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Tick-borne encephalitis virus (TBEV) and Powassan virus (POWV) are neurotropic tick-borne orthoflaviviruses. They cause mostly asymptomatic infections in hosts, but severe forms with CNS involvement can occur. Studying the early stages of viral infections in humans is challenging, and appropriate animal models are essential for understanding the factors determining the disease severity and for developing emergency prophylaxis and treatment options. In this work, we assessed the model of the early stages of TBEV and POWV mono- and co-infections in Macaca fascicularis . Serological, biochemical, and virological parameters were investigated to describe the infection, including its impact on animal behavior. Viremia, neutralizing antibody dynamics, and viral load in organs were chosen as the main parameters distinguishing early-stage orthoflavivirus infection. Levels of IFNα, monocyte count, and cognitive test scores were proposed as additional informative indicators. An assessment of a tick-borne encephalitis vaccine using this model showed that it provided partial protection against POWV infection in Macaca fascicularis without signs of antibody-dependent enhancement of infection.

Currently the only effective measure against tick-borne encephalitis (TBE) is vaccination. Despite the high efficacy of approved vaccines against TBE, rare cases of vaccine failures are well documented. Both host- and virus-related factors can account for such failures. In this work, we studied the influence of mouse strain and sex and the effects of cyclophosphamide-induced immunosuppression on the efficacy of an inactivated TBE vaccine. We also investigated how an increased proportion of non-infectious particles in the challenge TBE virus would affect the protectivity of the vaccine. The vaccine efficacy was assessed by mortality, morbidity, levels of viral RNA in the brain of surviving mice, and neutralizing antibody (NAb) titers against the vaccine strain and the challenge virus. Two-dose vaccination protected most animals against TBE symptoms and death, and protectivity depended on strain and sex of mice. Immunosuppression decreased the vaccine efficacy in a dose-dependent manner and changed the vaccine-induced NAb spectrum. The vaccination protected mice against TBE virus neuroinvasion and persistence. However, viral RNA was detected in the brain of some asymptomatic animals at 21 and 42 dpi. Challenge with TBE virus enriched with non-infectious particles led to lower NAb titers in vaccinated mice after the challenge but did not affect the protective efficacy.

A series of inhibitors of plant enzymes of the non-mevalonate pathway from herbicide research efforts at BASF were screened for antimalarial activity in a cell-based assay. A 1,3-diiminoisoindoline carbohydrazide was found to inhibit the growth of Plasmodium falciparum with an IC(50) value <100 nM. Synthesis of a variety of derivatives allowed an improvement of the initial antimalarial activity down to IC(50) =18 nM for the most potent compound, the establishment of a structure-activity relationship, and the evaluation of the cytotoxic profile of the diiminoisoindolines. Furthermore, interesting configurational and conformational aspects for this class of compounds were studied by computational and X-ray crystal structure analysis. Some of the compounds can act as tridentate ligands, forming 2:1 ligand-iron(III) complexes, which also display antimalarial activity in the nanomolar IC(50) range, paired with low cytotoxicity., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Reverse hydroxamate-based inhibitors of IspC, a key enzyme of the non-mevalonate pathway of isoprenoid biosynthesis and a validated antimalarial target, were synthesized and biologically evaluated. The binding mode of one derivative in complex with EcIspC and a divalent metal ion was clarified by X-ray analysis. Pilot experiments have demonstrated in vivo potential.

A library of 40,000 compounds was screened for inhibitors of 2-methylerythritol 2,4-cyclodiphosphate synthase (IspF) protein from Arabidopsis thaliana using a photometric assay. A thiazolopyrimidine derivative resulting from the high-throughput screen was found to inhibit the IspF proteins of Mycobacterium tuberculosis, Plasmodium falciparum, and A. thaliana with IC(50) values in the micromolar range. Synthetic efforts afforded derivatives that inhibit IspF protein from M. tuberculosis and P. falciparum with IC(50) values in the low micromolar range. Several compounds act as weak inhibitors in the P. falciparum red blood cell assay.

The intensely fluorescent lumazine protein is believed to be involved in the bioluminescence of certain marine bacteria. The sequence of the catalytically inactive protein resembles that of the enzyme riboflavin synthase. Its non-covalently bound fluorophore, 6,7-dimethyl-8-ribityllumazine, is the substrate of this enzyme and also the committed precursor of vitamin B(2). An extensive crystallization screen was performed using numerous single-site mutants of the lumazine protein from Photobacterium leiognathi in complex with its fluorophore and with riboflavin, respectively. Only the L49N mutant in complex with riboflavin yielded suitable crystals, allowing X-ray structure determination to a resolution of 2.5 A. The monomeric protein folds into two closely similar domains that are structurally related by pseudo-C(2) symmetry, whereby the entire domain topology resembles that of riboflavin synthase. Riboflavin is bound to a shallow cavity in the N-terminal domain of lumazine protein, whereas the C-terminal domain lacks a ligand.

2C-Methyl-D-erythritol-4-phosphate synthase, encoded by the ispC gene (also designated dxr), catalyzes the first committed step in the nonmevalonate isoprenoid biosynthetic pathway. The reaction involves the isomerization of 1-deoxy-D-xylulose 5-phosphate, giving a branched-chain aldose derivative that is subsequently reduced to 2C-methyl-D-erythritol 4-phosphate. The isomerization step has been proposed to proceed as an intramolecular rearrangement or a retroaldol-aldol sequence. We report the preparation of (13)C-labeled substrate isotopologs that were designed to optimize the detection of an exchange of putative cleavage products that might occur in the hypothetical retroaldol-aldol reaction sequence. In reaction mixtures containing large amounts of 2C-methyl-D-erythritol-4-phosphate synthase from Escherichia coli, Mycobacterium tuberculosis or Arabidopsis thaliana, and a mixture of [1-(13)C(1)]-2C-methyl-D-erythritol 4-phosphate and [3-(13)C(1)]2C-methyl-D-erythritol 4-phosphate, the reversible reaction could be followed over thousands of reaction cycles. No fragment exchange could be detected by NMR spectroscopy, and the frequency of exchange, if any, is less than 5 p.p.m. per catalytic cycle. Hydroxyacetone, the putative second fragment expected from the retroaldol cleavage, was not incorporated into the enzyme product. In contrast to other reports, IspC did not catalyze the isomerisation of 1-deoxy-D-xylulose 5-phosphate to give 1-deoxy-L-ribulose 5-phosphate under any conditions tested. However, we could show that the isomerization reaction proceeds at room temperature without a requirement for enzyme catalysis. Although a retroaldol-aldol mechanism cannot be ruled out conclusively, the data show that a retroldol-aldol reaction sequence would have to proceed with very stringent fragment containment that would apply to the enzymes from three genetically distant organisms.

4-Diphosphocytidyl-2C-methyl-D-erythritol kinase (IspE) catalyses the ATP-dependent conversion of 4-diphosphocytidyl-2C-methyl-D-erythritol (CDPME) to 4-diphosphocytidyl-2C-methyl-d-erythritol 2-phosphate with the release of ADP. This reaction occurs in the non-mevalonate pathway of isoprenoid precursor biosynthesis and because it is essential in important microbial pathogens and absent from mammals it represents a potential target for anti-infective drugs. We set out to characterize the biochemical properties, determinants of molecular recognition and reactivity of IspE and report the cloning and purification of recombinant Aquifex aeolicus IspE (AaIspE), kinetic data, metal ion, temperature and pH dependence, crystallization and structure determination of the enzyme in complex with CDP, CDPME and ADP. In addition, 4-fluoro-3,5-dihydroxy-4-methylpent-1-enylphosphonic acid (compound 1) was designed to mimic a fragment of the substrate, a synthetic route to 1 was elucidated and the complex structure determined. Surprisingly, this ligand occupies the binding site for the ATP alpha-phosphate not the binding site for the methyl-D-erythritol moiety of CDPME. Gel filtration and analytical ultracentrifugation indicate that AaIspE is a monomer in solution. The enzyme displays the characteristic alpha/beta galacto-homoserine-mevalonate-phosphomevalonate kinase fold, with the catalytic centre positioned in a deep cleft between the ATP- and CDPME-binding domains. Comparisons indicate a high degree of sequence conservation on the IspE active site across bacterial species, similarities in structure, specificity of substrate recognition and mechanism. The biochemical characterization, attainment of well-ordered and reproducible crystals and the models resulting from the analyses provide reagents and templates to support the structure-based design of broad-spectrum antimicrobial agents.

The enzymes of the non-mevalonate pathway for isoprenoid biosynthesis are attractive targets for the development of novel drugs against malaria and tuberculosis. This pathway is used exclusively by the corresponding pathogens, but not by humans. A series of water-soluble, cytidine-based inhibitors that were originally designed for the fourth enzyme in the pathway, IspD, were shown to inhibit the subsequent enzyme, the kinase IspE (from Escherichia coli). The binding mode of the inhibitors was verified by co-crystal structure analysis, using Aquifex aeolicus IspE. The crystal structures represent the first reported example of a co-crystal structure of IspE with a synthetic ligand and confirmed that ligand binding affinity originates mainly from the interactions of the nucleobase moiety in the cytidine binding pocket of the enzyme. In contrast, the appended benzimidazole moieties of the ligands adopt various orientations in the active site and establish only poor intermolecular contacts with the protein. Defined binding sites for sulfate ions and glycerol molecules, components in the crystallization buffer, near the well-conserved ATP-binding Gly-rich loop of IspE were observed. The crystal structures of A. aeolicus IspE nicely complement the one from E. coli IspE for use in structure-based design, namely by providing invaluable structural information for the design of inhibitors targeting IspE from Mycobacterium tuberculosis and Plasmodium falciparum. Similar to the enzymes from these pathogens, A. aeolicus IspE directs the OH group of a tyrosine residue into a pocket in the active site. In the E. coli enzyme, on the other hand, this pocket is lined by phenylalanine and has a more pronounced hydrophobic character.

The nonmevalonate isoprenoid pathway is an established target for antiinfective drug development. This paper describes high-throughput methods for the screening of 2C-methyl-D-erythritol synthase (IspC protein), 4-diphosphocytidyl-2C-methyl-D-erythritol synthase (IspD protein), 4-diphosphocytidyl-2C-methyl-D-erythritol kinase (IspE protein), and 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF protein) against large compound libraries. The assays use up to three auxiliary enzymes. They are all monitored photometrically at 340 nm and are robust as documented by Z-factors of >or=0.86. 13C NMR assays designed for hit verification via direct detection of the primary reaction product are also described. Enzyme-assisted methods for the preparation, on a multigram scale, of isoprenoid biosynthesis intermediates required as substrates for these assays are reported. Notably, these methods enable the introduction of single or multiple 13C labels as required for NMR-monitored assays. The preparation of 4-diphosphosphocytidyl-2C-methyl-D-erythritol 2-phosphate in multigram quantities is described for the first time.

Cyclobutane-type pyrimidine dimers generated by ultraviolet irradiation of DNA can be cleaved by DNA photolyase. The enzyme-catalysed reaction is believed to be initiated by the light-induced transfer of an electron from the anionic FADH- chromophore of the enzyme to the pyrimidine dimer. In this contribution, first infrared experiments using a novel E109A mutant of Escherichia coli DNA photolyase, which is catalytically active but unable to bind the second cofactor methenyltetrahydrofolate, are described. A stable blue-coloured form of the enzyme carrying a neutral FADH radical cofactor can be interpreted as an intermediate analogue of the light-driven DNA repair reaction and can be reduced to the enzymatically active FADH- form by red-light irradiation. Difference Fourier transform infrared (FT-IR) spectroscopy was used to monitor vibronic bands of the blue radical form and of the fully reduced FADH- form of the enzyme. Preliminary band assignments are based on experiments with 15N-labelled enzyme and on experiments with D2O as solvent. Difference FT-IR measurements were also used to observe the formation of thymidine dimers by ultraviolet irradiation and their repair by light-driven photolyase catalysis. This study provides the basis for future time-resolved FT-IR studies which are aimed at an elucidation of a detailed molecular picture of the light-driven DNA repair process.

Dihydroneopterin aldolase (DHNA) catalyses a retroaldol reaction yielding 6-hydroxymethyl-7,8-dihydropterin, a biosynthetic precursor of the vitamin, tetrahydrofolate. The enzyme is a potential target for antimicrobial and anti-parasite chemotherapy. A gene specifying a dihydroneopterin aldolase from Arabidopsis thaliana was expressed in a recombinant Escherichia coli strain. The recombinant protein was purified to apparent homogeneity and crystallised using polyethylenglycol as the precipitating agent. The crystal structure was solved by X-ray diffraction analysis at 2.2A resolution. The enzyme forms a D(4)-symmetric homooctamer. Each polypeptide chain is folded into a single domain comprising an antiparallel four-stranded beta-sheet and two long alpha-helices. Four monomers are arranged in a tetrameric ring, and two of these rings form a hollow cylinder. Well defined purine derivatives are found at all eight topologically equivalent active sites. The subunit fold of the enzyme is related to substructures of dihydroneopterin triphosphate epimerase, GTP cyclohydrolase I, and pyruvoyltetrahydropterin synthase, which are all involved in the biosynthesis of pteridine type cofactors, and to urate oxidase, although some members of that superfamily have no detectable sequence similarity. Due to structural and mechanistical differences of DHNA in comparison with class I and class II aldolases, a new aldolase class is proposed.

Dihydroneopterin aldolase (EC 4.1.2.25) is one of the enzymes of folate synthesis that remains to be cloned and characterized from plants. This enzyme catalyzes conversion of 7,8-dihydroneopterin (DHN) to 6-hydroxymethyl-7,8-dihydropterin, and is encoded by the folB gene in Escherichia coli. The E. coli FolB protein also mediates epimerization of DHN to 7,8-dihydromonapterin. Searches of the Arabidopsis genome detected three genes encoding substantially diverged FolB homologs (AtFolB1-3, sharing 57%-73% identity), for which cDNAs were isolated. A fourth cDNA specifying a FolB-like protein (LeFolB1) was obtained from tomato (Lycopersicon esculentum) by reverse transcription-PCR. When overproduced in E. coli, recombinant AtFolB1, AtFolB2, and LeFolB1 proteins all had both dihydroneopterin aldolase and epimerase activities, and carried out the aldol cleavage reaction on the epimerization product, 7,8-dihydromonapterin, as well as on DHN. AtFolB3, however, could not be expressed in active form. Size exclusion chromatography indicated that the plant enzyme is an octamer, like the bacterial enzyme. Quantifying expression of the Arabidopsis genes by real-time reverse transcription-PCR showed that AtFolB1 and AtFolB2 messages occur at low levels throughout the plant, whereas the AtFolB3 mRNA was detected only in siliques and only with an extremely low abundance. Sequence comparisons and phylogenetic analysis of FolB homologs from 16 plants indicated that their N-terminal regions are highly variable, and that most species have a small number of FolB genes that diverged after separation of the lineages leading to families. The substantial divergence of FolB homologs in Arabidopsis and other plants suggests that some of them may act on substrates other than DHN.

7,8-Dihydroneopterin aldolase catalyzes the formation of the tetrahydrofolate precursor, 6-hydroxymethyl-7,8-dihydropterin, and is a potential target for antimicrobial and anti-parasite chemotherapy. The last step of the enzyme-catalyzed reaction is believed to involve the protonation of an enol type intermediate. In order to study the stereochemical course of that reaction step, [1',2',3',6,7-13C5]dihydroneopterin was treated with aldolase in deuterated buffer. The resulting, partially deuterated [6alpha,6,7-13C3]6-hydroxymethyl-7,8-dihydropterin was converted to partially deuterated 6-(R)-[6,7,9,11-13C4]5,10-methylenetetrahydropteroate by a sequence of three enzyme-catalyzed reactions followed by treatment with [13C]formaldehyde. The product was analyzed by multinuclear NMR spectroscopy. The data show that the carbinol group of enzymatically formed 6-hydroxymethyl-dihydropterin contained 2H predominantly in the pro-S position.

Obelin is a photoprotein that emits light by Ca2+-binding. To develop a bioluminescent immunoassay based on the light emission property of obelin, we have expressed the apoobelin fusion protein with ZZ-domain of S. aureus protein A in E. coli by recombinant DNA techniques. The proZZ-obelin expressed was purified by one-step affinity chromatography on IgG-Agarose. The purified proZZ-obelin has both the luminescent activity of obelin and the IgG-binding ability of ZZ-domain. The specific activity of fusion protein was 8.5 X 10(15) photons per mg of protein.

A cDNA clone encoding the Ca(2+)-activated photoprotein, obelin (Obl), from Obelia longissima was sequenced. The nucleotide (nt) sequence contained two long overlapping open reading frames (ORFs), one of which encoded apoobelin (apoObl). The deduced amino acid (aa) sequence of apoObl revealed that this 195-aa protein has three EF-hand structures that are characteristic for Ca(2+)-binding domains. Strong aa homology was shown among apoObl, apoaequorin and apoclytin. The second ORF present in the obl cDNA consists of 139 codons and encodes a very basic protein with a calculated pI of 10.56 and a molecular mass of 16,153 Da.

The gyration radius (R0) of native streptokinase (SK) was found to be R0 = (40 +/- ) A by small-angle X-ray scattering. Experimental hydrodynamic characteristics of SK were S0(20),W = (2.8 +/- 0.1)S; D0(20),W = (6.0 +/- 0.5) x 10(-7) cm2/s; [n] = 0.12 dl/g. The molecular weight of the enzyme was found to be 44,000. The values of the form factor R0/Rsphere = 2.1 and the frictional ratio f/f0 = 1.5 indicate considerable anisometry of the SK molecule. Basing on the curves of small-angle X-ray scattering of SK modified with a synthetic linear copolymer of N-vinylpyrrolidone (P) at a molar ratio SK less than P, a structural model of the conjugate was proposed. The modified form consisted of a dense nucleus covered with a diffuse polymeric membrane. In accordance with the model, R0 of modified SK and of the whole conjugate were found to be R0nucleus = (34 +/- 2) A and R0conjugate = (114 +/- 5)A.

The ability to emulsify n-hexadecane was compared among ten strains of Candida lipolytica. The cultures were shown to differ in the activity of emulsification. The highest activity was recorded in the phase of growth deceleration. Substances involved in n-alkane emulsification were isolated.

The influence of aeration, pH and iron concentration on the growth of yeast C. lipolytica 704 on the hexadecane medium and on the synthesis of citric and isocitric acids was investigated. The yeast synthesized citric acids actively during intensive aeration. The acid formation was strongly dependent on the medium acidity: pH 6.0 was most favourable for the synthesis of citric acids. The Fe concentration influenced significantly the ratio of the acids synthesized. At a low concentration of iron (0.005 mg Fe/l) equal amounts of citrate and isocitrate were formed; at an increased concentration isocitrate was in predominant formation.

Bioemulsifiers and ether extracts from the cultural broth of yeast cells were used to study their effect of Candida lipolytica growth and ultrastructural organisation. When the emulsifiers and ether extracts were added to the growth medium, the lag phase was reduced but the growth rate did not change. The ether extracts increased the growth rate of C. lipolytica 374/2 and the final biomass yield of C. lipolytica 704. The ultrastructural organisation of C. lipolytica 374/2 cells changed under the action of the bioemulsifier added to the growth medium.

Experiments were carried out to examine the capacity of two strains of Candida lipolytica, producing citric and isocitric acids in the alkane and glucose containing media, to grow on different two- and three-carbon compounds. The strains did not grow on oxalate, glyoxalate, glycolate, malonate or propionate. When cultivated in the media containing acetate, ethanol, glycerol, glucose or hexadecane, supersynthesis of the acids started after complete consumption of the nitrogen source and resultant delay of the culture growth. Either strain discharged the two acids in a proportion that depended on the strain nature and the type of the carbon source. The mutant strain produced only citrate while the wild-type synthesized both citrate and isocitrate, the ratio of which was related to the nature of the carbon source utilized.