Expression, purification, crystallization and preliminary X-ray analysis of rice (<i>Oryza sativa</i>L.) Os4BGlu12 β-glucosidase

Sansenya, Sompong; Cairns, James R. Ketudat; Opassiri, Rodjana

doi:10.1107/s174430911000103x

Cited by 4 publications

(4 citation statements)

References 29 publications

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“…In the preparation of rOsTAGG2E, a thioredoxin tag fused at the N-terminal was eliminated, but an extra peptide comprised of 22 amino acid residues was attached before the mature enzyme. 21) The N-terminal of OsTAGG2 is far from the active site, as pointed out above, and this extension probably does not affect substrate specificity.…”

Section: )mentioning

confidence: 90%

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Identification of Rice β-Glucosidase with High Hydrolytic Activity towards Salicylic Acid β-D-Glucoside

Himeno¹,

Saburi²,

Wakuta³

et al. 2013

Bioscience, Biotechnology, and Biochemistry

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β-Glucosidases (EC 3.2.1.21) split β-glucosidic linkages at the non-reducing end of glucosides and oligosaccharides to release β-D-glucose. One of the important functions of plant β-glucosidase is deglucosylation of inactive glucosides of phytohormones to regulate levels of active hormones. Tuberonic acid is a jasmonate-related compound that shows tuber-inducing activity in the potato. We have identified two enzymes, OsTAGG1 and OsTAGG2, that have hydrolytic activity towards tuberonic acid β-D-glucoside in rice (Oryza sativa L.). The expression of OsTAGG2 is upregulated by wounding and by methyl jasmonate, suggesting that this isozyme is involved in responses to biotic stresses and wounding, but the physiological substrate of OsTAGG2 remains ambiguous. In this study, we produced recombinant OsTAGG2 in Pichia pastoris (rOsTAGG2P), and investigated its substrate specificity in detail. From 1 L of culture medium, 2.1 mg of purified recombinant enzyme was obtained by ammonium sulfate precipitation and Ni-chelating column chromatography. The specific activity of rOsTAGG2P (182 U/mg) was close to that of the native enzyme (171 U/mg), unlike recombinant OsTAGG2 produced in Escherichia coli, which had approximately 3-fold lower specific activity than the native enzyme. The optimum pH and temperature for rOsTAGG2P were pH 3.4 and 60 °C. After pH and heat treatments, the enzyme retained its original activity in a pH range of 3.4-9.8 and below 55 °C. Native OsTAGG2 and rOsTAGG2P showed 4.5-4.7-fold higher activities towards salicylic acid β-D-glucoside, an inactive storage-form of salicylic acid, than towards tuberonic acid β-D-glucoside (TAG), although OsTAGG2 was originally isolated from rice based on TAG-hydrolytic activity

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Section: )mentioning

confidence: 90%

“…19,20) Native OsTAGG2 and rOsTAGG2E were prepared as reported previously. 8,21) Construction of an expression plasmid for OsTAGG2. Total RNA was prepared from leaf sheath of the rice plant (O. sativa L. cv.…”

Section: Methodsmentioning

confidence: 99%

Identification of Rice β-Glucosidase with High Hydrolytic Activity towards Salicylic Acid β-D-Glucoside

Himeno¹,

Saburi²,

Wakuta³

et al. 2013

Bioscience, Biotechnology, and Biochemistry

Self Cite

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“…The modelled structures were subjected to short all-atomistic molecular dynamics (MD) simulations. Gromacs 2021.3 (Van Der Spoel et al, 2005) with AMBER-99ff-ILDN (Sansenya et al, 2010) force field were used for these MD simulations. The proteins were put in a cubic box with 1 nm distance between protein and the edges of the box, and solvated with TIP3P (transferable intermolecular potential with 3 points) water.…”

Section: Methodsmentioning

confidence: 99%

Localization of four class I glutaredoxins in the cytosol and the secretory pathway and characterization of their biochemical diversification

Schlößer,

Moseler,

Bodnar

et al. 2023

Preprint

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Class I glutaredoxins (GRXs) are catalytically active oxidoreductases and considered key proteins mediating reversible glutathionylation and deglutathionylation of protein thiols during development and stress responses. To narrow in on putative target proteins, it is mandatory to know the subcellular localization of the respective GRXs and to understand their catalytic activities and putative redundancy between isoforms in the same compartment. We show that GRXC1 and GRXC2 are cytosolic proteins with GRXC1 being attached to membranes through myristoylation. GRXC3 and GRXC4 are identified as type II membrane proteins along the early secretory pathway with their enzymatic function on the luminal side. Comparison of all four studied GRXs for their oxidoreductase function highlights biochemical diversification with GRXC1 and GRXC2 being better reductants than GRXC3 and GRXC4 with bis(2-hydroxyethyl) disulfide and oxidized roGFP2 as substrates. Vice versa, GRXC3 and GRXC4 are better oxidants of reduced roGFP2 in the reverse reaction. Analysis of electrostatic surface potentials mirrors the phylogenetic classification of class I GRXs but cannot fully account for the observed kinetic differences in their interaction with roGPF2. Despite localization of two class I GRXs each in the cytosol and the endomembrane system, the respective double null mutants are viable without obvious phenotypes.

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“…The modeled structures were subjected to short all-atomistic molecular dynamics (MD) simulations. Gromacs 2021.3 (Van Der Spoel et al, 2005 with AMBER-99ff-ILDN (Sansenya et al, 2010) force field were used for these MD simulations. The proteins were put in a cubic box with a 1 nm distance between the protein and the edges of the box, and solvated with TIP3P (transferable intermolecular potential with 3 points) water.…”

Section: Molecular Modeling and Electrostatic Calculationsmentioning

confidence: 99%

Localization of four class I glutaredoxins in the cytosol and the secretory pathway and characterization of their biochemical diversification

Schlößer,

Moseler,

Bodnar

et al. 2024

The Plant Journal

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SUMMARYClass I glutaredoxins (GRXs) are catalytically active oxidoreductases and considered key proteins mediating reversible glutathionylation and deglutathionylation of protein thiols during development and stress responses. To narrow in on putative target proteins, it is mandatory to know the subcellular localization of the respective GRXs and to understand their catalytic activities and putative redundancy between isoforms in the same compartment. We show that in Arabidopsis thaliana, GRXC1 and GRXC2 are cytosolic proteins with GRXC1 being attached to membranes through myristoylation. GRXC3 and GRXC4 are identified as type II membrane proteins along the early secretory pathway with their enzymatic function on the luminal side. Unexpectedly, neither single nor double mutants lacking both GRXs isoforms in the cytosol or the ER show phenotypes that differ from wild‐type controls. Analysis of electrostatic surface potentials and clustering of GRXs based on their electrostatic interaction with roGFP2 mirrors the phylogenetic classification of class I GRXs, which clearly separates the cytosolic GRXC1 and GRXC2 from the luminal GRXC3 and GRXC4. Comparison of all four studied GRXs for their oxidoreductase function highlights biochemical diversification with GRXC3 and GRXC4 being better catalysts than GRXC1 and GRXC2 for the reduction of bis(2‐hydroxyethyl) disulfide. With oxidized roGFP2 as an alternative substrate, GRXC1 and GRXC2 catalyze the reduction faster than GRXC3 and GRXC4, which suggests that catalytic efficiency of GRXs in reductive reactions depends on the respective substrate. Vice versa, GRXC3 and GRXC4 are faster than GRXC1 and GRXC2 in catalyzing the oxidation of pre‐reduced roGFP2 in the reverse reaction.

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Expression, purification, crystallization and preliminary X-ray analysis of rice (Oryza sativaL.) Os4BGlu12 β-glucosidase

Cited by 4 publications

References 29 publications

Identification of Rice β-Glucosidase with High Hydrolytic Activity towards Salicylic Acid β-D-Glucoside

Identification of Rice β-Glucosidase with High Hydrolytic Activity towards Salicylic Acid β-D-Glucoside

Localization of four class I glutaredoxins in the cytosol and the secretory pathway and characterization of their biochemical diversification

Localization of four class I glutaredoxins in the cytosol and the secretory pathway and characterization of their biochemical diversification

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