scholarly journals Assaying Paenibacillus alvei CsaB-Catalysed Ketalpyruvyltransfer to Saccharides by Measurement of Phosphate Release

Biomolecules ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 1732
Author(s):  
Fiona F. Hager-Mair ◽  
Cordula Stefanović ◽  
Charlie Lim ◽  
Katharina Webhofer ◽  
Simon Krauter ◽  
...  
Keyword(s):  
Cell Wall ◽  
Enzyme Assay ◽  
Colorimetric Detection ◽  
Limiting Factors ◽  
Biosynthesis Pathway ◽  
Phosphate Release ◽  
Acceptor Substrate ◽  
Paenibacillus Alvei ◽  
Colour Development ◽  
Assay Conditions

Ketalpyruvyltransferases belong to a widespread but little investigated class of enzymes, which utilise phosphoenolpyruvate (PEP) for the pyruvylation of saccharides. Pyruvylated saccharides play pivotal biological roles, ranging from protein binding to virulence. Limiting factors for the characterisation of ketalpyruvyltransferases are the availability of cognate acceptor substrates and a straightforward enzyme assay. We report on a fast ketalpyruvyltransferase assay based on the colorimetric detection of phosphate released during pyruvyltransfer from PEP onto the acceptor via complexation with Malachite Green and molybdate. To optimise the assay for the model 4,6-ketalpyruvyl::ManNAc-transferase CsaB from Paenibacillus alvei, a β-d-ManNAc-α-d-GlcNAc-diphosphoryl-11-phenoxyundecyl acceptor mimicking an intermediate of the bacterium’s cell wall glycopolymer biosynthesis pathway, upon which CsaB is naturally active, was produced chemo-enzymatically and used together with recombinant CsaB. Optimal assay conditions were 5 min reaction time at 37 °C and pH 7.5, followed by colour development for 1 h at 37 °C and measurement of absorbance at 620 nm. The structure of the generated pyruvylated product was confirmed by NMR spectroscopy. Using the established assay, the first kinetic constants of a 4,6-ketalpyuvyl::ManNAc-transferase could be determined; upon variation of the acceptor and PEP concentrations, a KM, PEP of 19.50 ± 3.50 µM and kcat, PEP of 0.21 ± 0.01 s−1 as well as a KM, Acceptor of 258 ± 38 µM and a kcat, Acceptor of 0.15 ± 0.01 s−1 were revealed. P. alvei CsaB was inactive on synthetic pNP-β-d-ManNAc and β-d-ManNAc-β-d-GlcNAc-1-OMe, supporting the necessity of a complex acceptor substrate.

scholarly journals Yeast Mnn9 is both a priming glycosyltransferase and an allosteric activator of mannan biosynthesis

Open Biology ◽  
2013 ◽  
Vol 3 (9) ◽  
pp. 130022 ◽  
Author(s):  
Alexander Striebeck ◽  
David A. Robinson ◽  
Alexander W. Schüttelkopf ◽  
Daan M. F. van Aalten
Keyword(s):  
Cell Wall ◽  
Fungal Pathogens ◽  
Enzyme Assay ◽  
Antibiotic Sensitivity ◽  
Site Directed Mutagenesis ◽  
Fungal Cell ◽  
Pol I ◽  
Conserved Amino Acids ◽  
Allosteric Activator ◽  
Mannan Synthesis

The fungal cell possesses an essential carbohydrate cell wall. The outer layer, mannan, is formed by mannoproteins carrying highly mannosylated O - and N -linked glycans. Yeast mannan biosynthesis is initiated by a Golgi-located complex (M-Pol I) of two GT-62 mannosyltransferases, Mnn9p and Van1p, that are conserved in fungal pathogens. Saccharomyces cerevisiae and Candida albicans mnn9 knockouts show an aberrant cell wall and increased antibiotic sensitivity, suggesting the enzyme is a potential drug target. Here, we present the structure of Sc Mnn9 in complex with GDP and Mn 2+ , defining the fold and catalytic machinery of the GT-62 family. Compared with distantly related GT-78/GT-15 enzymes, Sc Mnn9 carries an unusual extension. Using a novel enzyme assay and site-directed mutagenesis, we identify conserved amino acids essential for Sc Mnn9 ‘priming’ α-1,6-mannosyltransferase activity. Strikingly, both the presence of the Sc Mnn9 protein and its product, but not Sc Mnn9 catalytic activity, are required to activate subsequent Sc Van1 processive α-1,6-mannosyltransferase activity in the M-Pol I complex. These results reveal the molecular basis of mannan synthesis and will aid development of inhibitors targeting this process.

scholarly journals Biosynthesis of the Unique Wall Teichoic Acid of Staphylococcus aureus Lineage ST395

mBio ◽  
2014 ◽  
Vol 5 (2) ◽  
Author(s):  
Volker Winstel ◽  
Patricia Sanchez-Carballo ◽  
Otto Holst ◽  
Guoqing Xia ◽  
Andreas Peschel
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Biosynthesis Pathway ◽  
Glycerol Phosphate ◽  
Coagulase Negative Staphylococci ◽  
Content Type ◽  
Gram Positive Bacteria ◽  
Wall Teichoic Acids

ABSTRACT The major clonal lineages of the human pathogen Staphylococcus aureus produce cell wall-anchored anionic poly-ribitol-phosphate (RboP) wall teichoic acids (WTA) substituted with d-Alanine and N-acetyl-d-glucosamine. The phylogenetically isolated S. aureus ST395 lineage has recently been found to produce a unique poly-glycerol-phosphate (GroP) WTA glycosylated with N-acetyl-d-galactosamine (GalNAc). ST395 clones bear putative WTA biosynthesis genes on a novel genetic element probably acquired from coagulase-negative staphylococci (CoNS). We elucidated the ST395 WTA biosynthesis pathway and identified three novel WTA biosynthetic genes, including those encoding an α-O-GalNAc transferase TagN, a nucleotide sugar epimerase TagV probably required for generation of the activated sugar donor substrate for TagN, and an unusually short GroP WTA polymerase TagF. By using a panel of mutants derived from ST395, the GalNAc residues carried by GroP WTA were found to be required for infection by the ST395-specific bacteriophage Φ187 and to play a crucial role in horizontal gene transfer of S. aureus pathogenicity islands (SaPIs). Notably, ectopic expression of ST395 WTA biosynthesis genes rendered normal S. aureus susceptible to Φ187 and enabled Φ187-mediated SaPI transfer from ST395 to regular S. aureus. We provide evidence that exchange of WTA genes and their combination in variable, mosaic-like gene clusters have shaped the evolution of staphylococci and their capacities to undergo horizontal gene transfer events. IMPORTANCE The structural highly diverse wall teichoic acids (WTA) are cell wall-anchored glycopolymers produced by most Gram-positive bacteria. While most of the dominant Staphylococcus aureus lineages produce poly-ribitol-phosphate WTA, the recently described ST395 lineage produces a distinct poly-glycerol-phosphate WTA type resembling the WTA backbone of coagulase-negative staphylococci (CoNS). Here, we analyzed the ST395 WTA biosynthesis pathway and found new types of WTA biosynthesis genes along with an evolutionary link between ST395 and CoNS, from which the ST395 WTA genes probably originate. The elucidation of ST395 WTA biosynthesis will help to understand how Gram-positive bacteria produce highly variable WTA types and elucidate functional consequences of WTA variation.

scholarly journals Mutations in the Lipopolysaccharide Biosynthesis Pathway Interfere with Crescentin-Mediated Cell Curvature in Caulobacter crescentus

2010 ◽  
Vol 192 (13) ◽  
pp. 3368-3378 ◽  
Author(s):  
Matthew T. Cabeen ◽  
Michelle A. Murolo ◽  
Ariane Briegel ◽  
N. Khai Bui ◽  
Waldemar Vollmer ◽  
...  
Keyword(s):  
Cell Wall ◽  
Caulobacter Crescentus ◽  
Cell Envelope ◽  
Cellular Systems ◽  
Biosynthesis Pathway ◽  
Cell Morphogenesis ◽  
Division Site ◽  
Growth Properties ◽  
A Cell ◽  
Cellular Components

ABSTRACT Bacterial cell morphogenesis requires coordination among multiple cellular systems, including the bacterial cytoskeleton and the cell wall. In the vibrioid bacterium Caulobacter crescentus, the intermediate filament-like protein crescentin forms a cell envelope-associated cytoskeletal structure that controls cell wall growth to generate cell curvature. We undertook a genetic screen to find other cellular components important for cell curvature. Here we report that deletion of a gene (wbqL) involved in the lipopolysaccharide (LPS) biosynthesis pathway abolishes cell curvature. Loss of WbqL function leads to the accumulation of an aberrant O-polysaccharide species and to the release of the S layer in the culture medium. Epistasis and microscopy experiments show that neither S-layer nor O-polysaccharide production is required for curved cell morphology per se but that production of the altered O-polysaccharide species abolishes cell curvature by apparently interfering with the ability of the crescentin structure to associate with the cell envelope. Our data suggest that perturbations in a cellular pathway that is itself fully dispensable for cell curvature can cause a disruption of cell morphogenesis, highlighting the delicate harmony among unrelated cellular systems. Using the wbqL mutant, we also show that the normal assembly and growth properties of the crescentin structure are independent of its association with the cell envelope. However, this envelope association is important for facilitating the local disruption of the stable crescentin structure at the division site during cytokinesis.

scholarly journals Isothiocyanates as substrates for human glutathione transferases: structure-activity studies

1995 ◽  
Vol 311 (2) ◽  
pp. 453-459 ◽  
Author(s):  
R H Kolm ◽  
U H Danielson ◽  
Y Zhang ◽  
P Talalay ◽  
B Mannervik
Keyword(s):  
Enzyme Assay ◽  
Catalytic Properties ◽  
Thiol Group ◽  
Binding Energies ◽  
Glutathione Transferases ◽  
Structure Activity ◽  
Transition State Stabilization ◽  
Central Carbon ◽  
Methylene Groups ◽  
Assay Conditions

The catalytic properties of four human glutathione transferases (GSTs), A1-1, M1-1, M4-4 and P1-1, were examined with 14 isothiocyanate (R-NCS) substrates. The compounds include aliphatic and aromatic homologues, some of which are natural constituents of human food, namely sulphoraphane [1-isothiocyanato-4-(methylsulphinyl)butane], erucin [1-isothiocyanato-4-(methylthio)butane], erysolin [1-isothiocyanato-4-(methylsulphonyl)butane], benzyl-NCS, phenethyl-NCS and allyl-NCS. All isothiocyanates investigated were substrates for the four GSTs. The enzymes promote addition of the thiol group of GSH to the electrophilic central carbon of the isothiocyanate group to form dithiocarbamates [R-NH-C(=S)-SG] which have high UV absorption at 274 nm. Molar absorption coefficients and non-enzymic rate constants as well as standardized enzyme assay conditions for all compounds were established. Of the four isoenzymes investigated, GSTs M1-1 and P1-1 were generally the most efficient catalysts, whereas GST M4-4 was the least efficient. Isothiocyanates are among the GST substrates that are most rapidly conjugated. On the basis of rate-enhancement data and binding energies, the isothiocyanates were compared with 4-hydroxyalkenals, another class of natural GST substrates previously subjected to systematic kinetic analysis. The incremental transition-state stabilization attributable to an increased number of methylene groups in homologous alkyl isothiocyanates is similar to that previously noted for homologous 4-hydroxyalkenals.

scholarly journals Two-component system VraSR positively modulates the regulation of cell-wall biosynthesis pathway in Staphylococcus aureus

2004 ◽  
Vol 49 (3) ◽  
pp. 807-821 ◽  
Author(s):  
Makoto Kuroda ◽  
Hiroko Kuroda ◽  
Taku Oshima ◽  
Fumihiko Takeuchi ◽  
Hirotada Mori ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Cell Wall Biosynthesis ◽  
Biosynthesis Pathway ◽  
Two Component System ◽  
Component System ◽  
Two Component

Limitations to wheat starch digestion in growing broiler chickens: a brief review

2011 ◽  
Vol 51 (7) ◽  
pp. 583 ◽  
Author(s):  
Birger Svihus
Keyword(s):  
Cell Wall ◽  
Broiler Chickens ◽  
Glucose Absorption ◽  
Wheat Starch ◽  
Starch Digestibility ◽  
Limiting Factors ◽  
Wall Structure ◽  
Aleurone Layer ◽  
Starch Granules ◽  
Absorption Process

Starch digestibility may be suboptimal in broilers fed pelleted wheat-based diets. In the present review, the digestion and absorption process related to starch is presented, followed by a discussion of the effect of wheat characteristics and bird-related effects. Enzyme secretion or glucose absorption and metabolism have not been shown to be limiting factors. Suboptimal starch digestibility is primarily observed when a large proportion of wheat is included in the diet, and appears to be partly associated with characteristics of the wheat such as hardness and cell wall structure, which cause starch granules to remain entrapped in the protein matrix and the cell wall of the endosperm or aleurone layer. There are indications that low starch digestibility is negatively correlated with feed intake, and that such a feed over-consumption is linked to an under-developed gizzard.

scholarly journals Kinetics of Crude Peroxidase from the Rind of Watermelon Fruit

2021 ◽  
pp. 20-27
Author(s):  
O. M. Iniaghe ◽  
O. Ibukun ◽  
R. E. Giwa
Keyword(s):  
Enzyme Activity ◽  
Enzyme Assay ◽  
Crude Enzyme ◽  
Redox Mediators ◽  
Alternative Source ◽  
Optimal Activity ◽  
Edo State ◽  
Kinetics Of ◽  
Assay Conditions

Aims: To study the kinetics of crude peroxidase from the rind of watermelon fruit in various assay conditions. Study Design: In vitro enzyme assay. Place and Duration of Study: Department of Biochemistry, Faculty of Life Sciences, Ambrose Alli University, Ekpoma, Edo State, Nigeria between October 2015 and January 2016. Methodology: The activity of the crude peroxidase extracted from the rind of watermelon was determined by measuring the rate of oxidation of KI at 25oC in a 3.0 ml reaction mixture which contained 2.3 ml of 25 mM - 400 mM sodium acetate buffer (pH 3.5-6.0), 0.2 ml of 2 mM KI, 0.1ml of the crude peroxidase, and 0.2 ml of varying concentrations of chlorpromazine (0.01 mM - 0.1 mM). In all cases, 0.2 ml of 0.01 mM – 1 mM H2O2 was added last to initiate the reaction. Only one parameter was varied per assay. Assays were done in five replicates. The initial velocity of the crude peroxidase for KI oxidation was determined using the absorbance at 353 nm. Results: The concentration of H2O2 that generated an optimal activity for the crude peroxidase extracted was 0.2 mM, while a pH of 5.5 was optimal for the crude enzyme. The activity of the crude enzyme increased proportionately within a buffer concentration range of 25 mM and 400 mM. Chlorpromazine (0.01 mM - 0.1 mM) proportionately increased the enzyme activity, while promethazine within a range of 0.01 mM and 0.06 mM proportionally increased the enzyme activity. Further increase in promethazine concentration beyond 0.6 mM resulted in a decreased activity of the enzyme. Conclusion: This study suggests that the Rind of watermelon is an alternative source of peroxidase. The activity of this peroxidase can be enhanced by high buffer concentrations in the presence of some redox mediators like promethazine and chlorpromazine at a pH of 5.5.

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