Structure of a peptidoglycan-related polysaccharide from <Emphasis Type="Italic">Providencia alcalifaciens</Emphasis> O45

A polysaccharide was isolated from the opportunistic human pathogen Providencia alcalifaciens O45:H26 by extraction with aqueous phenol and studied by sugar and methylation analyses along with ¹H and ¹³C NMR spectroscopy, including two-dimensional ROESY and H-detected ¹H,¹³C HSQC experiments. The polysaccharide contains N-acetylglu-cosamine and N-acetylmuramic acid (D-GlcpNAc3Rlac) amidated with L-alanine and has the following structure:

$\to 4) - \beta - D - GlcpNAc - (1 \to 4) - \beta - D - GlcpNAc3(Rlac - L - Ala) - (1 \to .$

The polysaccharide possesses a remarkable structural similarity to the bacterial cell wall peptidoglycan. It is not unique to the strain studied but is common to strains of at least four P. alcalifaciens O-serogroups (O3, O24, O38, and O45). No evidence was obtained that the polysaccharide is associated with the LPS, and hence it might represent a bacterial capsule component.

     

Near-complete backbone resonance assignments of acid-denatured human cytochrome <Emphasis Type="Italic">c</Emphasis> in dimethylsulfoxide: a prelude to studying interactions with phospholipids

Human cytochrome c plays a central role in the mitochondrial electron transfer chain and in the intrinsic apoptosis pathway. Through the interaction with the phospholipid cardiolipin, cytochrome c triggers release of pro-apoptotic factors, including itself, from the mitochondrion into the cytosol of cells undergoing apoptosis. The cytochrome c/cardiolipin complex has been extensively studied through various spectroscopies, most recently with high-field solution and solid-state NMR spectroscopies, but there is no agreement between the various studies on key structural features of cytochrome c in its complex with cardiolipin. In the present study, we report backbone ¹H, ¹³C, ¹⁵N resonance assignments of acid-denatured human cytochrome c in the aprotic solvent dimethylsulfoxide. These have led to the assignment of a reference 2D ¹H-¹⁵N HSQC spectrum in which out of the 99 non-proline residues 87% of the backbone amides are assigned. These assignments are being used in an interrupted H/D exchange strategy to map the binding site of cardiolipin on human cytochrome c.     

<Superscript>15</Superscript>N photo-CIDNP MAS NMR analysis of reaction centers of <Emphasis Type="Italic">Chloracidobacterium thermophilum</Emphasis>

Photochemically induced dynamic nuclear polarization (photo-CIDNP) has been observed in the homodimeric, type-1 photochemical reaction centers (RCs) of the acidobacterium, Chloracidobacterium (Cab.) thermophilum, by ¹⁵N magic-angle spinning (MAS) solid-state NMR under continuous white-light illumination. Three light-induced emissive (negative) signals are detected. In the RCs of Cab. thermophilum, three types of (bacterio)chlorophylls have previously been identified: bacteriochlorophyll a (BChl a), chlorophyll a (Chl a), and Zn-bacteriochlorophyll a′ (Zn-BChl a′) (Tsukatani et al. in J Biol Chem 287:5720–5732, 2012). Based upon experimental and quantum chemical ¹⁵N NMR data, we assign the observed signals to a Chl a cofactor. We exclude Zn-BChl because of its measured spectroscopic properties. We conclude that Chl a is the primary electron acceptor, which implies that the primary donor is most likely Zn-BChl a′. Chl a and 8¹-OH Chl a have been shown to be the primary electron acceptors in green sulfur bacteria and heliobacteria, respectively, and thus a Chl a molecule serves this role in all known homodimeric type-1 RCs.     

Backbone chemical shift assignments for <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> peroxiredoxin Q in the reduced and oxidized states: a dramatic change in backbone dynamics

Peroxiredoxins (Prx) are ubiquitous enzymes that reduce peroxides as part of antioxidant defenses and redox signaling. While Prx catalytic activity and sensitivity to hyperoxidative inactivation depend on their dynamic properties, there are few examples where their dynamics has been characterized by NMR spectroscopy. Here, we provide a foundation for studies of the solution properties of peroxiredoxin Q from the plant pathogen Xanthomonas campestris (XcPrxQ) by assigning the observable ¹H^N, ¹⁵N, ¹³C^α, ¹³C^β, and ¹³C′ chemical shifts for both the reduced (dithiol) and oxidized (disulfide) states. In the reduced state, most of the backbone amide resonances (149/152, 98 %) can be assigned in the XcPrxQ ¹H–¹⁵N HSQC spectrum. In contrast, a remarkable 51 % (77) of these amide resonances are not visible in the ¹H–¹⁵N HSQC spectrum of the disulfide state of the enzyme, indicating a substantial change in backbone dynamics associated with the formation of an intramolecular C48–C84 disulfide bond.     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N and <Superscript>13</Superscript>C resonance assignments of the C-terminal domain of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> TolA protein

Vibrio cholerae is the bacterial causative agent of the human disease cholera. Non-pathogenic bacterium can be converted to pathogenic following infection by a filamentous phage, CTXΦ, that carries the cholera toxin encoding genes. A crucial step during phage infection requires a direct interaction between the CTXΦ minor coat protein (pIII^CTX) and the C-terminal domain of V. cholerae TolA protein (TolAIIIvc). In order to get a better understanding of TolA function during the infection process, we have initiated a study of the V. cholerae TolAIII domain by 2D and 3D heteronuclear NMR. With the exception of the His-tag (H123–H128), 97 % of backbone ¹H, ¹⁵N and ¹³C resonances were assigned and the side chain assignments for 92 % of the protein were obtained (BMRB deposit with accession number 25689).     

Real-time pure shift <Superscript>15</Superscript>N HSQC of proteins: a real improvement in resolution and sensitivity

Spectral resolution in proton NMR spectroscopy is reduced by the splitting of resonances into multiplets due to the effect of homonuclear scalar couplings. Although these effects are often hidden in protein NMR spectroscopy by low digital resolution and routine apodization, behind the scenes homonuclear scalar couplings increase spectral overcrowding. The possibilities for biomolecular NMR offered by new pure shift NMR methods are illustrated here. Both resolution and sensitivity are improved, without any increase in experiment time. In these experiments, free induction decays are collected in short bursts of data acquisition, with durations short on the timescale of J-evolution, interspersed with suitable refocusing elements. The net effect is real-time (t ₂) broadband homodecoupling, suppressing the multiplet structure caused by proton–proton interactions. The key feature of the refocusing elements is that they discriminate between the resonances of active (observed) and passive (coupling partner) spins. This can be achieved either by using band-selective refocusing or by the BIRD element, in both cases accompanied by a nonselective 180° proton pulse. The latter method selects the active spins based on their one-bond heteronuclear J-coupling to ¹⁵N, while the former selects a region of the ¹H spectrum. Several novel pure shift experiments are presented, and the improvements in resolution and sensitivity they provide are evaluated for representative samples: the N-terminal domain of PGK; ubiquitin; and two mutants of the small antifungal protein PAF. These new experiments, delivering improved sensitivity and resolution, have the potential to replace the current standard HSQC experiments.     

Two distinct <Emphasis Type="Italic">waxy</Emphasis> alleles impact the granule-bound starch synthase in sorghum

The granule-bound starch synthase (GBSS) is the enzyme responsible for amylose synthesis in starch granules. Loss of GBSS activity results in starch granules containing mostly amylopectin and little or no amylose, a phenotype described as waxy. Previously, two phenotypic classes of waxy alleles were identified in sorghum (Sorghum bicolor L. Moench) characterized by the absence (waxy ^a ; wx ^a ) or presence (waxy ^b ; wx ^b ) of the GBSS protein in the endosperm. To characterize these alleles, we examined endosperm architecture using scanning electron microscopy (SEM), assayed GBSS enzymatic activities, and identified DNA lesions associated with the mutations in the GBSS (Sb10g002140) gene. wx ^a , the allele present in B Tx630 and R Tx2907, contained a large insertion in the third exon, which was consistent with the absence of the GBSS protein previously observed. wx ^b , the allele present in B 9307 and B TxARG1, contained a missense mutation that resulted in conversion of glutamine 268 to histidine in a conserved domain in starch synthases. In wx ^b , GBSS activity was less than 25% that of the non-waxy line B Wheatland, and GBSS activity was not detected in wx ^a . SEM showed that endosperm architecture was very similar in both wx ^a and wx ^b alleles, but altered in comparison to non-waxy lines R Tx430 and B Wheatland. Both alleles may have a range of potential applications in grain sorghum because of low amylose content in their starch and the presence or absence of the GBSS protein. PCR based markers were developed for both the wx ^a and the wx ^b alleles to aid in molecular breeding of low amylose sorghum.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N NMR assignments of a bacterial immunoglobulin-like domain (group 2) of a protein of a bacterium <Emphasis Type="Italic">Paenarthrobacter aurescens</Emphasis> TC1

The bacterial immunoglobulin-like (Big) domain is one of the prevalent domain types, which facilitates cell–cell adhesion by assembling into multi-domain architectures. We selected a four Big_2 domain protein (named ‘Arig’) from a Gram positive, Paenarthrobacter aurescens TC1 (known earlier as Arthrobacter aurescens TC1). In an attempt to characterize structural and ligand-binding features of individual Big_2 domains, we have cloned, overexpressed, isolated and purified the second Big_2 domain of Arig along with a few of its adjacent Big_2 domain residues (residue 143 to 269) referred to as ‘Arig2’. The ¹³C/¹⁵N-doubly-labeled His-tagged Arig2 (133 residues long) showed an ordered conformation as revealed by the well dispersed 2D [¹⁵N-¹H]-HSQC spectrum. Subsequently, a suite of heteronuclear 3D NMR experiments has enabled almost complete ¹H, ¹³C and ¹⁵N NMR resonance assignments of Arig2.     

Synthesis and antibacterial activity of some new benzo[5,6]chromeno[2,3-<Emphasis Type="Italic">d</Emphasis>]pyrimidines

The synthesis and antibacterial activity of some new benzo[5,6]chromeno[2,3-d]pyrimidine derivatives are described. The title compounds were obtained by the reaction of 1H-benzo[f]chromenes with aliphatic and aromatic amines. The structures of all newly synthesized compounds were confirmed by IR, ¹HNMR, ¹³C NMR, and NOESY experiments. The compounds exhibited potent antibacterial activity against gram-positive and gram-negative bacterial species. 10-Methyl-12-(4-hydroxyphenyl)-10,12-dihydro-11H-benzo[5,6]chromeno[2,3-d] pyrimidin-11-imine displayed greater antibacterial activity against gramnegative bacterial species than did ciprofloxacinandamoxicillin.     

Laccase-Catalyzed Heterocoupling of Dihydroquercetin and <Emphasis Type="Italic">p</Emphasis>-Aminobenzoic Acid: Effect of the Reaction Product on Cultured Cells

Derivatization of the natural flavonoid dihydroquercetin with p-aminobenzoic acid was carried out in an ethyl acetate/citric buffer biphasic system using laccase from the fungus Trametes hirsuta. The main reaction product yield was ~68 mol %. The product was characterized by ¹H NMR, ¹³C NMR, and liquid chromatography-mass spectroscopy, and its structure was elucidated. The reaction product affected viability of cultured human rhabdomyosarcoma cells (RD cell line) in a dose-dependent manner and, therefore, can be of interest to pharmaceutical industry.     

Protein–nucleotide contacts in motor proteins detected by DNP-enhanced solid-state NMR

DNP (dynamic nuclear polarization)-enhanced solid-state NMR is employed to directly detect protein–DNA and protein–ATP interactions and identify the residue type establishing the intermolecular contacts. While conventional solid-state NMR can detect protein–DNA interactions in large oligomeric protein assemblies in favorable cases, it typically suffers from low signal-to-noise ratios. We show here, for the oligomeric DnaB helicase from Helicobacter pylori complexed with ADP and single-stranded DNA, that this limitation can be overcome by using DNP-enhanced spectroscopy. Interactions are established by DNP-enhanced ³¹P–¹³C polarization-transfer experiments followed by the recording of a 2D ¹³C–¹³C correlation experiment. The NMR spectra were obtained in less than 2 days and allowed the identification of residues of the motor protein involved in nucleotide binding.     

A highly branched storage polyglucan in the thermoacidophilic red microalga <Emphasis Type="Italic">Galdieria maxima</Emphasis> cells

The thermoacidophilic red alga Galdieria maxima is capable of heterotrophic growth. The content of carbohydrates in G. maxima grown heterotrophically increases by a factor of 4, reaching as much as 60% of cell dry weight. The increase in the level of carbohydrates in cells is due to accumulation of a storage α-glycan. According to a specific cleavage to glucose catalyzed by amyloglucosidase and the high positive specific optical rotation characteristic of polyglucans, this polysaccharide can be classified as a floridean starch. The data of ¹H NMR spectroscopy and the results of methylation showed that the average length of the unbranched regions of the polysaccharide molecule is six to seven glucose residues. The degree of branching of the starch molecule of G. maxima is greater than that of storage polysaccharides of other red algae, glycogens of yeast, and phytoglycogens of cyanobacteria.     

The conformation of the Congo-red ligand bound to amyloid fibrils HET-s(218–289): a solid-state NMR study

We have previously shown that Congo red (CR) binds site specifically to amyloid fibrils formed by HET-s(218–289) with the long axis of the CR molecule almost parallel to the fibril axis. HADDOCK docking studies indicated that CR adopts a roughly planar conformation with the torsion angle ? characterizing the relative orientation of the two phenyl rings being a few degrees. In this study, we experimentally determine the torsion angle ? at the center of the CR molecule when bound to HET-s(218–289) amyloid fibrils using solid-state NMR tensor-correlation experiments. The method described here relies on the site-specific ¹³C labeling of CR and on the analysis of the two-dimensional magic-angle spinning tensor-correlation spectrum of ¹³C₂-CR. We determined the torsion angle ? to be 19°.     

Backbone and side-chain resonance assignments for the tmRNA-binding protein,SmpB, from <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

Small protein B (SmpB) is an essential molecule in trans-translation which is a universal biological pathway for protein synthesis in bacteria. Trans-translation can release stalled ribosomes from defective mRNAs and target tag-protein fragments for degradation, and then restart protein synthesis. The SmpB-tmRNA complex coordinating with other components of the trans-translation system, plays vital roles in Mycobacterium tuberculosis under both stress conditions and non-replicating conditions. Thus, elucidation of molecular details and dynamic properties of the SmpB-tmRNA interaction is a crucial step towards effectively blocking trans-translation process to shorten the duration of tuberculosis treatment. Here, we report resonance assignments for ¹H, ¹³C and ¹⁵N of M. tuberculosis SmpB (MtSmpB, spanning residues 4–133) protein determined by a suite of 2D/3D heteronuclear NMR experiments along with predicted the secondary structure.     

Chemical shift assignments of Ribosomal protein S1 from <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

RpsA, also known as ribosomal protein S1, is an essential protein required for translation initiation of mRNAs when their Shine-Dalgarno sequence is degenerated (Sorensen et al. 1998). In addition, RpsA of Mycobacterium tuberculosis (M. tb) is involved in trans-translation, which is an effective system mediated by tmRNA-SmpB to release stalled ribosomes from mRNA in the presence of rare codons (Keiler 2008). Shi et al. found that POA binds to RpsA of Mtb and disrupts the formation of RpsA–tmRNA complex (Shi et al. 2011) and mutations at the C-terminus of RpsA confer PZA resistance. The previous work reported the pyrazinoic acid-binding domain of RpsA (Yang et al. Mol Microbiol 95:791–803, 2015). However, the HSQC spectra of the isolated S1 domain does not overlap with that of MtRpsA280-438, suggesting that substantial interactions occur between the flexible C-terminus and the S1 domain in MtRpsA .To further study the PZA resistance and how substantial interactions influence/affect protein structure, using heteronuclear NMR spectroscopy, we have completed backbone and side-chain ¹H, ¹⁵N, ¹³C chemical shift assignments of MtRpsA280-438 which contains S1 domain and the flexible C-terminus. These NMR resonance assignments provide the framework for detailed characterization of the solution-state protein structure determination, dynamic studies of this domain, as well as NMR-based drug discovery efforts.     

A tomato plastidic ATP/ADP transporter gene <Emphasis Type="Italic">SlAATP</Emphasis> increases starch content in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

A plastidic ATP/ADP transporter (AATP) is responsible for importing ATP from the cytosol into plastids. Increasing the ATP supply is a potential way to facilitate anabolic synthesis in heterotrophic plastids of plants. In this work, a gene encoding the AATP protein, named SlAATP, was successfully isolated from tomato. Expression of SlAATP was induced by exogenous sucrose treatment in tomato. The coding region of SlAATP was cloned into a binary vector under the control of 35S promoter and then transformed into Arabidopsis to obtain transgenic plants. Constitutive expression of SlAATP significantly increased the starch accumulation in the transgenic plants. Real-time quantitative PCR (qRT-PCR) analysis showed that constitutive expression of StAATP up-regulated the expression of phosphoglucomutase (AtPGM), ADP-glucose pyrophosphorylase (AtAGPase), granule-bound starch synthase (AtGBSS I and AtGBSS II), soluble starch synthases (AtSSS I, AtSSS II, AtSSS III and AtSSS IV) and starch branching enzyme (AtSBE I and AtSBE II) genes involved in starch biosynthesis in the transgenic Arabidopsis plants. Meanwhile, enzymatic analyses indicated that the major enzymes (AGPase, GBSS, SSS and SBE) involved in the starch biosynthesis exhibited higher activities in the transgenic plants compared to the wild-type (WT). These findings suggest that SlAATP may improve starch content of Arabidopsis by up-regulating the expression of the related genes and increasing the activities of the major enzymes invovled in starch biosynthesis. The manipulation of SlAATP expression might be used for increasing starch accumulation of plants in the future.     

Backbone and side-chain resonance assignments of <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> SUMO

One of the most debilitating diseases Malaria, in its different forms, is caused by protozoan of Plasmodium species. Deadliest among these forms is the “cerebral malaria” which is afflicted upon by Plasmodium falciparum. Plasmodium adopts numerous strategies including various post-translational modifications (PTMs) to infect and survive in the human host. These PTMs have proven their critical requirement in the Plasmodium biology. Recently, sumoylation has been characterized as one of the important PTMs and many of its putative substrates have been identified in Plasmodium. Sumoylation is the covalent attachment of SUMO protein to the substrate protein, which is mediated by an enzyme cascade involving activating (E1), conjugating (E2), and ligating enzymes (E3). Here, we report resonance assignment for ¹H, ¹³C and ¹⁵N of Plasmodium falciparum SUMO (Pf-SUMO) protein determined by various 2D and 3D heteronuclear NMR experiments along with predicted secondary structures.     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N, <Superscript>13</Superscript>C backbone resonance assignment of the C-terminal domain of enzyme I from <Emphasis Type="Italic">Thermoanaerobacter tengcongensis</Emphasis>

Phosphoenolpyruvate binding to the C-terminal domain (EIC) of enzyme I of the bacterial phosphotransferase system (PTS) initiates a phosphorylation cascade that results in sugar translocation across the cell membrane and controls a large number of essential pathways in bacterial metabolism. EIC undergoes an expanded to compact conformational equilibrium that is regulated by ligand binding and determines the phosphorylation state of the overall PTS. Here, we report the backbone ¹H, ¹⁵N and ¹³C chemical shift assignments of the 70 kDa EIC dimer from the thermophilic bacterium Thermoanaerobacter tengcongensis. Assignments were obtained at 70 °C by heteronuclear multidimensional NMR spectroscopy. In total, 90% of all backbone resonances were assigned, with 264 out of a possible 299 residues assigned in the ¹H–¹⁵N TROSY spectrum. The secondary structure predicted from the assigned backbone resonance using the program TALOS+ is in good agreement with the X-ray crystal structure of T. tengcongensis EIC. The reported assignments will allow detailed structural and thermodynamic investigations on the coupling between ligand binding and conformational dynamics in EIC.     

Chemical shift assignments of Mb1858 (24-155), a FHA domain-containing protein from <Emphasis Type="Italic">Mycobacterium bovis</Emphasis>

The forkhead-associated (FHA) domain is known as a phosphopeptide recognition domain embedded in regulatory proteins from both eukaryotes and bacteria with various biological functions. In this study, the gene encoding a predicted FHA domain from Mb1858 (residues V24-D155 from the 162 amino acid protein Mb1858) in Mycobacterium bovis was cloned, and U-¹³C/¹⁵N-labeled protein was prepared for backbone and side chain resonance assignments by NMR spectroscopy. These assignments are preliminary work towards the determination of the structure and phosphopeptide-binding properties using NMR methods, which will provide useful information about the function of Mb1858 protein.     

A new <Emphasis Type="Italic">Streptomyces</Emphasis> strain isolated from Saharan soil produces di-(2-ethylhexyl) phthalate,a metabolite active against methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

An actinomycete strain designated G60 was isolated from a Saharan soil sample in Ghardaïa, Algeria, by a dilution agar plating method using chitin-vitamin agar medium supplemented with penicillin. Morphological and chemical studies indicated that this strain belonged to the genus Streptomyces. Analysis of the 16S rDNA sequence showed an identity level within Streptomyces species, with S. coerulescens ISP 5146^T and S. bellus ISP 5185^T the most closely related (100 % for each). However, the comparison of the morphological and physiological characteristics of the strain with those of the two nearest species showed significant differences. Strain G60 had a very strong activity against pathogenic staphylococci, including methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300, other clinical isolates of MRSA and vancomycin resistant S. aureus (VRSA) S1. One antimicrobial compound was extracted by n-hexane from the ISP2 culture medium at 5 days of fermentation culture and purified by HPLC. The chemical structure of the compound was determined after spectroscopic (¹H NMR, ¹³C NMR, ¹H-¹H COSY and ¹H-¹³C HMBC spectra), and spectrometric (mass spectrum) analyses. The bioactive compound was identified as di-(2-ethylhexyl) phthalate.