scholarly journals KdgF, the missing link in the microbial metabolism of uronate sugars from pectin and alginate

2016 ◽  
Vol 113 (22) ◽  
pp. 6188-6193 ◽  
Author(s):  
Joanne K. Hobbs ◽  
Seunghyae M. Lee ◽  
Melissa Robb ◽  
Fraser Hof ◽  
Christopher Barr ◽  
...  
Keyword(s):  
Metabolic Pathway ◽  
Cell Walls ◽  
Marine Algae ◽  
Biofuel Production ◽  
Enzyme Assays ◽  
Efficient Production ◽  
Terrestrial Plants ◽  
X Ray ◽  
Pathogen Virulence

Uronates are charged sugars that form the basis of two abundant sources of biomass—pectin and alginate—found in the cell walls of terrestrial plants and marine algae, respectively. These polysaccharides represent an important source of carbon to those organisms with the machinery to degrade them. The microbial pathways of pectin and alginate metabolism are well studied and essentially parallel; in both cases, unsaturated monouronates are produced and processed into the key metabolite 2-keto-3-deoxygluconate (KDG). The enzymes required to catalyze each step have been identified within pectinolytic and alginolytic microbes; yet the function of a small ORF,kdgF, which cooccurs with the genes for these enzymes, is unknown. Here we show that KdgF catalyzes the conversion of pectin- and alginate-derived 4,5-unsaturated monouronates to linear ketonized forms, a step in uronate metabolism that was previously thought to occur spontaneously. Using enzyme assays, NMR, mutagenesis, and deletion ofkdgF,we show that KdgF proteins from both pectinolytic and alginolytic bacteria catalyze the ketonization of unsaturated monouronates and contribute to efficient production of KDG. We also report the X-ray crystal structures of two KdgF proteins and propose a mechanism for catalysis. The discovery of the function of KdgF fills a 50-y-old gap in the knowledge of uronate metabolism. Our findings have implications not only for the understanding of an important metabolic pathway, but also the role of pectinolysis in plant-pathogen virulence and the growing interest in the use of pectin and alginate as feedstocks for biofuel production.

Non-cellulosic structural polysaccharides in algal cell walls I. Xylan in siphoneous green algae

1964 ◽  
Vol 160 (980) ◽  
pp. 293-313 ◽  
Keyword(s):  
Relative Humidity ◽  
Cell Walls ◽  
Marine Algae ◽  
Transverse Section ◽  
Algal Cell ◽  
Repeat Distance ◽  
X Ray ◽  
Face View ◽  
Half Turn ◽  
Parallel Arrays

The cell walls of a number of marine algae, namely species of Bryopsis, Caulerpa, Udotea, Halimeda and Penicillus and of one freshwater alga, Dichotomosiphon , are examined using both chemical and physical techniques. It is shown that, with the possible exception of Bryopsis , cellulose is completely absent and that the walls contain instead β -l,3-linked xylan as the structural polysaccharide. Bryopsis contains, in addition, a glucan which is most abundant in the outer layers of the wall and which stains like cellulose. The xylan is microfibrillar but the microfibrils are more strongly adherent than they are in cellulose, and in some species appear in the electron microscope to be joined by short crossed rod-like bodies. The orientation of the microfibrils is found to vary, ranging from a net tendency to transverse orientation through complete randomness to almost perfect longitudinal alinement. The microfibrils are negatively birefringent, so that all walls seen in optical section, and all parallel arrays of microfibrils whether in face view or in section (except strictly transverse section) are negatively birefringent. With Bryopsis , the negative birefringence in face view is overcompensated by the positive birefringence of the incrusting glucan so that the true birefringence of the crystalline polysaccharide is observed only after the glucan is removed. The X-ray diagram of parallel arrays of microfibrils as found, for instance, in Penicillus dumetosus shows that the xylan chains are helically coiled, in harmony with the negative birefringence. It is deduced that the microfibrils consist of hexagonally packed, double-stranded helices. The diameter of the helices increases with increasing relative humidity, as water is taken into the lattice, from 13.7 Å in material dried over phosphorus pentoxide to a maximum of 1.54 Å at 65 % relative humidity when the xylan contains 30 % of its weight as water. The repeat distance along the helix axis ranges from 5.85 Å (dry) to 6.06 Å (wet), the length of a half turn of each helix containing three xylose residues. The incrusting substances in these walls often include a glucan which is said also to be 1,3-linked. The significance of the extensive differences between this xylan and cellulose are examined both as regards some of the physical properties of the respective cell walls and in relation to the taxonomic position of these plants.

scholarly journals The Role of Synthetic Biology in the Design of Microbial Cell Factories for Biofuel Production

2011 ◽  
Vol 2011 ◽  
pp. 1-9 ◽  
Author(s):  
Verónica Leticia Colin ◽  
Analía Rodríguez ◽  
Héctor Antonio Cristóbal
Keyword(s):  
Synthetic Biology ◽  
Industrial Applications ◽  
Microbial Cell ◽  
Biodiesel Production ◽  
Biofuel Production ◽  
Attractive Alternative ◽  
Efficient Production ◽  
Microbial Cell Factories ◽  
Cell Factories

Insecurity in the supply of fossil fuels, volatile fuel prices, and major concerns regarding climate change have sparked renewed interest in the production of fuels from renewable resources. Because of this, the use of biodiesel has grown dramatically during the last few years and is expected to increase even further in the future. Biodiesel production through the use of microbial systems has marked a turning point in the field of biofuels since it is emerging as an attractive alternative to conventional technology. Recent progress in synthetic biology has accelerated the ability to analyze, construct, and/or redesign microbial metabolic pathways with unprecedented precision, in order to permit biofuel production that is amenable to industrial applications. The review presented here focuses specifically on the role of synthetic biology in the design of microbial cell factories for efficient production of biodiesel.

scholarly journals PFN2 and NAA80 cooperate to efficiently acetylate the N-terminus of actin

2020 ◽  
Vol 295 (49) ◽  
pp. 16713-16731 ◽  
Author(s):  
Rasmus Ree ◽  
Laura Kind ◽  
Anna Kaziales ◽  
Sylvia Varland ◽  
Minglu Dai ◽  
...  
Keyword(s):  
Cell Shape ◽  
Analytical Ultracentrifugation ◽  
Enzyme Assays ◽  
Modus Operandi ◽  
X Ray ◽  
X Ray Scattering ◽  
N Terminus ◽  
Cytoskeletal Dynamics ◽  
Interaction Proteomics

The actin cytoskeleton is of profound importance to cell shape, division, and intracellular force generation. Profilins bind to globular (G-)actin and regulate actin filament formation. Although profilins are well-established actin regulators, the distinct roles of the dominant profilin, profilin 1 (PFN1), versus the less abundant profilin 2 (PFN2) remain enigmatic. In this study, we use interaction proteomics to discover that PFN2 is an interaction partner of the actin N-terminal acetyltransferase NAA80, and further confirm this by analytical ultracentrifugation. Enzyme assays with NAA80 and different profilins demonstrate that PFN2 binding specifically increases the intrinsic catalytic activity of NAA80. NAA80 binds PFN2 through a proline-rich loop, deletion of which abrogates PFN2 binding. Small-angle X-ray scattering shows that NAA80, actin, and PFN2 form a ternary complex and that NAA80 has partly disordered regions in the N-terminus and the proline-rich loop, the latter of which is partly ordered upon PFN2 binding. Furthermore, binding of PFN2 to NAA80 via the proline-rich loop promotes binding between the globular domains of actin and NAA80, and thus acetylation of actin. However, the majority of cellular NAA80 is stably bound to PFN2 and not to actin, and we propose that this complex acetylates G-actin before it is incorporated into filaments. In conclusion, we reveal a functionally specific role of PFN2 as a stable interactor and regulator of the actin N-terminal acetyltransferase NAA80, and establish the modus operandi for NAA80-mediated actin N-terminal acetylation, a modification with a major impact on cytoskeletal dynamics.

scholarly journals Biochemical Reconstruction of a Metabolic Pathway from a Marine Bacterium Reveals Its Mechanism of Pectin Depolymerization

2018 ◽  
Vol 85 (1) ◽  
Author(s):  
Joanne K. Hobbs ◽  
Andrew G. Hettle ◽  
Chelsea Vickers ◽  
Alisdair B. Boraston
Keyword(s):  
Carbon Source ◽  
Marine Bacteria ◽  
Cell Walls ◽  
Marine Bacterium ◽  
Canonical Pathway ◽  
Glycoside Hydrolases ◽  
Biofuel Production ◽  
Terrestrial Plants ◽  
Metabolic Intermediate ◽  
Plant Waste

ABSTRACTPectin is a complex uronic acid-containing polysaccharide typically found in plant cell walls, though forms of pectin are also found in marine diatoms and seagrasses. Genetic loci that target pectin have recently been identified in two phyla of marine bacteria. These loci appear to encode a pectin saccharification pathway that is distinct from the canonical pathway typically associated with phytopathogenic terrestrial bacteria. However, very few components of the marine pectin metabolism pathway have been experimentally validated. Here, we biochemically reconstructed the pectin saccharification pathway from a marinePseudoalteromonassp.in vitroand show that it results in the production of galacturonate and the key metabolic intermediate 5-keto-4-deoxyuronate (DKI). We demonstrate the sequential de-esterification and depolymerization of pectin into oligosaccharides and the synergistic action of glycoside hydrolases (GHs) to fully degrade these oligosaccharides into monosaccharides. Furthermore, we show that this pathway relies on enzymes belonging to GH family 105 to carry out the equivalent chemistry afforded by an exolytic polysaccharide lyase (PL) and KdgF in the canonical pectin pathway. Finally, we synthesize our findings into a model of marine pectin degradation and compare it with the canonical pathway. Our results underline the shifting view of pectin as a solely terrestrial polysaccharide and highlight the importance of marine pectin as a carbon source for suitably adapted marine heterotrophs. This alternate pathway has the potential to be exploited in the growing field of biofuel production from plant waste.IMPORTANCEMarine polysaccharides, found in the cell walls of seaweeds and other marine macrophytes, represent a vast sink of photosynthetically fixed carbon. As such, their breakdown by marine microbes contributes significantly to global carbon cycling. Pectin is an abundant polysaccharide found in the cell walls of terrestrial plants, but it has recently been reported that some marine bacteria possess the genetic capacity to degrade it. In this study, we biochemically characterized seven key enzymes from a marine bacterium that, together, fully degrade the backbone of pectin into its constituent monosaccharides. Our findings highlight the importance of pectin as a marine carbon source available to bacteria that possess this pathway. The characterized enzymes also have the potential to be utilized in the production of biofuels from plant waste.

scholarly journals Changes of Cadmium Storage Forms and Isotope Ratios in Rice During Grain Filling

2021 ◽  
Vol 12 ◽  
Author(s):  
Matthias Wiggenhauser ◽  
Anne-Marie Aucour ◽  
Philippe Telouk ◽  
Hester Blommaert ◽  
Géraldine Sarret
Keyword(s):  
Cell Walls ◽  
Grain Filling ◽  
Isotope Ratios ◽  
Flag Leaves ◽  
X Ray ◽  
S 100 ◽  
Cd Isotopes ◽  
Upland Conditions ◽  
X Ray Absorption

Rice poses a major source of the toxic contaminant cadmium (Cd) for humans. Here, we elucidated the role of Cd storage forms (i.e., the chemical Cd speciation) on the dynamics of Cd within rice. In a pot trial, we grew rice on a Cd-contaminated soil in upland conditions and sampled roots and shoots parts at flowering and maturity. Cd concentrations, isotope ratios, Cd speciation (X-ray absorption spectroscopy), and micronutrient concentrations were analyzed. During grain filling, Cd and preferentially light Cd isotopes were strongly retained in roots where the Cd storage form did not change (Cd bound to thiols, Cd–S = 100%). In the same period, no net change of Cd mass occurred in roots and shoots, and the shoots became enriched in heavy isotopes (Δ114/110Cdmaturity–flowering = 0.14 ± 0.04‰). These results are consistent with a sequestration of Cd in root vacuoles that includes strong binding of Cd to thiol containing ligands that favor light isotopes, with a small fraction of Cd strongly enriched in heavy isotopes being transferred to shoots during grain filling. The Cd speciation in the shoots changed from predominantly Cd–S (72%) to Cd bound to O ligands (Cd–O, 80%) during grain filling. Cd–O may represent Cd binding to organic acids in vacuoles and/or binding to cell walls in the apoplast. Despite this change of ligands, which was attributed to plant senescence, Cd was largely immobile in the shoots since only 0.77% of Cd in the shoots were transferred into the grains. Thus, both storage forms (Cd–S and Cd–O) contributed to the retention of Cd in the straw. Cd was mainly bound to S in nodes I and grains (Cd–S > 84%), and these organs were strongly enriched in heavy isotopes compared to straw (Δ114/110Cdgrains/nodes–straw = 0.66–0.72‰) and flag leaves (Δ114/110Cdgrains/nodes–flag leaves = 0.49–0.52‰). Hence, xylem to phloem transfer in the node favors heavy isotopes, and the Cd–S form may persist during the transfer of Cd from node to grain. This study highlights the importance of Cd storage forms during its journey to grain and potentially into the food chain.

Digital x-ray mapping of nanometer-size supported bimetallic catalysts

1988 ◽  
Vol 46 ◽  
pp. 530-531
Author(s):  
L. T. Germinario
Keyword(s):  
Background Radiation ◽  
Metal Cluster ◽  
Single Element ◽  
Beam Diameter ◽  
X Rays ◽  
X Ray ◽  
Major Interest ◽  
Induced Changes

Understanding the role of metal cluster composition in determining catalytic selectivity and activity is of major interest in heterogeneous catalysis. The electron microscope is well established as a powerful tool for ultrastructural and compositional characterization of support and catalyst. Because the spatial resolution of x-ray microanalysis is defined by the smallest beam diameter into which the required number of electrons can be focused, the dedicated STEM with FEG is the instrument of choice. The main sources of errors in energy dispersive x-ray analysis (EDS) are: (1) beam-induced changes in specimen composition, (2) specimen drift, (3) instrumental factors which produce background radiation, and (4) basic statistical limitations which result in the detection of a finite number of x-ray photons. Digital beam techniques have been described for supported single-element metal clusters with spatial resolutions of about 10 nm. However, the detection of spurious characteristic x-rays away from catalyst particles produced images requiring several image processing steps.

The role of silicon in forages: A quantitative X-Ray study

1987 ◽  
Vol 45 ◽  
pp. 416-417
Author(s):  
Janet H. Woodward ◽  
D. E. Akin
Keyword(s):  
Sodium Acetate ◽  
Dry Matter ◽  
Acetate Buffer ◽  
Plant Tissues ◽  
Sodium Acetate Buffer ◽  
X Ray ◽  
Dry Matter Digestibility ◽  
Percent Composition

Silicon (Si) is distributed throughout plant tissues, but its role in forages has not been clarified. Although Si has been suggested as an antiquality factor which limits the digestibility of structural carbohydrates, other research indicates that its presence in plants does not affect digestibility. We employed x-ray microanalysis to evaluate Si as an antiquality factor at specific sites of two cultivars of bermuda grass (Cynodon dactvlon (L.) Pers.). “Coastal” and “Tifton-78” were chosen for this study because previous work in our lab has shown that, although these two grasses are similar ultrastructurally, they differ in in vitro dry matter digestibility and in percent composition of Si.Two millimeter leaf sections of Tifton-7 8 (Tift-7 8) and Coastal (CBG) were incubated for 72 hr in 2.5% (w/v) cellulase in 0.05 M sodium acetate buffer, pH 5.0. For controls, sections were incubated in the sodium acetate buffer or were not treated.

Pathophysiological function of the mitochondria as a calcium reservoir: Quantitative x-ray microanalysis

1990 ◽  
Vol 48 (2) ◽  
pp. 164-165
Author(s):  
K. Teraoka ◽  
N. Kaneko ◽  
Y. Horikawa ◽  
T. Uchida ◽  
R. Matsuda ◽  
...  
Keyword(s):  
Bolus Injection ◽  
Contact Method ◽  
X Ray

The aim of this study was to elucidate the role of the mitochondria as a store of calcium(Ca) under the condition of pathophysiological Ca overload induced by a rise in extracellular Ca concentration and the administration of isoproterenol.Eight rats were employed, and hearts were perfused as in the Langendorff method with Krebs-Henseleit solution gassed with 95% O2 and 5% CO2. Tow specimens were perfused with 2mM Ca for 30 min, and 2 were perfused with 5.5 mM Ca for 20 min. 4 specimens were perfused with 2 mM Ca for 5 min, and of these 4, 2 were infused with 10-7 mM/kg/min. isoproterenol for 5 min, and 2 were given a bolus injection of 3 x 10-7 mM isoproterenol. After rapid-cryofixation by the metal-mirror contact method with a Reichert-Jung KF80/MM80, and cryosectioning at -160 to -180° C with a Reichert-Jung Ultracut Fc-4E, ultrathin specimens (100nm) were free-ze-dreid for several hours at 10-5 Torr in the JEOL FD 7000, and mitochondrial Ca was determined by quantitative x-ray micranalysis (JEOL 1200EX, LINK AN 10000S).

Ionic homeostasis and subcellular element compartmentation

1990 ◽  
Vol 48 (2) ◽  
pp. 150-151
Author(s):  
Ann LeFurgey ◽  
Peter Ingram ◽  
J.J. Blum ◽  
M.C. Carney ◽  
L.A. Hawkey ◽  
...  
Keyword(s):  
Leishmania Major ◽  
Ionic Homeostasis ◽  
X Ray ◽  
Functional Studies ◽  
Cell Compartments ◽  
X Ray Imaging ◽  
Resolution Electron ◽  
Multiple Cell ◽  
Role Of Zn

Subcellular compartments commonly identified and analyzed by high resolution electron probe x-ray microanalysis (EPXMA) include mitochondria, cytoplasm and endoplasmic or sarcoplasmic reticulum. These organelles and cell regions are of primary importance in regulation of cell ionic homeostasis. Correlative structural-functional studies, based on the static probe method of EPXMA combined with biochemical and electrophysiological techniques, have focused on the role of these organelles, for example, in maintaining cell calcium homeostasis or in control of excitation-contraction coupling. New methods of real time quantitative x-ray imaging permit simultaneous examination of multiple cell compartments, especially those areas for which both membrane transport properties and element content are less well defined, e.g. nuclei including euchromatin and heterochromatin, lysosomes, mucous granules, storage vacuoles, microvilli. Investigations currently in progress have examined the role of Zn-containing polyphosphate vacuoles in the metabolism of Leishmania major, the distribution of Na, K, S and other elements during anoxia in kidney cell nuclel and lysosomes; the content and distribution of S and Ca in mucous granules of cystic fibrosis (CF) nasal epithelia; the uptake of cationic probes by mltochondria in cultured heart ceils; and the junctional sarcoplasmic retlculum (JSR) in frog skeletal muscle.

Silicon Substitution for Calcium in the Shell of the Fingernail Clam, Musculium Transversum Studied By X-Ray Analysis

1980 ◽  
Vol 38 ◽  
pp. 560-561
Author(s):  
Judith A. Murphy ◽  
Anthony Paparo ◽  
Richard Sparks
Keyword(s):  
Carbonic Anhydrase ◽  
River Water ◽  
Food Chains ◽  
Well Water ◽  
Shell Formation ◽  
X Ray ◽  
Molluscan Shell ◽  
Fingernail Clams

Fingernail clams (Muscu1ium transversum) are dominant bottom-dwelling animals in some waters of the midwest U.S. These organisms are key links in food chains leading from nutrients in water and mud to fish and ducks which are utilized by man. In the mid-1950’s, fingernail clams disappeared from a 100-mile section of the Illinois R., a tributary of the Mississippi R. Some factor(s) in the river and/or sediment currently prevent clams from recolonizing areas where they were formerly abundant. Recently, clams developed shell deformities and died without reproducing. The greatest mortality and highest incidence of shell deformities appeared in test chambers containing the highest proportion of river water to well water. The molluscan shell consists of CaCO3, and the tissue concerned in its secretion is the mantle. The source of the carbonate is probably from metabolic CO2 and the maintenance of ionized Ca concentration in the mantle is controlled by carbonic anhydrase. The Ca is stored in extracellular concentric spherical granules(0.6-5.5μm) which represent a large amount of inertCa in the mantle. The purpose of this investigation was to examine the role of raw river water and well water on shell formation in the fingernail clam.

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