MTH187 from Methanobacterium thermoautotrophicum has three HEAT-like Repeats

With the completion of genome sequencing projects, there are a large number of proteins for which we have little or no functional information. Since protein function is closely related to three-dimensional conformation, structural proteomics is one avenue where the role of proteins with unknown function can be investigated. In the present structural project, the structure of MTH187 has been determined by solution-state NMR spectroscopy. This protein of 12.4 kDa is one of the 424 non-membrane proteins that were cloned and purified for the structural proteomic project of Methanobacterium thermoautotrophicum [Christendat, D., Yee, A., Dharamsi, A., Kluger, Y., Gerstein, M., Arrowsmith, C.H. and Edwards, A.M. (2000) Prog. Biophys. Mol. Biol., 73, 339–345]. Methanobacterium thermoautotrophicum is a thermophilic archaeon that grows optimally at 65 °C. A particular characteristic of this microorganism is its ability to generate methane from carbon dioxide and hydrogen [Smith, D.R., Doucette-Stamm, L.A., Deloughery, C., Lee, H., Dubois, J., Aldredge, T., Bashirzadeh, R., Blakely, D., Cook, R., Gilbert, K., Harrison, D., Hoang, L., Keagle, P., Lumm, W., Pothier, B., Qiu, D., Spadafora, R., Vicaire, R., Wang, Y., Wierzbowski, J., Gibson, R., Jiwani, N., Caruso, A., Bush, D., Reeve, J. N. et al. (1997) J. Bacteriol., 179, 7135–7155].Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users. Structure data have been deposited at PDB (1TE4) and NMR data at BMRB (5629).Olivier Julien and Isabelle Gignac - Both authors contributed equally to this work.     

Structure from NMR and molecular dynamics: Distance restraining inhibits motion in the essential subspace

Summary We address the question how well proteins can be modelled on the basis of NMR data, when these data are incorporated into the protein model using distance restraints in a molecular dynamics simulation. We found, using HPr as a model protein, that distance restraining freezes the essential motion of proteins, as defined by Amadei et al. [Amadei, A., Linssen, A.B.M. and Berendsen, H.J.C. (1993) Protein Struct. Funct. Genet., 17, 412–425]. We discuss how modelling protocols can be improved in order to solve this problem.     

Materials for the fungus flora of Japan (53)

Two interesting ascomycetes are described as new to Japan:Ascotricha amesii (Xylariaceae), isolated from garden soil in Osaka; andAuxarthron reticulatum, isolated from forest soil in Tokyo. ADicyma anamorph is associated with the former species. (52): Tubaki, K., Tokumasu, S. and Konno, M., Nippon Kingakukai Kaiho38: 249–250. 1997.     

Protein structure calculation with data imputation: the use of substitute restraints

The amount of experimental restraints e.g., NOEs is often too small for calculating high quality three-dimensional structures by restrained molecular dynamics. Considering this as a typical missing value problem we propose here a model based data imputation technique that should lead to an improved estimation of the correct structure. The novel automated method implemented in AUREMOL makes a more efficient use of the experimental information to obtain NMR structures with higher accuracy. It creates a large set of substitute restraints that are used either alone or together with the experimental restraints. The new approach was successfully tested on three examples: firstly, the Ras-binding domain of Byr2 from Schizosaccharomyces pombe, the mutant HPr (H15A) from Staphylococcus aureus, and a X-ray structure of human ubiquitin. In all three examples, the quality of the resulting final bundles was improved considerably by the use of additional substitute restraints, as assessed quantitatively by the calculation of RMSD values to the “true” structure and NMR R-factors directly calculated from the original NOESY spectra or the published diffraction data.     

Simultaneous determination of disulphide bridge topology and three-dimensional structure using ambiguous intersulphur distance restraints: Possibilities and limitations

Knowledge of the native disulphide bridge topology allows the introduction of conformational restraints between remote parts of the peptide chain. This information is therefore of great importance for the successful determination of the three-dimensional structure of cysteine-rich proteins by NMR spectroscopy. In this paper we investigate the limitations of using ambiguous intersulphur restraints [Nilges, M. (1995) J. Mol. Biol., 245, 645–660] associated with NMR experimental information to determine the native disulphide bridge pattern. Using these restraints in a simulated annealing protocol we have determined the correct topology of numerous examples, including a protein with seven disulphide bridges (phospholipase A₂) and a protein in which 25% of the total number of residues are cysteines (-conotoxin GIIIB). We have also characterised the behaviour of the method when only limited experimental data is available, and find that the proposed protocol permits disulphide bridge determination even with a small number of restraints (around 5 NOEs – including a long-range restraint – per residue). In addition, we have shown that under these conditions the use of a reduced penalty function allows the identification of misassigned NOE restraints. These results indicate that the use of ambiguous intersulphur distances with the proposed simulated annealing protocol is a general method for the determination of disulphide bridge topology, particularly interesting in the first steps of NMR study of cysteine-rich proteins. Comparison with previously proposed protocols indicates that the presented method is more reliable and the interpretation of results is straightforward.     

Bioactive metabolites from Alternaria brassicicola ML-P08, an endophytic fungus residing in Malus halliana

A total of 48 strains were isolated from the normal tissues of Malus halliana and the EtOAc extracts of their cultures were subjected to primary antimicrobial screening against four test bacteria and three fungi. As a result, 22 strains exhibited antimicrobial activity against at least one test microbe. Among them, Alternaria brassicicola ML-P08 showing strong activity (MICs: 0.31–2.50 mg/ml) was selected for further investigation on its secondary metabolites. Bioassay-guided fractionation of the EtOAc extract of its liquid culture afforded seven compounds, which were identified as alternariol (1), alternariol 9-methyl ether (2), altechromone A (3), herbarin A (4), cerevisterol (5), 3β,5α-dihydroxy-(22E,24R)-ergosta-7,22-dien-6-one (6) and 3β-hydroxy-(22E,24R)-ergosta-5,8,22-trien-7-one (7), respectively, by spectral means (MS, IR, ¹H- and ¹³C-NMR). In vitro antimicrobial assay showed that compound 3 was substantially active against Bacillus subtilis, Escherichia coli, Pseudomonas fluorescens and Candida albicans with the MICs of 3.9, 3.9, 1.8, and 3.9 μg/ml, respectively. Compound 4 also showed pronounced antifungal activity against Trichophyton rubrum and C. albicans with MICs of both 15.6 μg/ml. In addition, compound 1 exhibited strong xanthine oxidase inhibitory activity with the IC₅₀ of 15.5 μM, comparable to that of positive control, allopurinol (IC₅₀: 10.7 μM).     

Taxonomic studies in the Miconieae (Melastomataceae). VII. Miconia howardiana,a new speices from Hispaniola

Miconia howardiana, which is known only from a diverse moist montane forest in the vicinity of Loma Trocha de Pey (or “Monteada Nueva”), the easternmost peak of the Sierra de Baoruco, is described and illustrated. It is compared to phenetically similar (and probably phylogenetically related) species of theMiconia favosa complex, i.e.,M. favosa, M. xenotricha, M. campanensis M. Sintenisii, M. foveolata, andM. pycnoneura. The species of this complex are characterized by bullate leaves with frequently cordate bases and often six secondary veins. Most species also have large berries and flowers, 5-locular ovaries, large inflorescence bracts and bracteoles, and more or less dendritic multicellular hairs.     

A Voucher Specimen for <Emphasis Type="Italic">Macaca munzala</Emphasis>: Interspecific Affinities,Evolution, and Conservation of a Newly Discovered Primate

Sinha, A., Datta, A., Madhusudan, M. D., & Mishra, C. (2005. Macaca munzala: A new species from western Arunachal Pradesh, northeastern India. International Journal of Primatology, 26, 977–989) discovered Arunachal macaques (Macaca munzala), a species new to science, in the eastern Himalaya of Arunachal Pradesh in northeastern India. They depicted the holotype and paratypes of the species in photographs, and a specimen of the species had been unavailable for preservation and examination. In March 2005, we obtained an entire specimen of an adult male Macaca munzala, which we propose as a voucher specimen for the species. We provide detailed morphological and anatomical measurements of the specimen and examine its affinities with other macaques. Macaca munzala appears to be unique among macaques in craniodental size and structure, baculum, and aspects of caudal structure, while exhibiting affinities with the other members of the sinica-group to which it belongs. We summarize our insights on the origins and phylogeny of Macaca munzala. Finally, we review the current conservation status of the macaques, which are threatened by extensive hunting in the only 2 districts of Arunachal Pradesh where they are documented to occur.     

Novelties in Mascagnia (Malpighiaceae)

Seven new species ofMascagnia are described:M. tomentosa from southern Mexico and Central America;M. arenicola from the Guianas;M. riparia, M. tucuruensis, andM. velutina from Brazil;M. boliviana from Bolivia; andM. australis from Argentina and Paraguay. These novelties were previously included in the “M. sepium-complex,” an omniumgatherum comprising superficially similar yellow-flowered taxa.     

Purification and characterization of UDP-glucose: anthocyanin 3′,5′-<Emphasis Type="Italic">O</Emphasis>-glucosyltransferase from <Emphasis Type="Italic">Clitoria ternatea</Emphasis>

A UDP-glucose: anthocyanin 3′,5′-O-glucosyltransferase (UA3′5′GT) (EC 2.4.1.-) was purified from the petals of Clitoria ternatea L. (Phaseoleae), which accumulate polyacylated anthocyanins named ternatins. In the biosynthesis of ternatins, delphinidin 3-O-(6″-O-malonyl)-β-glucoside (1) is first converted to delphinidin 3-O-(6″-O-malonyl)-β-glucoside-3′-O-β-glucoside (2). Then 2 is converted to ternatin C5 (3), which is delphinidin 3-O-(6″-O-malonyl)-β-glucoside-3′,5′-di-O-β-glucoside. UA3′5′GT is responsible for these two steps by transferring two glucosyl groups in a stepwise manner. Its substrate specificity revealed the regioselectivity to the anthocyanin′s 3′- or 5′-OH groups. Its kinetic properties showed comparable k _cat values for 1 and 2, suggesting the subequality of these anthocyanins as substrates. However, the apparent K _m value for 1 (3.89 × 10⁻⁵ M), which is lower than that for 2 (1.38 × 10⁻⁴ M), renders the k _cat/K _m value for 1 smaller, making 1 catalytically more efficient than 2. Although the apparent K _m value for UDP-glucose (6.18 × 10⁻³ M) with saturated 2 is larger than that for UDP-glucose (1.49 × 10⁻³ M) with saturated 1, the k _cat values are almost the same, suggesting the UDP-glucose binding inhibition by 2 as a product. UA3′5′GT turns the product 2 into a substrate possibly by reversing the B-ring of 2 along the C2-C1′ single bond axis so that the 5′-OH group of 2 can point toward the catalytic center. K. Kogawa, N. Kato, K. Kazuma, and N. Noda contributed equally to this work.     

New species of sap beetles (Coleoptera: Nitidulidae: Nitidulini) from the Baltic and Bitterfeld ambers

A new genus, Microsoronia, gen. nov., and new species of this genus, M. hoffeinsorum, sp. nov. from the Bitterfeld amber and M. kerneggeri sp. nov., M. nigerrima sp. nov., and M. interfax, sp. nov. from the Baltic amber, are described. The earliest known member of the genus Phenolia, P. (Lasiodites) angustitibialis, sp. nov., is described from the Baltic amber. The systematic position of these two genera, their possible evolution, as well as the possible ecology and bionomics of their members are discussed. It is shown that “Phenolia” incapax Scudder, 1890 should be included in the family Peltidae, rather than Nitidulidae.     

Zapoteca quichoi (Leguminosae, Mimosoideae), a new species from southeastern Mexico

Zapoteca quichoi, a new species endemic to southern Tabasco, Mexico, is described and illustrated. This species exhibits morphological characteristics that place it within subgenus Amazonica, together with Z. amazonica and Z. microcephala, from Peru and Colombia, respectively.

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Resumen  Se describe e ilustra Zapoteca quichoi (Leguminosae, Mimosoideae), una nueva especie de Tabasco, México. Esta especie exhibe características morfológicas que la ubican dentro del subgénero Amazonica, junto con Z. amazonica y Z. microcephala, especies nativas de Perú y Colombia respectivamente.

     

Pictolejeunea reginac, a new species of Lejeuneaceae (Hepaticae) from Venezuela

Pictolejeunea reginae, a new species from Venezuela is described and illustrated. It is the type of the new subgenusNeopitolejeunea Ilkiu-Borges, which is characterized by entire underleaves. A key to the two subgenera and four known species ofPictolejeunea is provided.

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Ilkiu-Borges, A. L. (Museu Paraense Emílio Goeldi, C. P. 399, Belém, Pará, CEP 66040-170. Brasil: email: ilkiuborges@hotmail.com).Pictolejeunea reginae, a new species of Lejeuneaceae (Hepaticae) from Venezuela. Brittonia 54: 318–321. 2002.—Pictolejeunea reginae, uma nova espécie encontrada na Venezuela, é descrita e ilustrada. A espécie é tratada como tipo do novo subgêneroNeopictolejeunea Ilkiu-Borges, caracterizada pelos anfigastros inteiros. é apresentada uma chave para os dois subgêneros e as quatro espécies dePictolejeunea.

     

Antifouling Activity of Bromotyrosine-Derived Sponge Metabolites and Synthetic Analogues

Eighteen brominated sponge-derived metabolites and synthetic analogues were analyzed for antilarval settlement of Balanus improvisus. Only compounds exhibiting oxime substituents including bastadin-3 (4), −4 (1), −9 (2), and −16 (3), hemibastadin-1 (6), aplysamine-2 (5), and psammaplin A (10) turned out to inhibit larval settling at 1 to 10 μM. Analogues of hemibastadin-1 (6) were synthesized and tested for structure activity studies. Debromohemibastadin-1 (8) inhibited settling of B. improvisus, albeit at lower concentrations than hemibastadin-1 (6). Both 6 and 8 also induced cyprid mortality. 5,5′-dibromohemibastadin-1 (7) proved to be nontoxic, but settlement inhibition was observed at 10 μM. Tyrosinyltyramine (9), lacking the oxime function, was not antifouling active and was non-toxic at 100 μM. Hemibastadin-1 (6) and the synthetic products showed no general toxicity when tested against brine shrimp larvae. In contrast to the lipophilic psammaplin A (10), the hydrophilic sulfated psammaplin A derivative (11) showed no antifouling activity even though it contains an oxime group. We therefore hypothesize that the compound needs to cross membranes (probably by diffusion) and that the target for psammaplin A lies intracellularly.     

Pressure-dependent <Superscript>13</Superscript>C chemical shifts in proteins: origins and applications

Pressure-dependent ¹³C chemical shifts have been measured for aliphatic carbons in barnase and Protein G. Up to 200 MPa (2 kbar), most shift changes are linear, demonstrating pressure-independent compressibilities. CH₃, CH₂ and CH carbon shifts change on average by +0.23, −0.09 and −0.18 ppm, respectively, due to a combination of bond shortening and changes in bond angles, the latter matching one explanation for the γ-gauche effect. In addition, there is a residue-specific component, arising from both local compression and conformational change. To assess the relative magnitudes of these effects, residue-specific shift changes for protein G were converted into structural restraints and used to calculate the change in structure with pressure, using a genetic algorithm to convert shift changes into dihedral angle restraints. The results demonstrate that residual ¹³Cα shifts are dominated by dihedral angle changes and can be used to calculate structural change, whereas ¹³Cβ shifts retain significant dependence on local compression, making them less useful as structural restraints.     

New lichenicolous, muscicolous, corticolous and lignicolous taxa of Burgoa s. l. and Marchandiomyces s. l. (anamorphic Basidiomycota), a new genus for Omphalinafoliacea, and a catalogue and a key to the non-lichenized, bulbilliferous basidiomycetes

A catalogue and a key to the non-lichenized, bulbilliferous basidiomycetes are given. The new genus Burgella is described for the lichenicolous B. flavoparmeliae, phylogenetically close to Sistotrema oblongisporum and Multiclavula. The genera Pneumatospora and Tricellulortus are placed in synonymy of Minimedusa, the new combination M. obcoronata is proposed, and the new facultative lichenicolous M. pubescens is described. The new facultative lichenicolous Burgoa angulosa is phylogenetically close to the generic type B. verzuoliana, whilst the new B. moriformis and B. splendens are provisionally included in the genus Burgoa. A Burgoa-like species in the Ceratobasidiaceae is left unnamed. Two new species of Marchandiomyces, M. buckii and M. nothofagicola, are described. As Marchandiomyces aurantiacus is phylogenetically more close to Erythricium than to Marchandiomyces, it is proposed to exclude it from that genus and to use the holomorphic generic name Marchandiobasidium for both anamorph and teleomorph of this species. The new genus Marchandiomphalina is introduced for the lichenized Omphalina foliacea, a taxon phylogenetically close to Marchandiobasidium. Taxonomic novelties Burgella Diederich & Lawrey; B. flavoparmeliae Diederich & Lawrey; Burgoa angulosa Diederich, Lawrey & Etayo; B. moriformis Diederich, Ertz & Coppins; B. splendens Diederich & Coppins; Marchandiomphalina Diederich, Lawrey & Binder; Marchandiomphalina foliacea (P. M. J?rg.) Diederich, Lawrey & Binder; Marchandiomyces buckii Diederich & Lawrey; M. nothofagicola Diederich & Lawrey; Minimedusa pubescens Diederich, Lawrey & Heylen; M. obcoronata (B. Sutton, Kuthub. & Muid) Diederich & Lawrey     

Exhaustive enumeration of protein conformations using experimental restraints.

We present an efficient new algorithm that enumerates all possible conformations of a protein that satisfy a given set of distance restraints. Rapid growth of all possible self-avoiding conformations on the diamond lattice provides construction of alpha-carbon representations of a protein fold. We investigated the dependence of the number of conformations on pairwise distance restraints for the proteins crambin, pancreatic trypsin inhibitor, and ubiquitin. Knowledge of between one and two contacts per monomer is shown to be sufficient to restrict the number of candidate structures to approximately 1,000 conformations. Pairwise RMS deviations of atomic position comparisons between pairs of these 1,000 structures revealed that these conformations can be grouped into about 25 families of structures. These results suggest a new approach to assessing alternative protein folds given a very limited number of distance restraints. Such restraints are available from several experimental techniques such as NMR, NOESY, energy transfer fluorescence spectroscopy, and crosslinking experiments. This work focuses on exhaustive enumeration of protein structures with emphasis on the possible use of NOESY-determined distance restraints.     

Schistosoma mansoni:The Circulating Cathodic Antigen Forms an Abundant Product of 41/42 kDa in the Urine of Infected Patients

Abdeen, H. H., Attallah, A.-F. M., El-Mohamady, H. I., Harrison, R. A., and Mansour, M. M. 1998.Schistosoma mansoni: The circulating cathodic antigen forms an abundant product of 41/42 kDa in the urine of infected patients.Experimental Parasitology90, 286–289.     

Influence of oxidative and nitrosative stress on accumulation of diphosphate intermediates of the non-mevalonate pathway of isoprenoid biosynthesis in corynebacteria and mycobacteria

Artificial generation of oxygen superoxide radicals in actively growing cultures of Mycobacterium tuberculosis, Myc. smegmatis, and Corynebacterium ammoniagenes is followed by accumulation in the bacterial cells of substantial amounts of 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MEcDP) — an intermediate of the non-mevalonate pathway of isoprenoid biosynthesis (MEP) — most possibly due to the interaction of the oxygen radicals with the 4Fe-4S group in the active center and inhibition of the enzyme (E)-4-oxy-3-methylbut-2-enyl diphosphate synthase (IspG). Cadmium ions known to inhibit IspG enzyme in chloroplasts (Rivasseau, C., Seemann, M., Boisson, A. M., Streb, P., Gout, E., Douce, R., Rohmer, M., and Bligny, R. (2009) Plant Cell Environ., 32, 82–92), when added to culture of Myc. smegmatis, substantially increase accumulation of MEcDP induced by oxidative stress with no accumulation of other organic phosphate intermediates in the cell. Corynebacterium ammoniagenes, well-known for its ability to synthesize large amounts of MEcDP, was also shown to accumulate this unique cyclodiphosphate in actively growing culture when NO at low concentration is artificially generated in the medium. A possible role of the MEP-pathway of isoprenoid biosynthesis and a role of its central intermediate MEcDP in bacterial response to nitrosative and oxidative stress is discussed.