Structural elucidation of fungal polysaccharides isolated from the cell wall of Plectosphaerella cucumerina and Verticillium spp

The structure of acidic fungal polysaccharides isolated from the cell wall of Plectosphaerella cucumerina, Verticillium dahliae, and V. albo-atrum has been investigated by chemical analysis, methylation analysis, and 1D and 2D 1H and 13C NMR spectroscopy. The polysaccharides have an idealized repeating block of the type: [carbohydrates: see text] linked to a small mannan core (<15%), where n=13, m=13, p=5, and q=8 for P. cucumerina, and n=16, m=16, p=6, and q <1 for both Verticillium species.     

PhoR/PhoP two component regulatory system affects biocontrol capability of Bacillus subtilis NCD-2

The Bacillus subtilis strain NCD-2 is an important biocontrol agent against cotton verticillium wilt and cotton sore shin in the field, which are caused by Verticillium dahliae Kleb and Rhizoctonia solani Kuhn, respectively. A mutant of strain NCD-2, designated M216, with decreased antagonism to V. dahliae and R. solani, was selected by mini-Tn10 mutagenesis and in vitro virulence screening. The inserted gene in the mutant was cloned and identified as the phoR gene, which encodes a sensor kinase in the PhoP/PhoR two-component system. Compared to the wild-type strain, the APase activities of the mutant was decreased significantly when cultured in low phosphate medium, but no obvious difference was observed when cultured in high phosphate medium. The mutant also grew more slowly on organic phosphate agar and lost its phosphatidylcholine-solubilizing ability. The suppression of cotton seedling damping-off in vivo and colonization of the rhizosphere of cotton also decreased in the mutant strain when compared with the wild type strain. All of these characteristics could be partially restored by complementation of the phoR gene in the M216 mutant.     

Dimeric phenylphenalenones from Musa acuminata and various Haemodoraceae species. Crystal structure of anigorootin

Three fused octacyclic phenylphenalenone dimers were isolated from Musa acuminata: Anigorootin, which was first isolated from Anigozanthos flavidus and hitherto represented the only compound of that type, the new 4'-hydroxy-anigorootin, and 4',4"-di-hydroxy-anigorootin, which is a revised structure. The crystal structure of anigorootin was determined by X-ray crystallography. 3,3'-Bis-hydroxyanigorufone, a dimer of the conventional type known from Anigozanthos preissii, was also found in Musa acuminata. Phytochemical analysis of several Haemodoraceae species revealed the occurrence of anigorootin, 3,3'-bis-hydroxyanigorufone, and the novel metabolite 3,3'-bis-anigorufone. The occurrence of the same compounds in Musaceae and Haemodoraceae indicates the close chemotaxonomic relationship of both plant families.     

In vitro anti-influenza screening of several Euphorbiaceae species: structure of a bioactive Cyanoglucoside from Codiaeum variegatum

A bio-guided screening against influenza A virus (FLUAV) was carried out with seven Euphorbiaceae species. The results showed that chromatographic fractions from Phyllantus niruri, Euphorbia pulcherrima and Codiaeum variegatum had relevant anti-FLUAV activity, although only chromatographical subfractions from C. variegatum kept the activity. From this plant, the active compound against FLUAV was isolated. Its structure was assigned as 2-(3,4,5)-trihydroxy-6-hydroxymethyltetrahydropyran-2-yloxymethyl)acrylonitrile (1) on the basis of NMR, mass spectrometry and X-ray diffraction analysis. The compound displayed virucidal activity without impairment of haemagglutination properties of the used virus strain. This is the first report indicating antiviral activity of a cyanoglucoside.     

A lignan from the root of Ecbolium linneanum Kurz

From the chloroform extract of the root of Ecbolium linneanum Kurz., a furofuran type of unsymmetrical lignan named as Ecbolin A was isolated. The structure was established by spectroscopic methods and confirmed by single crystal X-ray diffraction studies.     

Microsecond hydrophobic collapse in the folding of Escherichia coli dihydrofolate reductase, an alpha/beta-type protein

Using small-angle X-ray scattering combined with a continuous-flow mixing device, we monitored the microsecond compaction dynamics in the folding of Escherichia coli dihydrofolate reductase, an alpha/beta-type protein. A significant collapse of the radius of gyration from 30 A to 23.2 A occurs within 300 micros after the initiation of refolding by a urea dilution jump. The subsequent folding after the major chain collapse occurs on a considerably longer time-scale. The protein folding trajectories constructed by comparing the development of the compactness and the secondary structure suggest that the specific hydrophobic collapse model rather than the framework model better explains the experimental observations. The folding trajectory of this alpha/beta-type protein is located between those of alpha-helical and beta-sheet proteins, suggesting that native structure determines the folding landscape.     

The 1.6A X-ray structure of the unusual c-type cytochrome, cytochrome cL, from the methylotrophic bacterium Methylobacterium extorquens

The structure of cytochrome cL from Methylobacterium extorquens has been determined by X-ray crystallography to a resolution of 1.6 A. This unusually large, acidic cytochrome is the physiological electron acceptor for the quinoprotein methanol dehydrogenase in the periplasm of methylotrophic bacteria. Its amino acid sequence is completely different from that of other cytochromes but its X-ray structure reveals a core that is typical of class I cytochromes c, having alpha-helices folded into a compact structure enclosing the single haem c prosthetic group and leaving one edge of the haem exposed. The haem is bound through thioether bonds to Cys65 and Cys68, and the fifth ligand to the haem iron is provided by His69. Remarkably, the sixth ligand is provided by His112, and not by Met109, which had been shown to be the sixth ligand in solution. Cytochrome cL is unusual in having a disulphide bridge that tethers the long C-terminal extension to the body of the structure. The crystal structure reveals that, close to the inner haem propionate, there is tightly bound calcium ion that is likely to be involved in stabilization of the redox potential, and that may be important in the flow of electrons from reduced pyrroloquinoline quinone in methanol dehydrogenase to the haem of cytochrome cL. As predicted, both haem propionates are exposed to solvent, accounting for the unusual influence of pH on the redox potential of this cytochrome.     

Cyathenosin A, a spiropyranosyl derivative of protocatechuic acid from Cyathea phalerata

Cyathenosin A, a spiropyranosyl derivative of protocatechuic acid was isolated from the stem pith of Cyathea phalerata Mart. Its structure was determined by MS, 1D and 2D NMR spectroscopic analyses and confirmed by single crystal X-ray analysis. Cyathenosin A is the first example of a naturally occurring compound containing a spirocyclic orthoester pyranosidic structure.     

An antifungal and plant growth promoting metabolite from a sterile dark ectotrophic fungus

A sterile dark ectotrophic fungus isolated from roots of an Australian native grass, Neurachne alopecuroidea produces compound 1 in liquid cultures. The structure of the metabolite was determined by spectroscopic and X-ray diffraction studies. The metabolite shows activity against phytopathogens and plant growth promoting activity, properties that are also expressed in vivo by the ectotrophic fungus.     

Antifungal diterpenes from Hypoestes serpens (Acanthaceae)

Two new diterpenes, fusicoserpenol A and dolabeserpenoic acid A, with antifungal activity, were isolated from leaves of Hypoestes serpens (Acanthaceae). Their structures were elucidated by means of spectrometric methods including 1D and 2D NMR experiments and MS analysis. X-ray crystallographic analysis confirmed the structure of fusicoserpenol A and established the relative configuration.     

Solid and solution state conformation of 1L-1-O-acetyl-2,3:5,6-di-O-isopropylidene-chiro-inositol

The X-ray crystal structure of 1L-1-O-acetyl-2,3:5,6-di-O-isopropylidene-chiro-inositol is described. The inositol ring deviates considerably from the ideal chair conformation to a flattened chair. A comparison of its conformation in solution with that in solid was made by the use of 1H NMR. This conformational analysis revealed that the title compound adopts similar conformations in solid state and in solution states irrespective of solvent polarity.     

Tricalysiosides H-O: Ent-kaurane glucosides from the leaves of Tricalysia dubia

Eight ent-kaurane glucosides, named tricalysiosides H-O, were isolated from Tricalysia dubia. Tricalysioside H possessed a hydroxyl group at the 1-position, to which the glucose moiety was attached. The structure was first elucidated by means of spectroscopic data analysis and finally confirmed by X-ray crystallography. Since acid hydrolysis of 1 gave D-glucose, the aglycone was proved to have an enantio-kaurane type skeleton. The structures of tricalysiosides I-O were mainly elucidated from analysis of spectroscopic evidence.     

Crystal structure of 1L-1,2:4,5-di-O-isopropylidene-allo-inositol: a comparison of its conformation in solid and solution states

The X-ray crystal structure of 1l-1,2:4,5-di-O-isopropylidene-allo-inositol, is described. The inositol ring deviate slightly from the ideal chair conformation to a flattened chair. A comparison of its conformation in solution with that in solid was made by the use of 1H NMR. This conformational analysis revealed that the title compound adopts similar conformations in solid state and in solution in low polar solvents like benzene and CHCl3 while in high polar solvents such as Me2SO, the solid state conformation is not retained.     

X-ray structure of floridoside isolated from the marine red algae Dilsea carnosa

The natural floridoside (2-O-alpha-d-galactopyranosylglycerol) was isolated from Dilsea carnosa, and its structure has been determined by single-crystal X-ray diffraction analysis. The solved structure is in agreement with the previously solved crystal structure of floridoside [Simon-Colin, C.; Michaud, F.; Léger, J.-M.; Deslandes E. Carbohydr. Res.2003, 338, 2413-2416] and demonstrates for the first time the presence of floridoside in the red algae Dilsea carnosa.     

Crystal structure of exo-inulinase from Aspergillus awamori: the enzyme fold and structural determinants of substrate recognition

Exo-inulinases hydrolyze terminal, non-reducing 2,1-linked and 2,6-linked beta-d-fructofuranose residues in inulin, levan and sucrose releasing beta-d-fructose. We present the X-ray structure at 1.55A resolution of exo-inulinase from Aspergillus awamori, a member of glycoside hydrolase family 32, solved by single isomorphous replacement with the anomalous scattering method using the heavy-atom sites derived from a quick cryo-soaking technique. The tertiary structure of this enzyme folds into two domains: the N-terminal catalytic domain of an unusual five-bladed beta-propeller fold and the C-terminal domain folded into a beta-sandwich-like structure. Its structural architecture is very similar to that of another member of glycoside hydrolase family 32, invertase (beta-fructosidase) from Thermotoga maritima, determined recently by X-ray crystallography The exo-inulinase is a glycoprotein containing five N-linked oligosaccharides. Two crystal forms obtained under similar crystallization conditions differ by the degree of protein glycosylation. The X-ray structure of the enzyme:fructose complex, at a resolution of 1.87A, reveals two catalytically important residues: Asp41 and Glu241, a nucleophile and a catalytic acid/base, respectively. The distance between the side-chains of these residues is consistent with a double displacement mechanism of reaction. Asp189, which is part of the Arg-Asp-Pro motif, provides hydrogen bonds important for substrate recognition.     

Multiple assembly states of lumazine synthase: a model relating catalytic function and molecular assembly

Lumazine synthases have been observed in the form of pentamers, dimers of pentamers, icosahedral capsids consisting of 60 subunits and larger capsids with unknown molecular structure. Here we describe the analysis of the assembly of native and mutant forms of lumazine synthases from Bacillus subtilis and Aquifex aeolicus at various pH values and in the presence of different buffers using small angle X-ray scattering and electron microscopy. Both wild-type lumazine synthases are able to form capsids with a diameter of roughly 160 A and larger capsids with diameters of around 300 A. The relative abundance of smaller and larger capsids is strongly dependent on buffer and pH. Both forms can co-exist and are in some cases accompanied by other incomplete or deformed capsids. Several mutants of the B. subtilis lumazine synthase, in which residues in or close to the active site were replaced, as well as an insertion mutant of A. aeolicus lumazine synthase form partially or exclusively larger capsids with a diameter of about 300 A. The mutations also reduce or inhibit enzymatic activity, suggesting that the catalytic function of the enzyme is tightly correlated with its quaternary structure. The data show that multiple assembly forms are a general feature of lumazine synthases.     

Structure and action of the N-oxygenase AurF from Streptomyces thioluteus

Nitro groups are found in a number of bioactive compounds. Most of them arise by a stepwise mono-oxygenation of amino groups. One of the involved enzymes is AurF participating in the biosynthesis of aureothin. Its structure was established at 2.1 A resolution showing a homodimer with a binuclear manganese cluster. The enzyme preparation, which yielded the analyzed crystals, showed activity using in vitro and in vivo assays. Chain fold and cluster are homologous with ribonucleotide reductase subunit R2 and related enzymes. The two manganese ions and an iron content of about 15% were established by anomalous X-ray diffraction. A comparison of the cluster with more common di-iron clusters suggested an additional histidine in the coordination sphere to cause the preference for manganese over iron. There is no oxo-bridge. The substrate p-amino-benzoate was modeled into the active center. The model is supported by mutant activity measurements. It shows the geometry of the reaction and explains the established substrate spectrum.     

X-ray structure of putative acyl-ACP desaturase DesA2 from Mycobacterium tuberculosis H37Rv

Genome sequencing showed that two proteins in Mycobacterium tuberculosis H37Rv contain the metal binding motif (D/E)X(2)HX(approximately 100)(D/E)X(2)H characteristic of the soluble diiron enzyme superfamily. These putative acyl-ACP desaturase genes desA1 and desA2 were cloned from genomic DNA and expressed in Escherichia coli BL21(DE3). DesA1 was found to be insoluble, but in contrast, DesA2 was a soluble protein amenable to biophysical characterization. Here, we report the 2.0 A resolution X-ray structure of DesA2 determined by multiple anomalous dispersion (MAD) phasing from a Se-met derivative and refinement against diffraction data obtained on the native protein. The X-ray structure shows that DesA2 is a homodimeric protein with a four-helix bundle core flanked by five additional helices that overlay with 192 structurally equivalent amino acids in the structure of stearoyl-ACP Delta9 desaturase from castor plant with an rms difference 1.42 A. In the DesA2 crystals, one metal (likely Mn from the crystallization buffer) was bound in high occupancy at the B-site of the conserved metal binding motif, while the A-site was not occupied by a metal ion. Instead, the amino group of Lys-76 occupied this position. The relationships between DesA2 and known diiron enzymes are discussed.     

Structure determination of fibrillarin from the hyperthermophilic archaeon Pyrococcus furiosus

The methyltransferase fibrillarin is the catalytic component of ribonucleoprotein complexes that direct site-specific methylation of precursor ribosomal RNA and are critical for ribosome biogenesis in eukaryotes and archaea. Here we report the crystal structure of a fibrillarin ortholog from the hyperthermophilic archaeon Pyrococcus furiosus at 1.97A resolution. Comparisons of the X-ray structures of fibrillarin orthologs from Methanococcus jannashii and Archaeoglobus fulgidus reveal nearly identical backbone configurations for the catalytic C-terminal domain with the exception of a unique loop conformation at the S-adenosyl-l-methionine (AdoMet) binding pocket in P. furiosus. In contrast, the N-terminal domains are divergent which may explain why some forms of fibrillarin apparently homodimerize (M. jannashii) while others are monomeric (P. furiosus and A. fulgidus). Three positively charged amino acids surround the AdoMet-binding site and sequence analysis indicates that this is a conserved feature of both eukaryotic and archaeal fibrillarins. We discuss the possibility that these basic residues of fibrillarin are important for RNA-guided rRNA methylation.