scholarly journals Novel class of glutathione transferases from cyanobacteria exhibit high catalytic activities towards naturally occurring isothiocyanates

2007 ◽  
Vol 406 (1) ◽  
pp. 115-123 ◽  
Author(s):  
Eric Wiktelius ◽  
Gun Stenberg
Keyword(s):  
Catalytic Properties ◽  
Structural Features ◽  
Glutathione Transferases ◽  
Substrate Specificities ◽  
Catalytic Activities ◽  
Genome Databases ◽  
Naturally Occurring ◽  
Physiological Relevance ◽  
Associated Proteins

In the present paper, we report a novel class of GSTs (glutathione transferases), called the Chi class, originating from cyanobacteria and with properties not observed previously in prokaryotic enzymes. GSTs constitute a widespread multifunctional group of proteins, of which mammalian enzymes are the best characterized. Although GSTs have their origin in prokaryotes, few bacterial representatives have been characterized in detail, and the catalytic activities and substrate specificities observed have generally been very modest. The few well-studied bacterial GSTs have largely unknown physiological functions. Genome databases reveal that cyanobacteria have an extensive arsenal of glutathione-associated proteins. We have studied two cyanobacterial GSTs which are the first examples of bacterial enzymes that are as catalytically efficient as the best mammalian enzymes. GSTs from the thermophile Thermosynechococcus elongatus BP-1 and from Synechococcus elongatus PCC 6301 were found to catalyse the conjugation of naturally occurring plant-derived isothiocyanates to glutathione at high rates. The cyanobacterial GSTs studied are smaller than previously described members of this enzyme family, but display many of the typical structural features that are characteristics of GSTs. They are also active towards several classical substrates, but at the same moderate rates that have been observed for other GSTs derived from prokaryotes. The cloning, expression and characterization of two cyanobacterial GSTs are described. The possible significance of the observed catalytic properties is discussed in the context of physiological relevance and GST evolution.

scholarly journals Botulinum Neurotoxin F Subtypes Cleaving the VAMP-2 Q58–K59 Peptide Bond Exhibit Unique Catalytic Properties and Substrate Specificities

Toxins ◽  
2018 ◽  
Vol 10 (8) ◽  
pp. 311 ◽  
Author(s):  
Stefan Sikorra ◽  
Martin Skiba ◽  
Martin Dorner ◽  
Jasmin Weisemann ◽  
Mirjam Weil ◽  
...  
Keyword(s):  
Botulinum Neurotoxin ◽  
Biochemical Characterization ◽  
Catalytic Properties ◽  
Peptide Bond ◽  
Nerve Terminals ◽  
Recent Past ◽  
Substrate Specificities ◽  
Biochemical Analyses ◽  
Domain Level

In the recent past, about 40 botulinum neurotoxin (BoNT) subtypes belonging to serotypes A, B, E, and F pathogenic to humans were identified among hundreds of independent isolates. BoNTs are the etiological factors of botulism and represent potential bioweapons; however, they are also recognized pharmaceuticals for the efficient counteraction of hyperactive nerve terminals in a variety of human diseases. The detailed biochemical characterization of subtypes as the basis for development of suitable countermeasures and possible novel therapeutic applications is lagging behind the increase in new subtypes. Here, we report the primary structure of a ninth subtype of BoNT/F. Its amino-acid sequence diverges by at least 8.4% at the holotoxin and 13.4% at the enzymatic domain level from all other known BoNT/F subtypes. We found that BoNT/F9 shares the scissile Q58/K59 bond in its substrate vesicle associated membrane protein 2 with the prototype BoNT/F1. Comparative biochemical analyses of four BoNT/F enzymatic domains showed that the catalytic efficiencies decrease in the order F1 > F7 > F9 > F6, and vary by up to a factor of eight. KM values increase in the order F1 > F9 > F6 ≈ F7, whereas kcat decreases in the order F7 > F1 > F9 > F6. Comparative substrate scanning mutagenesis studies revealed a unique pattern of crucial substrate residues for each subtype. Based upon structural coordinates of F1 bound to an inhibitor polypeptide, the mutational analyses suggest different substrate interactions in the substrate binding channel of each subtype.

scholarly journals Human α-l-iduronidase. Catalytic properties and an integrated role in the lysosomal degradation of heparan sulphate

1992 ◽  
Vol 282 (3) ◽  
pp. 899-908 ◽  
Author(s):  
C Freeman ◽  
J J Hopwood
Keyword(s):  
Enzyme Activity ◽  
Enzyme Activities ◽  
Human Liver ◽  
Skin Fibroblast ◽  
Catalytic Properties ◽  
Heparan Sulphate ◽  
Structural Features ◽  
Lysosomal Degradation ◽  
Catalytic Activities ◽  
Wide Range

The kinetic parameters (Km and kcat) of human liver alpha-L-iduronidase were determined with a variety of heparin-derived disaccharide and tetrasaccharide substrates. More structurally complex substrates, in which several aspects of the aglycone structure of the natural substrates heparin and heparan sulphate were maintained, were hydrolysed with catalytic efficiencies up to 255 times that observed for the simplest disaccharide substrate to be hydrolysed. The major aglycone structure that influenced both substrate binding and enzyme activity was the presence of a C-6 sulphate ester on the residue adjacent to the iduronic acid residue being hydrolysed. Sulphate ions and a number of substrate and product analogues were potent inhibitors of enzyme activity. Human liver alpha-L-iduronidase activity towards 4-methylumbelliferyl alpha-L-iduronide at pH 4.8 had two Km values of 37 microM and 1.92 mM with corresponding kcat. values of 299 and 650 mol of product formed/min per mol of enzyme respectively, which may explain the wide range of Km values previously reported for alpha-L-iduronidase activity toward its substrate. Skin fibroblast alpha-L-iduronidase activity towards the heparin-derived oligosaccharides was influenced by the same substrate aglycone structural features as was observed for the human liver enzyme. A comparison was made of the effect of substrate aglycone structure upon catalytic activities of the enzymes which act to degrade the highly sulphated regions of heparan sulphate. A model was proposed whereby the substrate is directed from alpha-L-iduronidase to subsequent enzyme activities to ensure the efficient degradation of heparan sulphate.

scholarly journals Botulinum Neurotoxin F Subtypes Cleaving the VAMP-2 Q58-K59 Peptide Bond Exhibit Unique Catalytic Properties and Substrate Specificities

2018 ◽  
Author(s):  
Stefan Sikorra ◽  
Martin Skiba ◽  
Martin B. Dorner ◽  
Jasmin Weisemann ◽  
Mirjam Weil ◽  
...  
Keyword(s):  
Botulinum Neurotoxin ◽  
Biochemical Characterization ◽  
Catalytic Properties ◽  
Peptide Bond ◽  
Nerve Terminals ◽  
Recent Past ◽  
Substrate Specificities ◽  
Biochemical Analyses ◽  
Domain Level

In the recent past about 40 botulinum neurotoxin (BoNT) subtypes belonging to serotypes A, B, E, and F pathogenic to humans were identified among hundreds of independent isolates. BoNTs are the etiological factors of botulism and represent potential bioweapons, but are also recognized pharmaceuticals for efficient counteraction of hyperactive nerve terminals in a variety of human diseases. The detailed biochemical characterization of subtypes as the basis for development of suitable countermeasures and possible novel therapeutic applications is lagging behind the increase in new subtypes. Here we report the primary structure of a ninth subtype of BoNT/F. Its amino acid sequence diverges by at least 8.4% at the holotoxin and 13.4% on the enzymatic domain level from all other known BoNT/F subtypes. We found that BoNT/F9 shares the scissile Q58/K59 bond in its substrate vesicle associated membrane protein 2 with the prototype BoNT/F1. Comparative biochemical analyses of four BoNT/F enzymatic domains showed that the catalytic efficiencies decrease in the order F1 > F7 > F9 > F6 and vary by up to factor eight. KM values increase in the order F1 > F9 > F6 ≈ F7, whereas kcat decreases in the order F7 > F1 > F9 > F6. Comparative substrate scanning mutagenesis studies revealed a unique pattern of crucial substrate residues for each subtype. Based upon structural coordinates of F1 bound to an inhibitor polypeptide the mutational analyses suggest different substrate interactions in the substrate binding channel of each subtype.

scholarly journals Characterization of Affinity-Purified Isoforms ofAcinetobacter calcoaceticusY1 Glutathione Transferases

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Chin-Soon Chee ◽  
Irene Kit-Ping Tan ◽  
Zazali Alias
Keyword(s):  
Hydrogen Peroxide ◽  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Ethacrynic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Acinetobacter Calcoaceticus ◽  
Glutathione Transferases ◽  
Glutathione S Transferase ◽  
Substrate Specificities

Glutathione transferases (GST) were purified from locally isolated bacteria,Acinetobacter calcoaceticusY1, by glutathione-affinity chromatography and anion exchange, and their substrate specificities were investigated. SDS-polyacrylamide gel electrophoresis revealed that the purified GST resolved into a single band with a molecular weight (MW) of 23 kDa. 2-dimensional (2-D) gel electrophoresis showed the presence of two isoforms, GST1 (pI 4.5) and GST2 (pI 6.2) with identical MW. GST1 was reactive towards ethacrynic acid, hydrogen peroxide, 1-chloro-2,4-dinitrobenzene, andtrans,trans-hepta-2,4-dienalwhile GST2 was active towards all substrates except hydrogen peroxide. This demonstrated that GST1 possessed peroxidase activity which was absent in GST2. This study also showed that only GST2 was able to conjugate GSH to isoproturon, a herbicide. GST1 and GST2 were suggested to be similar to F0KLY9 (putative glutathione S-transferase) and F0KKB0 (glutathione S-transferase III) ofAcinetobacter calcoaceticusstrain PHEA-2, respectively.

Skeletal elements of crinoid echinoderms: High-resolution structural and microanalytical results

1983 ◽  
Vol 41 ◽  
pp. 194-195
Author(s):  
D. F. Blake ◽  
L. F. Allard ◽  
D. R. Peacor
Keyword(s):  
High Resolution ◽  
Marine Invertebrates ◽  
Sea Urchins ◽  
Structural Features ◽  
Sea Stars ◽  
High Resolution Tem ◽  
Relationship Of ◽  
The Relationship ◽  
Insight Into

Echinodermata is a phylum of marine invertebrates which has been extant since Cambrian time (c.a. 500 m.y. before the present). Modern examples of echinoderms include sea urchins, sea stars, and sea lilies (crinoids). The endoskeletons of echinoderms are composed of plates or ossicles (Fig. 1) which are with few exceptions, porous, single crystals of high-magnesian calcite. Despite their single crystal nature, fracture surfaces do not exhibit the near-perfect {10.4} cleavage characteristic of inorganic calcite. This paradoxical mix of biogenic and inorganic features has prompted much recent work on echinoderm skeletal crystallography. Furthermore, fossil echinoderm hard parts comprise a volumetrically significant portion of some marine limestones sequences. The ultrastructural and microchemical characterization of modern skeletal material should lend insight into: 1). The nature of the biogenic processes involved, for example, the relationship of Mg heterogeneity to morphological and structural features in modern echinoderm material, and 2). The nature of the diagenetic changes undergone by their ancient, fossilized counterparts. In this study, high resolution TEM (HRTEM), high voltage TEM (HVTEM), and STEM microanalysis are used to characterize tha ultrastructural and microchemical composition of skeletal elements of the modern crinoid Neocrinus blakei.

High Resolution Replication of Organoclay Surfaces

1982 ◽  
Vol 40 ◽  
pp. 576-577
Author(s):  
W. W. Barker ◽  
W. E. Rigsby ◽  
V. J. Hurst ◽  
W. J. Humphreys
Keyword(s):  
Clay Mineral ◽  
Organic Molecule ◽  
Organic Molecules ◽  
X Ray Diffraction ◽  
Xrd Pattern ◽  
X Ray ◽  
Naturally Occurring ◽  
Silicate Layers ◽  
Mineral Identification

Experimental clay mineral-organic molecule complexes long have been known and some of them have been extensively studied by X-ray diffraction methods. The organic molecules are adsorbed onto the surfaces of the clay minerals, or intercalated between the silicate layers. Natural organo-clays also are widely recognized but generally have not been well characterized. Widely used techniques for clay mineral identification involve treatment of the sample with H2 O2 or other oxidant to destroy any associated organics. This generally simplifies and intensifies the XRD pattern of the clay residue, but helps little with the characterization of the original organoclay. Adequate techniques for the direct observation of synthetic and naturally occurring organoclays are yet to be developed.

Ultrastructural identification of three types of sensory setae on copepod antennae

1995 ◽  
Vol 53 ◽  
pp. 956-957
Author(s):  
T. M. Weatherby ◽  
P.H. Lenz
Keyword(s):  
Phase Locking ◽  
Membrane Surface ◽  
Reaction Times ◽  
High Sensitivity ◽  
Structural Features ◽  
Calanoid Copepods ◽  
Marine Food Webs ◽  
Dendritic Membrane ◽  
Ultrastructural Characterization

Crustaceans, as well as other arthropods, are covered with sensory setae and hairs, including mechanoand chemosensory sensillae with a ciliary origin. Calanoid copepods are small planktonic crustaceans forming a major link in marine food webs. In conjunction with behavioral and physiological studies of the antennae of calanoids, we undertook the ultrastructural characterization of sensory setae on the antennae of Pleuromamma xiphias.Distal mechanoreceptive setae exhibit exceptional behavioral and physiological performance characteristics: high sensitivity (<10 nm displacements), fast reaction times (<1 msec latency) and phase locking to high frequencies (1-2 kHz). Unusual structural features of the mechanoreceptors are likely to be related to their physiological sensitivity. These features include a large number (up to 3000) of microtubules in each sensory cell dendrite, arising from or anchored to electron dense rods associated with the ciliary basal body microtubule doublets. The microtubules are arranged in a regular array, with bridges between and within rows. These bundles of microtubules extend far into each mechanoreceptive seta and terminate in a staggered fashion along the dendritic membrane, contacting a large membrane surface area and providing a large potential site of mechanotransduction.

Quantitative nano-characterization of heterogeneous catalysts

1995 ◽  
Vol 53 ◽  
pp. 398-399
Author(s):  
P.A. Crozier ◽  
M. Pan
Keyword(s):  
Heterogeneous Catalysts ◽  
Low Dose ◽  
Imaging Techniques ◽  
Structural Features ◽  
Catalyst Characterization ◽  
New Developments ◽  
Double Phase ◽  
Phase Systems ◽  
Dose Imaging

Heterogeneous catalysts can be of varying complexity ranging from single or double phase systems to complicated mixtures of metals and oxides with additives to help promote chemical reactions, extend the life of the catalysts, prevent poisoning etc. Although catalysis occurs on the surface of most systems, detailed descriptions of the microstructure and chemistry of catalysts can be helpful for developing an understanding of the mechanism by which a catalyst facilitates a reaction. Recent years have seen continued development and improvement of various TEM, STEM and AEM techniques for yielding information on the structure and chemistry of catalysts on the nanometer scale. Here we review some quantitative approaches to catalyst characterization that have resulted from new developments in instrumentation.HREM has been used to examine structural features of catalysts often by employing profile imaging techniques to study atomic details on the surface. Digital recording techniques employing slow-scan CCD cameras have facilitated the use of low-dose imaging in zeolite structure analysis and electron crystallography. Fig. la shows a low-dose image from SSZ-33 zeolite revealing the presence of a stacking fault.

A Pyridinic Fe-N4 Macrocycle Effectively Models the Active Sites in Fe/N-Doped Carbon Electrocatalysts

2020 ◽  
Author(s):  
Travis Marshall-Roth ◽  
Nicole J. Libretto ◽  
Alexandra T. Wrobel ◽  
Kevin Anderton ◽  
Nathan D. Ricke ◽  
...  
Keyword(s):  
Oxygen Reduction ◽  
Active Sites ◽  
Catalytic Properties ◽  
Reduction Reaction ◽  
Iron Phthalocyanine ◽  
Electrochemical Studies ◽  
Onset Potential ◽  
Nitrogen Doped Carbon ◽  
Iron Active Sites

Iron- and nitrogen-doped carbon (Fe-N-C) materials are leading candidates to replace platinum in fuel cells, but their active site structures are poorly understood. A leading postulate is that iron active sites in this class of materials exist in an Fe-N<sub>4</sub> pyridinic ligation environment. Yet, molecular Fe-based catalysts for the oxygen reduction reaction (ORR) generally feature pyrrolic coordination and pyridinic Fe-N<sub>4</sub> catalysts are, to the best of our knowledge, non-existent. We report the synthesis and characterization of a molecular pyridinic hexaazacyclophane macrocycle, (phen<sub>2</sub>N<sub>2</sub>)Fe, and compare its spectroscopic, electrochemical, and catalytic properties for oxygen reduction to a prototypical Fe-N-C material, as well as iron phthalocyanine, (Pc)Fe, and iron octaethylporphyrin, (OEP)Fe, prototypical pyrrolic iron macrocycles. N 1s XPS signatures for coordinated N atoms in (phen<sub>2</sub>N<sub>2</sub>)Fe are positively shifted relative to (Pc)Fe and (OEP)Fe, and overlay with those of Fe-N-C. Likewise, spectroscopic XAS signatures of (phen<sub>2</sub>N<sub>2</sub>)Fe are distinct from those of both (Pc)Fe and (OEP)Fe, and are remarkably similar to those of Fe-N-C with compressed Fe–N bond lengths of 1.97 Å in (phen<sub>2</sub>N<sub>2</sub>)Fe that are close to the average 1.94 Å length in Fe-N-C. Electrochemical studies establish that both (Pc)Fe and (phen<sub>2</sub>N<sub>2</sub>)Fe have relatively high Fe(III/II) potentials at ~0.6 V, ~300 mV positive of (OEP)Fe. The ORR onset potential is found to directly correlate with the Fe(III/II) potential leading to a ~300 mV positive shift in the onset of ORR for (Pc)Fe and (phen<sub>2</sub>N<sub>2</sub>)Fe relative to (OEP)Fe. Consequently, the ORR onset for (phen<sub>2</sub>N<sub>2</sub>)Fe and (Pc)Fe is within 150 mV of Fe-N-C. Unlike (OEP)Fe and (Pc)Fe, (phen<sub>2</sub>N<sub>2</sub>)Fe displays excellent selectivity for 4-electron ORR with <4% maximum H<sub>2</sub>O<sub>2</sub> production, comparable to Fe-N-C materials. The aggregate spectroscopic and electrochemical data establish (phen<sub>2</sub>N<sub>2</sub>)Fe as a pyridinic iron macrocycle that effectively models Fe-N-C active sites, thereby providing a rich molecular platform for understanding this important class of catalytic materials.<p><b></b></p>

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