PqsBC,a Condensing Enzyme in the Biosynthesis of the Pseudomonas aeruginosa Quinolone Signal: CRYSTAL STRUCTURE,INHIBITION, AND REACTION MECHANISM*?

Pseudomonas aeruginosa produces a number of alkylquinolone-type secondary metabolites best known for their antimicrobial effects and involvement in cell-cell communication. In the alkylquinolone biosynthetic pathway, the β-ketoacyl-(acyl carrier protein) synthase III (FabH)-like enzyme PqsBC catalyzes the condensation of octanoyl-coenzyme A and 2-aminobenzoylacetate (2-ABA) to form the signal molecule 2-heptyl-4(1H)-quinolone. PqsBC, a potential drug target, is unique for its heterodimeric arrangement and an active site different from that of canonical FabH-like enzymes. Considering the sequence dissimilarity between the subunits, a key question was how the two subunits are organized with respect to the active site. In this study, the PqsBC structure was determined to a 2 Å resolution, revealing that PqsB and PqsC have a pseudo-2-fold symmetry that unexpectedly mimics the FabH homodimer. PqsC has an active site composed of Cys-129 and His-269, and the surrounding active site cleft is hydrophobic in character and approximately twice the volume of related FabH enzymes that may be a requirement to accommodate the aromatic substrate 2-ABA. From physiological and kinetic studies, we identified 2-aminoacetophenone as a pathway-inherent competitive inhibitor of PqsBC, whose fluorescence properties could be used for in vitro binding studies. In a time-resolved setup, we demonstrated that the catalytic histidine is not involved in acyl-enzyme formation, but contributes to an acylation-dependent increase in affinity for the second substrate 2-ABA. Introduction of Asn into the PqsC active site led to significant activity toward the desamino substrate analog benzoylacetate, suggesting that the substrate 2-ABA itself supplies the asparagine-equivalent amino function that assists in catalysis.     

Heme oxygenase, a novel target for the treatment of hypertension and obesity?

Heme oxygenase (HO) is the rate-limiting enzyme in the metabolism of heme-releasing bioactive molecules carbon monoxide (CO), biliverdin, and iron, each with beneficial cardiovascular actions. Biliverdin is rapidly reduced to bilirubin, a potent antioxidant, by the enzyme biliverdin reductase, and iron is rapidly sequestered by ferritin in the cell. Several studies have demonstrated that HO-1 induction can attenuate the development of hypertension as well as lower blood pressure in established hypertension in both genetic and experimental models. HO-1 induction can also reduce target organ injury and can be beneficial in cardiovascular diseases, such as heart attack and stroke. Recent studies have also identified a beneficial role for HO-1 in the regulation of body weight and metabolism in diabetes and obesity. Chronic HO-1 induction lowers body weight and corrects hyperglycemia and hyperinsulinemia. Chronic HO-1 induction also modifies the phenotype of adipocytes in obesity from one of large, cytokine producing to smaller, adiponectin producing. Finally, chronic induction of HO-1 increases oxygen consumption, CO(2), and heat production and activity in obese mice. This review will discuss the current understanding of the actions of the HO system to lower blood pressure and body weight and how HO or its metabolites may be ideal candidates for the development of drugs that can both reduce blood pressure and lower body weight.     

Insights into the Structure and Function of Ciliary and Flagellar Doublet Microtubules: TEKTINS,Ca2+-BINDING PROTEINS,AND STABLE PROTOFILAMENTS*?

Cilia and flagella are conserved, motile, and sensory cell organelles involved in signal transduction and human disease. Their scaffold consists of a 9-fold array of remarkably stable doublet microtubules (DMTs), along which motor proteins transmit force for ciliary motility and intraflagellar transport. DMTs possess Ribbons of three to four hyper-stable protofilaments whose location, organization, and specialized functions have been elusive. We performed a comprehensive analysis of the distribution and structural arrangements of Ribbon proteins from sea urchin sperm flagella, using quantitative immunobiochemistry, proteomics, immuno-cryo-electron microscopy, and tomography. Isolated Ribbons contain acetylated α-tubulin, β-tubulin, conserved protein Rib45, >95% of the axonemal tektins, and >95% of the calcium-binding proteins, Rib74 and Rib85.5, whose human homologues are related to the cause of juvenile myoclonic epilepsy. DMTs contain only one type of Ribbon, corresponding to protofilaments A11-12-13-1 of the A-tubule. Rib74 and Rib85.5 are associated with the Ribbon in the lumen of the A-tubule. Ribbons contain a single ∼5-nm wide filament, composed of equimolar tektins A, B, and C, which interact with the nexin-dynein regulatory complex. A summary of findings is presented, and the functions of Ribbon proteins are discussed in terms of the assembly and stability of DMTs, ciliary motility, and other microtubule systems.     

The Structure of CcmP,a Tandem Bacterial Microcompartment Domain Protein from the β-Carboxysome,Forms a Subcompartment Within a Microcompartment

The carboxysome is a bacterial organelle found in all cyanobacteria; it encapsulates CO₂ fixation enzymes within a protein shell. The most abundant carboxysome shell protein contains a single bacterial microcompartment (BMC) domain. We present in vivo evidence that a hypothetical protein (dubbed CcmP) encoded in all β-cyanobacterial genomes is part of the carboxysome. We show that CcmP is a tandem BMC domain protein, the first to be structurally characterized from a β-carboxysome. CcmP forms a dimer of tightly stacked trimers, resulting in a nanocompartment-containing shell protein that may weakly bind 3-phosphoglycerate, the product of CO₂ fixation. The trimers have a large central pore through which metabolites presumably pass into the carboxysome. Conserved residues surrounding the pore have alternate side-chain conformations suggesting that it can be open or closed. Furthermore, CcmP and its orthologs in α-cyanobacterial genomes form a distinct clade of shell proteins. Members of this subgroup are also found in numerous heterotrophic BMC-associated gene clusters encoding functionally diverse bacterial organelles, suggesting that the potential to form a nanocompartment within a microcompartment shell is widespread. Given that carboxysomes and architecturally related bacterial organelles are the subject of intense interest for applications in synthetic biology/metabolic engineering, our results describe a new type of building block with which to functionalize BMC shells.     

NMR assignments of cd-HO, a 24 kDa heme oxygenase from Corynebacterium diphtheria

We are employing a number of selective in vitro and in vivo methods including NMR to screen compounds that bind to heme oxygenases from pathogenic bacteria. We report the nearly complete HN, N, CO, Cα and Cβ chemical shift assignments of a 215-amino acid HO from Corynebacterium diphtheria in three forms, apo cd-HO-G135A, apo cd-HO and CO-bound ferrous holo cd-HO; these assignments will enable us to identify residues on cd-HO that are perturbed upon binding to selected compounds, and to help with the development of inhibitors specific to the bacterial proteins.     

Significance of Arg3, Arg54, and Tyr58 of l-aspartate α-decarboxylase from Corynebacterium glutamicum in the process of self-cleavage

Purpose of work

We have elucidated the significance of three key amino acid residues of l-aspartate α-decarboxylase that act remotely from its cleavage site for its functional self-cleavage as well as for its catalytic activity. These results provide useful fundamental information for engineering l-aspartate α-decarboxylase. l-Aspartate α-decarboxylase (ADC) from Corynebacterium glutamicum, and encoded by panD, was cloned and expressed in Escherichia coli and then purified. Three amino acid residues were found to be related to ADC self-cleavage. Mutating R3 to either A, Q, N, L, D, or E produced only the unprocessed pro-enzyme. Although mutating R54 and Y58 into A or K and A or T, respectively, partly influenced ADC self-cleavage, the specific activity of each of the four ßmutants decreased to 3.5, 4, 2.4, and 2.6 U mg^?1, respectively, compared with a specific activity of 690 U mg^?1 for the wild-type enzyme. Thus, R3 triggers ADC self-cleavage and completes the modification of the active site with assistance by R54 and Y58. These results will help to engineer ADC for improved industrial applications.     

Crystal Structures of Penicillin-Binding Protein 3 (PBP3) from Methicillin-Resistant Staphylococcus aureus in the Apo and Cefotaxime‐Bound Forms

Staphylococcus aureus is a widespread Gram‐positive opportunistic pathogen, and a methicillin‐resistant form (MRSA) is particularly difficult to treat clinically. We have solved two crystal structures of penicillin‐binding protein (PBP) 3 (PBP3) from MRSA, the apo form and a complex with the β-lactam antibiotic cefotaxime, and used electrospray mass spectrometry to measure its sensitivity to a variety of penicillin derivatives. PBP3 is a class B PBP, possessing an N-terminal non-penicillin‐binding domain, sometimes called a dimerization domain, and a C-terminal transpeptidase domain. The model shows a different orientation of its two domains compared to earlier models of other class B PBPs and a novel, larger N-domain. Consistent with the nomenclature of “dimerization domain”, the N-terminal region forms an apparently tight interaction with a neighboring molecule related by a 2-fold symmetry axis in the crystal structure. This dimer form is predicted to be highly stable in solution by the PISA server, but mass spectrometry and analytical ultracentrifugation provide unequivocal evidence that the protein is a monomer in solution.     

Elucidating the Role of Disulfide Bond on Amyloid Formation and Fibril Reversibility of Somatostatin-14: RELEVANCE TO ITS STORAGE AND SECRETION*?

The storage of protein/peptide hormones within subcellular compartments and subsequent release are crucial for their native function, and hence these processes are intricately regulated in mammalian systems. Several peptide hormones were recently suggested to be stored as amyloids within endocrine secretory granules. This leads to an apparent paradox where storage requires formation of aggregates, and their function requires a supply of non-aggregated peptides on demand. The precise mechanism behind amyloid formation by these hormones and their subsequent release remain an open question. To address this, we examined aggregation and fibril reversibility of a cyclic peptide hormone somatostatin (SST)-14 using various techniques. After proving that SST gets stored as amyloid in vivo, we investigated the role of native structure in modulating its conformational dynamics and self-association by disrupting the disulfide bridge (Cys³–Cys¹⁴) in SST. Using two-dimensional NMR, we resolved the initial structure of somatostatin-14 leading to aggregation and further probed its conformational dynamics in silico. The perturbation in native structure (S-S cleavage) led to a significant increase in conformational flexibility and resulted in rapid amyloid formation. The fibrils formed by disulfide-reduced noncyclic SST possess greater resistance to denaturing conditions with decreased monomer releasing potency. MD simulations reveal marked differences in the intermolecular interactions in SST and noncyclic SST providing plausible explanation for differential aggregation and fibril reversibility observed experimentally in these structural variants. Our findings thus emphasize that subtle changes in the native structure of peptide hormone(s) could alter its conformational dynamics and amyloid formation, which might have significant implications on their reversible storage and secretion.     

Expression and characterization of a thermostable β-xylosidase from the hyperthermophile, Thermotoga maritima

A thermostable beta-xylosidase from a hyperthermophilic bacterium, Thermotoga maritima, was over-expressed in Escherichia coli using the T7 polymerase expression system. The expressed beta-xylosidase was purified in two steps, heat treatment and immobilized metal affinity chromatography, and gave a single band on SDS-PAGE. The maximum activity on p-nitrophenyl beta-D-xylopyranoside was at 90 degrees C and pH 6.1. The purified enzyme had a half-life of over 22-min at 95 degrees C, and retained over 57% of its activity after holding a pH ranging from 5.4 to 8.5 for 1 h at 80 degrees C. Among all tested substrates, the purified enzyme had specific activities of 275, 50 and 29 U mg(-1) on pNPX, pNPAF, and pNPG, respectively. The apparent Michaelis constant of the beta-xylosidase was 0.13 mM for p NPX with a V (max) of 280 U mg(-1). When the purified beta-xylosidase was added to xylanase, corncob xylan was hydrolized completely to xylose.     

Dinoflagellate cysts,Acritarchs and Tasmanitids from the uppermost Callovian of England and Scotland: With a reconsideration of the “xanthidium pilosum” problem

Four assemblages of fossil organic-walled microphytoplankton from the Upper Callovian (Lamberti Zone), three from England and one from Scotland, are described. They comprise at least 50 species of dinoflagellate cysts, 18 species of acritarchs and 2 species of tasmanitids. After a review of the «Xanthidium pilosum problem, a revised diagnosis is proposed for Sentusidinium pilosum (Ehrenberg) and three new species, Sentusidinium creberbarbatum, S. sparsibarbatum and S. erythrocomum are proposed. The new family name Scriniodiniaceae is substituted for the now invalid family name Endoscriniaceae. The new genus Escharisphaeridia is proposed to contain two existing species, E. pococki (Sarjeant) and E. dictydia (Sarjeant). The present content of the genus Compositosphaeridium is examined; the new combination C. polonicum (Górka) is proposed and a new species, C. bulgaricum, is erected. One further new species of dinoflagellate cyst, Prolixosphaeridium anasillum, is proposed. The new term «peniplate is proposed for paraplates whose boundaries are penitabular in situation.One new species of acritarch, Solisphaeridium?rhachos, is described and the new combination Polygonium aster (Sarjeant) is proposed. Evidence for reworking of some of the species of Micrhystridium present in the Hackness Rock assemblage is noted, though it is considered that the majority of species in that assemblage are indigenous. It is considered that all four assemblages are derived from a single phytoplankton province, the differences between them resulting from environmental factors such as water depth or distance from a shoreline.     

Biochemical Characterization and Crystal Structures of a Fungal Family 3 β-Glucosidase,Cel3A from Hypocrea jecorina

Cellulase mixtures from Hypocrea jecorina are commonly used for the saccharification of cellulose in biotechnical applications. The most abundant β-glucosidase in the mesophilic fungus Hypocrea jecorina is HjCel3A, which hydrolyzes the β-linkage between two adjacent molecules in dimers and short oligomers of glucose. It has been shown that enhanced levels of HjCel3A in H. jecorina cellulase mixtures benefit the conversion of cellulose to glucose. Biochemical characterization of HjCel3A shows that the enzyme efficiently hydrolyzes (1,4)- as well as (1,2)-, (1,3)-, and (1,6)-β-d-linked disaccharides. For crystallization studies, HjCel3A was produced in both H. jecorina (HjCel3A) and Pichia pastoris (Pp-HjCel3A). Whereas the thermostabilities of HjCel3A and Pp-HjCel3A are the same, Pp-HjCel3A has a higher degree of N-linked glycosylation. Here, we present x-ray structures of HjCel3A with and without glucose bound in the active site. The structures have a three-domain architecture as observed previously for other glycoside hydrolase family 3 β-glucosidases. Both production hosts resulted in HjCel3A structures that have N-linked glycosylations at Asn²⁰⁸ and Asn³¹⁰. In H. jecorina-produced HjCel3A, a single N-acetylglucosamine is present at both sites, whereas in Pp-HjCel3A, the P. pastoris-produced HjCel3A enzyme, the glycan chains consist of 8 or 4 saccharides. The glycosylations are involved in intermolecular contacts in the structures derived from either host. Due to the different sizes of the glycosylations, the interactions result in different crystal forms for the two protein forms.     

Avenacosidase from oat: purification,sequence analysis and biochemical characterization of a new member of the BGA family of β-glucosidases

A protein consisting of 60 kDa subunits (As-P60) was isolated from etiolated oat seedlings (Avena sativa L.) and characterized as avenacosidase, a -glucosidase that belongs to a preformed defence system of oat against fungal infection. The enzyme is highly aggregated; it consists of 300–350 kDa aggregates and multimers thereof. Dissociation by freezing/thawing leads to complete loss of enzyme activity. The specificity of the enzyme was investigated with para-nitrophenyl derivatives which serve as substrates, in decreasing order -fucoside, -glucoside, -galactoside, -xyloside. The corresponding orthonitrophenyl glycosides are less well accepted. No hydrolysis was found with -glycosides and -thioglucoside. An anti-As-P60 antiserum was prepared and used for isolation of a cDNA clone coding for As-P60. A presequence of 55 amino acid residues was deduced from comparison of the cDNA sequence with the N-terminal sequence determined by Edman degradation of the mature protein. The presequence has the characteristics of a stroma-directing signal peptide; localization of As-P60 in plastids of oat seedlings was confirmed by western blotting. The amino acid sequence revealed significant homology (>39% sequence identity) to -glucosidases that are constituents of a defence mechanism in dicotyledonous plants. 34% sequence identity was even found with mammalian and bacterial -glucosidases of the BGA family. Avenacosidase extends the occurrence of this family of -glucosidases to monocotyledonous plants.     

Structures,intermolecular interactions,and chemical hardness of binary water–organic solvents: a molecular dynamics study

The evolution of structural properties, thermodynamics and averaged (dynamic) total hardness values as a function of the composition of binary water–organic solvents, was rationalized in view of the intermolecular interactions. The organic solvents considered were ethanol, acetonitrile, and isopropanol at 0.25, 0.5, 0.75, and 1 mass fractions, and the results were obtained using molecular dynamics simulations. The site-to-site radial distribution functions reveal a well-defined peak for the first coordination shell in all solvents. A characteristic peak of the second coordination shell exists in aqueous mixtures of acetonitrile, whereas in the water–alcohol solvents, a second peak develops with the increase in alcohol content. From the computed coordination numbers, averaged hydrogen bonds and their lifetimes, we found that water mixed with acetonitrile largely preserves its structural features and promotes the acetonitrile structuring. Both the water and alcohol structures in their mixtures are disturbed and form hydrogen bonds between molecules of different kinds. The dynamic hardness values are obtained as the average over the total hardness values of 1200 snapshots per solvent type, extracted from the equilibrium dynamics. The dynamic hardness profile has a non-linear evolution with the liquid compositions, similarly to the thermodynamic properties of these non-ideal solvents.

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Graphical abstract Computed dynamic total hardness, as a function of the cosolvent mass fraction for water–ethanol (EtOH), water–isopropanol (2PrOH) and water–acetonitrile (AN)

     

Characterization of the long-wavelength opsin from Mecoptera and Siphonaptera: does a flea see?

Mecoptera and Siphonaptera represent two insect orders that have largely been overlooked in the study of insect vision. Recent phylogenetic evidence demonstrates that Mecoptera (scorpionflies) is paraphyletic, with the order Siphonaptera (fleas) nesting as sister to the family Boreidae (snow fleas), showing an evolutionary trend towards reduction in gross eye morphology within fleas. We provide the first molecular characterization of long-wavelength opsins from these three lineages (opsin gene from fleas [FL-Opsin], the Boreidae [B-Opsin], and a mecopteran family [M-Opsin]) and assess the effects of loss of visual acuity on the structure and function of the opsin gene. Phylogenetic analysis implies a physiological sensitivity in the red-green spectrum for these opsins. Analysis of intron splice sites reveals a high degree of similarity between FL-Opsin and B-Opsin as well as conserved splice sites across insect blue-green and long-wavelength opsins. Calculated rates of evolution and tests for destabilizing selection indicate that FL-Opsin, B-Opsin, and M-Opsin are evolving at similar rates with no radical selective pressures, implying conservative evolution and functional constraint across all three lineages.     

1′-Chloropannarin,a new depsidone from Argopsis friesiana: Notes on the structure of pannarin and on the chemistry of the lichen genus Argopsis

A new depsidone 1′-chloropannarin has been isolated from the lichen Argopsis friesiana Müll. Arg. and synthesized by chlorination of pannarin. A revised structure of pannarin, and notes on the chemical constitution of Argopsis friesiana Mill. Arg. A. megalospora Th. Fr., are given.     

Establishment and characterization of a cell line, MT-213-VGH, isolated from a mixed müllerian tumor of the uterus

OBJECTIVE: To establish a cell line from a woman with malignant mixed müllerian tumor of the uterus and to examine the biologic properties of this cell line (MT-213-VGH). STUDY DESIGN: Cells were cloned by the limiting dilution method. Histologic staining of mixed müllerian (mesodermal) tumor (MMMT) cells was performed with May-Grünwald-Giemsa and hematoxylin and eosin stain. After more than 20 passages, cells were used to estimate the population-doubling time and colony-forming efficiency of MMMT cells. The cell line exhibited considerable variation in the degree of sensitivity to diverse chemotherapy drugs in vitro. RESULTS: MMMT cells containing antigens for vimentin and myoglobin were detected, but those for CA-125, carcinoembryonic antigen, cytoskeleton, desmin, epithelial membrane antigen and fibronectin were not found. In addition, MT-213-VGH cells contained a mucinous substance; its chromosome model number is 45. This cell line showed differential sensitivities to chemotherapeutic agents, such as bleomycin, cisplatin, 5-fluorouracil and vinblastine. CONCLUSION: The establishment and availability of the number cell line MT-213-VGH for a malignant mixed müllerian tumor of the uterus should assist in research on new methods of managing this type of gynecologic cancer.