Catalytic properties of the Gas family β-(1,3)-glucanosyltransferases active in fungal cell-wall biogenesis as determined by a novel fluorescent assay

BGTs [β-(1,3)-glucanosyltransglycosylases; EC 2.4.1.-] of the GH72 (family 72 of glycosylhydrolases) are GPI (glycosylphosphatidylinositol)-anchored proteins that play an important role in the biogenesis of fungal cell walls. They randomly cleave glycosidic linkages in β-(1,3)-glucan chains and ligate the polysaccharide portions containing newly formed reducing ends to C3(OH) at non-reducing ends of other β-(1,3)-glucan molecules. We have developed a sensitive fluorescence-based method for the assay of transglycosylating activity of GH72 enzymes. In the new assay, laminarin [β-(1,3)-glucan] is used as the glucanosyl donor and LamOS (laminarioligosaccharides) fluorescently labelled with SR (sulforhodamine) serve as the acceptors. The new fluorescent assay was employed for partial biochemical characterization of the heterologously expressed Gas family proteins from the yeast Saccharomyces cerevisiae. All the Gas enzymes specifically used laminarin as the glucanosyl donor and a SR–LamOS of DP (degree of polymerization) ≥5 as the acceptors. Gas proteins expressed in distinct stages of the yeast life cycle showed differences in their pH optima. Gas1p and Gas5p, which are expressed during vegetative growth, had the highest activity at pH 4.5 and 3.5 respectively, whereas the sporulation-specific Gas2p and Gas4p were most active between pH 5 and 6. The novel fluorescent assay provides a suitable tool for the screening of potential glucanosyltransferases or their inhibitors.

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The novel purification and biochemical characterization of a reversible CYP24A1:adrenodoxin complex

The Journal of Steroid Biochemistry and Molecular Biology ◽

10.1016/j.jsbmb.2012.11.001 ◽

2013 ◽

Vol 136 ◽

pp. 47-53 ◽

Cited By ~ 4

Author(s):

Kimberly A. Hartfield ◽

C. David Stout ◽

Andrew J. Annalora

Keyword(s):

Biochemical Characterization ◽

The Novel

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Purification and biochemical characterization of a β-cyanoalanine synthase expressed in germinating seeds of Sorghum bicolor (L.) moench

Turkish Journal of Biochemistry ◽

10.1515/tjb-2017-0214 ◽

2018 ◽

Vol 43 (6) ◽

pp. 638-650

Author(s):

Ruth Ololade Amiola ◽

Adedeji Nelson Ademakinwa ◽

Zainab Adenike Ayinla ◽

Esther Nkechi Ezima ◽

Femi Kayode Agboola

Keyword(s):

Kinetic Parameters ◽

Biochemical Characterization ◽

Catalytic Properties ◽

Specific Activity ◽

Biochemical Properties ◽

Subunit Molecular Weight ◽

Cyanoalanine Synthase ◽

Germinating Seeds ◽

Sorghum Bicolor L

Abstract Background β-Cyanoalanine synthase plays essential roles in germinating seeds, such as in cyanide homeostasis. Methods β-Cyanoalanine synthase was isolated from sorghum seeds, purified using chromatographic techniques and its biochemical and catalytic properties were determined. Results The purified enzyme had a yield of 61.74% and specific activity of 577.50 nmol H2S/min/mg of protein. The apparent and subunit molecular weight for purified β-cyanoalanine synthase were 58.26±2.41 kDa and 63.4 kDa, respectively. The kinetic parameters with sodium cyanide as substrate were 0.67±0.08 mM, 17.60±0.50 nmol H2S/mL/min, 2.97×10−1 s−1 and 4.43×102 M−1 s−1 for KM, Vmax, kcat and kcat/KM, respectively. With L-cysteine as substrate, the kinetic parameters were 2.64±0.37 mM, 63.41±4.04 nmol H2S/mL/min, 10.71×10−1 s−1 and 4.06×102 M−1 s−1 for KM, Vmax, kcat and kcat/KM, respectively. The optimum temperature and pH for activity were 35°C and 8.5, respectively. The enzyme retained more than half of its activity at 40°C. Inhibitors such as HgCl2, EDTA, glycine and iodoacetamide reduced enzyme activity. Conclusion The biochemical properties of β-cyanoalanine synthase in germinating sorghum seeds highlights its roles in maintaining cyanide homeostasis.

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A Single Xyloglucan Xylosyltransferase is Sufficient for Generation of the XXXG Xylosylation Pattern of Xyloglucan

Plant and Cell Physiology ◽

10.1093/pcp/pcab113 ◽

2021 ◽

Author(s):

Ruiqin Zhong ◽

Dennis R Phillips ◽

Zheng-Hua Ye

Keyword(s):

Recombinant Proteins ◽

Cell Walls ◽

Degree Of Polymerization ◽

Primary Cell ◽

Hek293 Cells ◽

Biochemical Mechanism ◽

The Third ◽

Insight Into

Abstarct Xyloglucan is the most abundant hemicellulose in the primary cell walls of dicots. Dicot xyloglucan is the XXXG-type consisting of repeating units of three consecutive xylosylated Glc residues followed by one unsubstituted Glc. Its xylosylation is catalyzed by xyloglucan 6-xylosyltransferases (XXTs) and there exist five XXTs (AtXXT1-5) in Arabidopsis. While AtXXT1and AtXXT2 have been shown to add the first two Xyl residues in the XXXG repeat, which XXTs are responsible for the addition of the third Xyl residue remains elusive although AtXXT5 was a proposed candidate. In this report, we generated recombinant proteins of all five Arabidopsis XXTs and one rice XXT (OsXXT1) in the mammalian HEK293 cells and investigated their ability to sequentially xylosylate Glc residues to generate the XXXG xylosylation pattern. We found that like AtXXT1/2, AtXXT4 and OsXXT1 could efficiently xylosylate the cellohexaose (G6) acceptor to produce mono- and di-xylosylated G6, whereas AtXXT5 was only barely capable of adding one Xyl onto G6. When AtXXT1-catalyzed products were used as acceptors, AtXXT1/2/4 and OsXXT1 but not AtXXT5 were able to xylosylate additional Glc residues to generate tri- and tetra-xylosylated G6. Further characterization of the tri- and tetra-xylosylated G6 revealed that they had the sequence of GXXXGG and GXXXXG with three and four consecutive xylosylated Glc residues, respectively. In addition, we have found that although tri-xylosylation occurred on G6, cello-oligomers with a degree of polymerization of 3 to 5 could only be mono- and di-xylosylated. Together, these results indicate that each of AtXXT1/2/4 and OsXXT1 is capable of sequentially adding Xyl onto three contiguous Glc residues to generate the XXXG xylosylation pattern and these findings provide new insight into the biochemical mechanism underlying xyloglucan biosynthesis.

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Biochemical characterization of two chitinases from Bacillus cereus sensu lato B25 with antifungal activity against Fusarium verticillioides P03

FEMS Microbiology Letters ◽

10.1093/femsle/fnaa218 ◽

2020 ◽

Author(s):

Estefanía Morales-Ruiz ◽

Ricardo Priego-Rivera ◽

Alejandro Miguel Figueroa-López ◽

Jesús Eduardo Cazares-Álvarez ◽

Ignacio E Maldonado-Mendoza

Keyword(s):

Bacillus Cereus ◽

Biochemical Characterization ◽

Fusarium Verticillioides ◽

Expression System ◽

Phytopathogenic Fungi ◽

Fungal Cell ◽

Synthetic Pesticides ◽

Bacillus Cereus Sensu Lato ◽

Dual Substrate

Abstract Bacterial chitinases are a subject of intense scientific research due to their biotechnological applications, particularly their use as biological pesticides against phytopathogenic fungi as a green alternative to avoid the use of synthetic pesticides. Bacillus cereus sensu lato B25 is a rhizospheric bacterium that is a proven antagonist of Fusarium verticillioides, a major fungal pathogen of maize. This bacterium produces two chitinases that degrade the fungal cell wall and inhibit its growth. In this work, we used a heterologous expression system to purify both enzymes to investigate their biochemical traits in terms of Km, Vmax, optimal pH and temperature. ChiA and ChiB work as exochitinases, but ChiB exhibited a dual substrate activity and it is also an endochitinase. In this work, the direct addition of these chitinases inhibited fungal conidial germination and therefore they may play a major role in the antagonism against F. verticillioides.

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Molecular and biochemical characterization of the thermoactive family 1 pectate lyase from the hyperthermophilic bacterium Thermotoga maritima

Biochemical Journal ◽

10.1042/bj20021595 ◽

2003 ◽

Vol 370 (2) ◽

pp. 651-659 ◽

Cited By ~ 42

Author(s):

Leon D. KLUSKENS ◽

Gert-Jan W.M. van ALEBEEK ◽

Alphons G.J. VORAGEN ◽

Willem M. de VOS ◽

John van der OOST

Keyword(s):

Thermal Inactivation ◽

Biochemical Characterization ◽

Thermotoga Maritima ◽

Pectate Lyase ◽

Major Product ◽

Degree Of Polymerization ◽

Extracellular Medium ◽

Native Form ◽

Hyperthermophilic Bacterium

The ability of the hyperthermophilic bacterium Thermotoga maritima to grow on pectin as a sole carbon source coincides with the secretion of a pectate lyase A (PelA) in the extracellular medium. The pelA gene of T. maritima was functionally expressed in Escherichia coli as the first heterologously produced thermophilic pectinase, and purified to homogeneity. Gel filtration indicated that the native form of PelA is tetrameric. Highest activity (422units/mg, with a Km of 0.06mM) was demonstrated on polygalacturonic acid (PGA), whereas pectins with an increasing degree of methylation were degraded at a decreasing rate. In the tradition of pectate lyases, PelA demonstrated full dependency on Ca2+ for stability and activity. The enzyme is highly thermoactive and thermostable, operating optimally at 90°C and pH9.0, with a half-life for thermal inactivation of almost 2h at 95°C, and an apparent melting temperature of 102.5°C. Detailed characterization of the product formation with PGA indicated that PelA has a unique eliminative exo-cleavage pattern liberating unsaturated trigalacturonate as the major product, in contrast with unsaturated digalacturonate for other exopectate lyases known. The unique exo-acting mode of action was supported by progression profiles of PelA on oligogalacturonides (degree of polymerization, 3—8) and the examination of the bond cleavage frequencies.

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Biochemical characterization of the novel endo -β-mannanase At Man5-2 from Arabidopsis thaliana

Plant Science ◽

10.1016/j.plantsci.2015.10.002 ◽

2015 ◽

Vol 241 ◽

pp. 151-163 ◽

Cited By ~ 3

Author(s):

Yang Wang ◽

Shoaib Azhar ◽

Rosaria Gandini ◽

Christina Divne ◽

Ines Ezcurra ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Biochemical Characterization ◽

The Novel

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Biochemical characterization of the novel α-1, 3-galactosyltransferase WclR from Escherichia coli O3

Carbohydrate Research ◽

10.1016/j.carres.2016.04.012 ◽

2016 ◽

Vol 430 ◽

pp. 36-43 ◽

Cited By ~ 17

Author(s):

Chao Chen ◽

Bin Liu ◽

Yongchang Xu ◽

Natalia Utkina ◽

Dawei Zhou ◽

...

Keyword(s):

Escherichia Coli ◽

Biochemical Characterization ◽

The Novel

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Structural and biochemical characterization of the novel serpin Iripin-5 from Ixodes ricinus

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798321007920 ◽

2021 ◽

Vol 77 (9) ◽

pp. 1183-1196 ◽

Cited By ~ 2

Author(s):

Barbora Kascakova ◽

Jan Kotal ◽

Larissa Almeida Martins ◽

Zuzana Berankova ◽

Helena Langhansova ◽

...

Keyword(s):

Ixodes Ricinus ◽

Biochemical Characterization ◽

Neutrophil Migration ◽

Stable State ◽

Host Immunity ◽

The Novel ◽

Defence Mechanisms ◽

Reactive Centre Loop ◽

Main Substrate

Iripin-5 is the main Ixodes ricinus salivary serpin, which acts as a modulator of host defence mechanisms by impairing neutrophil migration, suppressing nitric oxide production by macrophages and altering complement functions. Iripin-5 influences host immunity and shows high expression in the salivary glands. Here, the crystal structure of Iripin-5 in the most thermodynamically stable state of serpins is described. In the reactive-centre loop, the main substrate-recognition site of Iripin-5 is likely to be represented by Arg342, which implies the targeting of trypsin-like proteases. Furthermore, a computational structural analysis of selected Iripin-5–protease complexes together with interface analysis revealed the most probable residues of Iripin-5 involved in complex formation.

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