMP and the substrate FBP exhibit an unusual binding mode when in complex with cy- FBP/ SBPase. Binding mode analysis suggested that AMP bound to the allosteric sites near the interface across the up/down subunit pairs C1 C4 and C2 C3 in the center of the tetramer, while FBP binds opposite to the interface between the horizontal subunit pairs C1 C2 or C3 C4. We identified a series of residues important for FBP and AMP binding, and suggest formation of a disulfide linkage between Cys75 and Cys99. Further analysis indicates that cy- FBP/ SBPase may be regulated through ligand binding and alteration of the structure of the enzyme complex. The interactions between ligands and cy- FBP/ SBPase are different from those of ligand-bound structures of other FBPase family members, and thus provide new insight into the molecular mechanisms of structure and catalysis of cy- FBP/ SBPase. Our studies provide insight into the evolution of this enzyme family, and may help in the design of inhibitors aimed at preventing toxic cyanobacterial blooms. Database Structural data have ben submitted to the Protein Data Bank under accession numbers and . Structured digital abstract and by (). [ABSTRACT FROM AUTHOR]

The last couple of decades have seen an extraordinary transformation in our knowledge and understanding of the multifarious biological roles of inositol phospholipids. Herein, I briefly consider two topics. The first is the role that recently acquired biochemical and genomic information - especially from archaeons - has played in illuminating the possible evolutionary origins of the biological employment of inositol in lipids, and some questions that these studies raise about the 'classical' biosynthetic route to phosphatidylinositol. The second is the growing recognition of the importance in eukaryotic cells of phosphatidylinositol 3,5-bisphosphate. Phosphatidylinositol 3,5-bisphosphate only entered our phosphoinositide consciousness quite recently, but it is speedily gathering a plethora of roles in diverse cellular processes and diseases thereof. These include: control of endolysosomal vesicular trafficking and of the activity of ion channels and pumps in the endolysosomal compartment; control of constitutive and stimulated protein traffic to and from plasma membrane subdomains; control of the nutrient and stress-sensing target of rapamycin complex 1 pathway (TORC1); and regulation of key genes in some central metabolic pathways. [ABSTRACT FROM AUTHOR]

A correction to the article related to the Department to Biochemistry is presented.

A correction to the article "Dual P2Y12 receptor signaling in thrombin-stimulated platelets - involvement of phosphoinositide 3-kinase b but not c isoform in Ca2+ mobilization and procoagulant activity" that was published in a previous issue is presented.

A correction to the article "Huntingtin Inclusion Bodies are Iron-dependent Centers of Oxidative Events," by W.J.J. Firdaus is presented.

Neurosporaxanthin (β-apo-4′-carotenoic acid) biosynthesis has been studied in detail in the fungus Fusarium fujikuroi. The genes and enzymes for this biosynthetic pathway are known until the last enzymatic step, the oxidation of the aldehyde group of its precursor, β-apo-4′-carotenal. On the basis of sequence homology to Neurospora crassa YLO-1, which mediates the formation of apo-4′-lycopenoic acid from the corresponding aldehyde substrate, we cloned the carD gene of F. fujikuroi and investigated the activity of the encoded enzyme. In vitro assays performed with heterologously expressed protein showed the formation of neurosporaxanthin and other apocarotenoid acids from the corresponding apocarotenals. To confirm this function in vivo, we generated an Escherichia coli strain producing β-apo-4′-carotenal, which was converted into neurosporaxanthin upon expression of carD. Moreover, the carD function was substantiated by its targeted disruption in a F. fujikuroi carotenoid-overproducing strain, which resulted in the loss of neurosporaxanthin and the accumulation of β-apo-4′-carotenal, its derivative β-apo-4′-carotenol, and minor amounts of other carotenoids. Intermediates accumulated in the Δ carD mutant suggest that the reactions leading to neurosporaxanthin in Neurospora and Fusarium are different in their order. In contrast to ylo-1 in N. crassa, carD mRNA content is enhanced by light, but to a lesser extent than other enzymatic genes of the F. fujikuroi carotenoid pathway. Furthermore, carD mRNA levels were higher in carotenoid-overproducing mutants, supporting a functional role for CarD in F. fujikuroi carotenogenesis. With the genetic and biochemical characterization of CarD, the whole neurosporaxanthin biosynthetic pathway of F. fujikuroi has been established. Database The carD gene sequence has been deposited in the EMBL Data Bank under accession number [ABSTRACT FROM AUTHOR]

A correction to an article on biochemistry previously published in the "FEBS Journal" is presented.

A correction to the article "The Accession Number for Partial Histone H1.a2 Sequence Data That Was Deposited in the UniProt Knowledge Base" that was published in the previous issue is presented.

It is assumed that protein fibrils manifested in amyloidosis result from an aggregation reaction involving small misfolded protein sequences being in an ‘oligomeric’ or ‘prefibrillar’ state. This review covers recent optical spectroscopic studies of amyloid protein misfolding, oligomerization and amyloid fibril growth. Although amyloid fibrils have been studied using established protein-characterization techniques throughout the years, their oligomeric precursor states require sensitive detection in real-time. Here, fluorescent staining is commonly performed using thioflavin T and other small fluorescent molecules such as 4-(dicyanovinyl)- julolidine and 1-amino-8-naphtalene sulphonate that have high affinity to hydrophobic patches. Thus, populated oligomeric intermediates and related ‘prefibrillar structures’ have been reported for several human amyloidogenic systems, including amyloid-beta protein, prion protein, transthyretin, α-synuclein, apolipoprotein C-II and insulin. To obtain information on the progression of the intermediate states, these were monitored in terms of fluorescence parameters, such as anisotropy, and quantum efficiency changes upon protein binding. Recently, new antibody stains have allowed precise monitoring of the oligomer size and distributions using multicolor labelling and single molecule detection. Moreover, a pentameric thiophene derivative (p-FTAA) was reported to indicate early precursors during A-beta1-40 fibrillation, and was also demonstrated in real-time visualization of cerebral protein aggregates in transgenic AD mouse models by multiphoton microscopy. Conclusively, molecular probes and optical spectroscopy are now entering a phase enabling the in vivo interrogation of the role of oligomers in amyloidosis. Such techniques used in parallel with in vitro experiments, of increasing detail, will probably couple structure to pathogenesis in the near future. [ABSTRACT FROM AUTHOR]

Transcription factors are DNA-binding proteins that regulate key biological processes. Their interactions with DNA are commonly analyzed with gel-based electrophoretic mobility shift assay (EMSA) using radioactively labeled probes. Within various fields of research, there exists an increasing demand to develop assays with faster sample throughput combined with improved sensitivity, increased analytical range, and precise quantification. Here, we describe the development and performance of a 384-well plate immunoassay, termed TransLISA, which is a novel homogeneous assay for rapid and sensitive quantification of the DNA-binding activity of transcription factors in cell and tissue lysates. TransLISA outperforms EMSAs, because it eliminates the need to use radioactive chemicals and allows fast and precise quantification of DNA-binding activity of transcription factors from large number of samples simultaneously. We have used TransLISA to demonstrate the DNA-binding activity of heat shock factor 1, representing a well-known model of inductive transcriptional regulatory responses, but the method is easily adaptable for the study of any transcription factor. Thus, TransLISA can replace EMSAs and may be used in various applications and research fields where quantitative, cost-effective and large-scale measurements of the DNA-binding activity of transcription factors are required, including screening of responses in multiple treatments in cellular and molecular biology, evolutionary research, environmental monitoring, and drug discovery. [ABSTRACT FROM AUTHOR]

The multifunctional tissue transglutaminase 2 (TG2) has a four-domain structure with several Ca2+-regulated biochemical activities, including transglutamylation and GTP hydrolysis. The structure of the Ca2+-binding form of the human enzyme is not known, and its Ca2+-binding sites have not been fully characterized. By mutagenesis, we have targeted its active site Cys, three sites based on homology to Ca2+-binding residues of epidermal transglutaminase and factor XIIIa (S1–S3), and two regions with negative surface potentials (S4 and S5). CD spectroscopy, antibody-binding assay and GTPase activity measurements indicated that the amino acid substitutions did not cause major structural alterations. Calcium-45 equilibrium dialysis and isothermal calorimetric titration showed that both wild-type and active site-deleted enzymes (C277S) bind six Ca2+. Each of the S1–S5 mutants binds fewer than six Ca2+, S1 is a strong Ca2+-binding site, and mutation of one site resulted in the loss of more than one bound Ca2+, suggesting cooperativity among sites. All mutants were deficient in transglutaminase activity, and GTP inhibited remnant activities. Like those of the wild-type enzyme, the GTPase activities of the mutants were inhibited by Ca2+, except in the case of the S4 and S5 mutants, which exhibited increased activity. TG2 is the major autoantigen in celiac disease, and testing the reactivity of mutants with autoantibodies from celiac disease patients revealed that S4 strongly determines antigenicity. It can be concluded that five of the Ca2+-binding sites of TG2 influence its transglutaminase activity, two sites are involved in the regulation of GTPase activity, and one determines antigenicity for autoantibodies in celiac patients. [ABSTRACT FROM AUTHOR]

There is growing concern about the safety of engineered nanoparticles, which are produced for various industrial applications. Quantum dots are colloidal semiconductor nanoparticles that have unique luminescence characteristics and the potential to become attractive tools for medical imaging. However, some of these particles can cause oxidative stress and induce cell death. The objective of this study was to explore quantum dot-induced metabolic changes, which could occur without any apparent cellular damage. We provide evidence that both uncoated and ZnS-coated quantum dots can induce the accumulation of lipids (increase in cytoplasmic lipid droplet formation) in two cell culture models: glial cells in primary mouse hypothalamic cultures and rat pheochromocytoma PC12 cells. Glial cells treated with CdTe quantum dots accumulated newly synthesized lipids in a phosphoinositide 3-kinase-dependent manner, which was consistent with the growth factor-dependent accumulation of lipids in PC12 cells treated with CdTe and CdSe/ZnS quantum dots. In PC12 cells, quantum dots, as well as the hypoxia mimetic CoCl2, induced the up-regulation of hypoxia-inducible transcription factor-1α and the down-regulation of the β-oxidation of fatty acids, both of which could contribute to the accumulation of lipids. On the basis of our results, we propose a model illustrating how nanoparticles, such as quantum dots, could trigger the formation of intracellular lipid droplets, and we suggest that metabolic measurements, such as the determination of fat oxidation in tissues, which are known sites of nanoparticle accumulation, could provide useful measures of nanoparticle safety. Such assays would expand the current platform of tests for the determination of the biocompatibility of nanomaterials. [ABSTRACT FROM AUTHOR]

HP1287 ( tenA) from Helicobacter pylori is included among the genes that play a relevant role in bacterium colonization and persistence. The gene has been cloned and its product, protein TenA, has been expressed and purified. The crystal structures of the wild-type protein and the mutant F47Y have been determined at resolutions of 2.7 and 2.4 Å, respectively. The molecular model, a homotetramer with 222 symmetry, shows that the H. pylori TenA structure belongs to the thiaminase II class of proteins. These enzymes were recently found to be involved in a salvage pathway for the synthesis of the thiamin precursor hydroxypyrimidine, which constitutes a building block in thiamin biosynthesis, in particular in bacteria living in the soil. By contrast, enzymatic measurements on TenA from H. pylori indicate that the activity on the putative substrate 4-amino-5-aminomethyl-2-methylpyrimidine is very modest. Moreover, in the present study, we demonstrate that the mutation at residue 47, a position where a phenylalanine occurs in all the strains of H. pylori sequenced to date, is not sufficient to explain the very low catalytic activity toward the expected substrate. As a result of differences in the colonization environment of H. pylori as well as the TenA structural and catalytic peculiar features, we suggest a possible pivotal role for the H. pylori enzyme in the thiamin biosynthetic route, which is in agreement with the relevance of this protein in the stomach colonization process. Structured digital abstract • : TenA (uniprotkb: ) and TenA (uniprotkb: ) bind ( ) by x-ray crystallography ( ) [ABSTRACT FROM AUTHOR]

This minireview covers the research carried out in recent years into different aspects of the function of the flavoproteins involved in cyanobacterial photosynthetic electron transfer from photosystem I to NADP+, flavodoxin and ferredoxin–NADP+ reductase. Interactions that stabilize protein–flavin complexes and tailor the midpoint potentials in these proteins, as well as many details of the binding and electron transfer to protein and ligand partners, have been revealed. In addition to their role in photosynthesis, flavodoxin and ferredoxin–NADP+ reductase are ubiquitous flavoenzymes that deliver NAD(P)H or low midpoint potential one-electron donors to redox-based metabolisms in plastids, mitochondria and bacteria. They are also the basic prototypes for a large family of diflavin electron transferases with common functional and structural properties. Understanding their mechanisms should enable greater comprehension of the many physiological roles played by flavodoxin and ferredoxin–NADP+ reductase, either free or as modules in multidomain proteins. Many aspects of their biochemistry have been extensively characterized using a combination of site-directed mutagenesis, steady-state and transient kinetics, spectroscopy and X-ray crystallography. Despite these considerable advances, various key features of the structural–function relationship are yet to be explained in molecular terms. Better knowledge of these systems and their particular properties may allow us to envisage several interesting applications of these proteins beyond their physiological functions. [ABSTRACT FROM AUTHOR]

The article presents abstracts on symposium on biochemistry topics which include Epigenetics: DNA Methylation and far Beyond, Protein Structure and Interactions, and Metabolites in Interactions.

The rare 6-deoxysugard-rhamnose is a component of bacterial cell surface glycans, including thed-rhamnose homopolymer produced by Pseudomonas aeruginosa, called A-band O polysaccharide. GDP-d-rhamnose synthesis from GDP-d-mannose is catalyzed by two enzymes. The first is a GDP-d-mannose-4,6-dehydratase (GMD). The second enzyme, RMD, reduces the GMD product (GDP-6-deoxy-d- lyxo-hexos-4-ulose) to GDP-d-rhamnose. Genes encoding GMD and RMD are present in P. aeruginosa, and genetic evidence indicates they act in A-band O-polysaccharide biosynthesis. Details of their enzyme functions have not, however, been previously elucidated. We aimed to characterize these enzymes biochemically, and to determine the structure of RMD to better understand what determines substrate specificity and catalytic activity in these enzymes. We used capillary electrophoresis and NMR analysis of reaction products to precisely define P. aeruginosa GMD and RMD functions. P. aeruginosa GMD is bifunctional, and can catalyze both GDP-d-mannose 4,6-dehydration and the subsequent reduction reaction to produce GDP-d-rhamnose. RMD catalyzes the stereospecific reduction of GDP-6-deoxy-d- lyxo-hexos-4-ulose, as predicted. Reconstitution of GDP-d-rhamnose biosynthesis in vitro revealed that the P. aeruginosa pathway may be regulated by feedback inhibition in the cell. We determined the structure of RMD from Aneurinibacillus thermoaerophilus at 1.8 Å resolution. The structure of A. thermoaerophilus RMD is remarkably similar to that of P. aeruginosa GMD, which explains why P. aeruginosa GMD is also able to catalyze the RMD reaction. Comparison of the active sites and amino acid sequences suggests that a conserved amino acid side chain (Arg185 in P. aeruginosa GMD) may be crucial for orienting substrate and cofactor in GMD enzymes. [ABSTRACT FROM AUTHOR]

Mathematical models of biological processes have various applications: to assist in understanding the functioning of a system, to simulate experiments before actually performing them, to study situations that cannot be dealt with experimentally, etc. Some parameters in the model can be directly obtained from experiments or from the literature. Others have to be inferred by comparing model results to experiments. In this minireview, we discuss the identifiability of models, both intrinsic to the model and taking into account the available data. Furthermore, we give an overview of the most frequently used approaches to search the parameter space. [ABSTRACT FROM AUTHOR]

Ca2+, as a messenger of signal transduction, regulates numerous target molecules via Ca2+-induced conformational changes. Investigation into the determinants for Ca2+-induced conformational change is often impeded by cooperativity between multiple metal-binding sites or protein oligomerization in naturally occurring proteins. To dissect the relative contributions of key determinants for Ca2+-dependent conformational changes, we report the design of a single-site Ca2+-binding protein (CD2.trigger) created by altering charged residues at an electrostatically sensitive location on the surface of the host protein rat Cluster of Differentiation 2 (CD2). CD2.trigger binds to Tb3+ and Ca2+ with dissociation constants of 0.3 ± 0.1 and 90 ± 25 μm, respectively. This protein is largely unfolded in the absence of metal ions at physiological pH, but Tb3+ or Ca2+ binding results in folding of the native-like conformation. Neutralization of the charged coordination residues, either by mutation or protonation, similarly induces folding of the protein. The control of a major conformational change by a single Ca2+ ion, achieved on a protein designed without reliance on sequence similarity to known Ca2+-dependent proteins and coupled metal-binding sites, represents an important step in the design of trigger proteins. [ABSTRACT FROM AUTHOR]

Lipid A is a biological component of the lipo-oligosaccharide of a human pathogen, Moraxella catarrhalis. No other acyltransferases except for UDP-GlcNAc acyltransferase, responsible for lipid A biosynthesis in M. catarrhalis, have been identified. By bioinformatics, two late acyltransferase genes, lpxX and lpxL, responsible for lipid A biosynthesis were identified, and knockout mutants of each gene in M. catarrhalis strain O35E were constructed and named O35ElpxX and O35ElpxL. Structural analysis of lipid A from the parental strain and derived mutants showed that O35ElpxX lacked two decanoic acids (C10:0), whereas O35ElpxL lacked one dodecanoic (lauric) acid (C12:0), suggesting that lpxX encoded decanoyl transferase and lpxL encoded dodecanoyl transferase. Phenotypic analysis revealed that both mutants were similar to the parental strain in their toxicity in vitro. However, O35ElpxX was sensitive to the bactericidal activity of normal human serum and hydrophobic reagents. It had a reduced growth rate in broth and an accelerated bacterial clearance at 3 h ( P < 0.01) or 6 h ( P < 0.05) after an aerosol challenge in a murine model of bacterial pulmonary clearance. O35ElpxL presented similar patterns to those of the parental strain, except that it was slightly sensitive to the hydrophobic reagents. These results indicate that these two genes, particularly lpxX, encoding late acyltransferases responsible for incorporation of the acyloxyacyl-linked secondary acyl chains into lipid A, are important for the biological activities of M. catarrhalis. [ABSTRACT FROM AUTHOR]

Adenosylcobalamin-dependent diol and glycerol dehydratases are isofunctional enzymes and undergo mechanism-based inactivation by a physiological substrate glycerol during catalysis. Inactivated holoenzymes are reactivated by their own reactivating factors that mediate the ATP-dependent exchange of an enzyme-bound, damaged cofactor for free adenosylcobalamin through intermediary formation of apoenzyme. The reactivation takes place in two steps: (a) ADP-dependent cobalamin release and (b) ATP-dependent dissociation of the resulting apoenzyme–reactivating factor complexes. The in vitro experiments with purified proteins indicated that diol dehydratase-reactivating factor (DDR) cross-reactivates the inactivated glycerol dehydratase, whereas glycerol dehydratase-reactivating factor (GDR) did not cross-reactivate the inactivated diol dehydratase. We investigated the molecular basis of their specificities in vitro by using purified preparations of cognate and noncognate enzymes and reactivating factors. DDR mediated the exchange of glycerol dehydratase-bound cyanocobalamin for free adeninylpentylcobalamin, whereas GDR cannot mediate the exchange of diol dehydratase-bound cyanocobalamin for free adeninylpentylcobalamin. As judged by denaturing PAGE, the glycerol dehydratase–DDR complex was cross-formed, although the diol dehydratase–GDR complex was not formed. There were no specificities of reactivating factors in the ATP-dependent dissociation of enzyme–reactivating factor complexes. Thus, it is very likely that the specificities of reactivating factors are determined by the capability of reactivating factors to form complexes with apoenzymes. A modeling study based on the crystal structures of enzymes and reactivating factors also suggested why DDR cross-forms a complex with glycerol dehydratase, and why GDR does not cross-form a complex with diol dehydratase. [ABSTRACT FROM AUTHOR]

Pseudouridine synthase RluD converts uridines at positions 1911, 1915, and 1917 of 23S rRNA to pseudouridines. These nucleotides are located in the functionally important helix-loop 69 of 23S rRNA. RluD is the only pseudouridine synthase that is required for normal growth in Escherichia coli. We have analyzed substrate specificity of RluD in vivo. Mutational analyses have revealed: (a) RluD isomerizes uridine in vivo only at positions 1911, 1915, and 1917, regardless of the presence of uridine at other positions in the loop of helix 69 of 23S rRNA variants; (b) substitution of one U by C has no effect on the conversion of others (i.e. formation of pseudouridines at positions 1911, 1915, and 1917 are independent of each other); (c) A1916 is the only position in the loop of helix 69, where mutations affect the RluD specific pseudouridine formation. Pseudouridines were determined in the ribosomal particles from a ribosomal large subunit defective strain (RNA helicase DeaD–). An absence of pseudouridines in the assembly precursor particles suggests that RluD directed isomerization of uridines occurs as a late step during the assembly of the large ribosomal subunit. [ABSTRACT FROM AUTHOR]