Structural and Enzymatic Characterization of Os3BGlu6, a Rice <i>β</i>-Glucosidase Hydrolyzing Hydrophobic Glycosides and (1→3)- and (1→2)-Linked Disaccharides

Seshadri, Supriya; Akiyama, Takashi; Opassiri, Rodjana; Kuaprasert, Buabarn; Cairns, James R. Ketudat

doi:10.1104/pp.109.139436

Cited by 73 publications

(60 citation statements)

References 46 publications

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“…CsBGlu12 pH Optimum, in Vitro Enzyme Assays, and Enzyme Kinetics-The optimum pH for CsBGlu12 was determined as described previously (35). Briefly, 1 g of enzyme was incubated in a reaction volume of 140 l containing 100 mM universal buffer (citric acid disodium hydrogen phosphate).…”

Section: Methodsmentioning

confidence: 99%

“…), as described previously (35). The reactions were stopped after an overnight incubation (30°C) by boiling for 5 min.…”

Section: Methodsmentioning

confidence: 99%

“…Glucose release was measured by the glucose oxidase assay to monitor hydrolysis of glucosides, as described previously (35). The kinetic constants K m and V max values were calculated with non-linear regression analysis using GraphPad Prism 6 software.…”

Section: Methodsmentioning

confidence: 99%

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Functional Characterization of CsBGlu12, a β-Glucosidase from Crocus sativus, Provides Insights into Its Role in Abiotic Stress through Accumulation of Antioxidant Flavonols

Baba

Vishwakarma

Ashraf

2017

Journal of Biological Chemistry

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Edited by Joseph JezGlycosylation and deglycosylation are impressive mechanisms that allow plants to regulate the biological activity of an array of secondary metabolites. Although glycosylation improves solubility and renders the metabolites suitable for transport and sequestration, deglycosylation activates them to carry out biological functions. Herein, we report the functional characterization of CsBGlu12, a ␤-glucosidase from Crocus sativus. CsBGlu12 has a characteristic glucoside hydrolase 1 family (␣/␤) 8 triose-phosphate isomerase (TIM) barrel structure with a highly conserved active site. In vitro enzyme activity revealed that CsBGlu12 catalyzes the hydrolysis of flavonol ␤-glucosides and cello-oligosaccharides. Site-directed mutagenesis of any of the two conserved catalytic glutamic acid residues (Glu 200 and Glu 414 ) of the active site completely abolishes the ␤-glucosidase activity. Transcript analysis revealed that Csbglu12 is highly induced in response to UV-B, dehydration, NaCl, methyl jasmonate, and abscisic acid treatments indicating its possible role in plant stress response. Transient overexpression of CsBGlu12 leads to the accumulation of antioxidant flavonols in Nicotiana benthamiana and confers tolerance to abiotic stresses. Antioxidant assays indicated that accumulation of flavonols alleviated the accretion of reactive oxygen species during abiotic stress conditions. ␤-Glucosidases are known to play a role in abiotic stresses, particularly dehydration through abscisic acid; however, their role through accumulation of reactive oxygen species (ROS) scavenging flavonols has not been established. Furthermore, only one ␤-glucosidase 12 homolog has been characterized so far. Therefore, this work presents an important report on characterization of CsBGlu12 and its role in abiotic stress through ROS scavenging.Plants are sessile organisms that cannot escape their predators and the harsh environmental conditions. However, during the course of evolution, plants have learned to defend themselves and adapt to different types of biotic and abiotic stresses by synthesizing a diverse assortment of secondary metabolites. Many of these secondary metabolites are stored in inactive glycosylated forms. The glycosylation chemically stabilizes and enhances the solubility of the metabolites and renders them fit for storage in the vacuole and more so to protect the plant from the noxious effects of its own defense system (1). These glyconjugates are activated by the hydrolysis of the ␤-glucosidic bond by a class of enzymes called ␤-glucosidases. ␤-Glucosidases (EC 3.2.1.2.1) belong to family 1 of glycoside hydrolases and perform diverse and vital functions in plants, which include, but are not limited to, the activation of lignin precursors (2), release of glucose from oligosaccharides (3), release of phytohormones from inactive glycosides (4), and activation of several defense compounds (5-9). This process of glycosylation and deglycosylation offers an efficient mechanism that regulates the homeostasis of secon...

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Section: Methodsmentioning

confidence: 99%

“…), as described previously (35). The reactions were stopped after an overnight incubation (30°C) by boiling for 5 min.…”

Section: Methodsmentioning

confidence: 99%

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Functional Characterization of CsBGlu12, a β-Glucosidase from Crocus sativus, Provides Insights into Its Role in Abiotic Stress through Accumulation of Antioxidant Flavonols

Baba

Vishwakarma

Ashraf

2017

Journal of Biological Chemistry

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“…Five cis-peptide bonds were found at Gly11-Gly12, Tyr180-Pro181, Ala287-Glu288, Trp416-Thr417 and Ser423-Trp424 of SPy1599. The occurrence of such bonds is characteristic of glycoside hydrolase family 1 (as discussed, for example, in Seshadri et al, 2009).…”

Section: Spy1599mentioning

confidence: 99%

Structure and activity of theStreptococcus pyogenesfamily GH1 6-phospho-β-glucosidase SPy1599

Stepper

Dabin

Eklöf

et al. 2012

Acta Crystallogr D Biol Cryst

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The group A streptococcus Streptococcus pyogenes is the causative agent of a wide spectrum of invasive infections, including necrotizing fasciitis, scarlet fever and toxic shock syndrome. In the context of its carbohydrate chemistry, it is interesting that S. pyogenes (in this work strain M1 GAS SF370) displays a spectrum of oligosaccharide‐processing enzymes that are located in close proximity on the genome but that the in vivo function of these proteins remains unknown. These proteins include different sugar transporters (SPy1593 and SPy1595), both GH125 α‐1,6‐ and GH38 α‐1,3‐mannosidases (SPy1603 and SPy1604), a GH84 β‐hexosaminidase (SPy1600) and a putative GH2 β‐galactosidase (SPy1586), as well as SPy1599, a family GH1 `putative β‐glucosidase'. Here, the solution of the three‐dimensional structure of SPy1599 in a number of crystal forms complicated by unusual crystallographic twinning is reported. The structure is a classical (β/α)8‐barrel, consistent with CAZy family GH1 and other members of the GH‐A clan. SPy1599 has been annotated in sequence depositions as a β‐glucosidase (EC 3.2.1.21), but no such activity could be found; instead, three‐dimensional structural overlaps with other enzymes of known function suggested that SPy1599 contains a phosphate‐binding pocket in the active site and has possible 6‐phospho‐β‐glycosidase activity. Subsequent kinetic analysis indeed showed that SPy1599 has 6‐phospho‐β‐glucosidase (EC 3.2.1.86) activity. These data suggest that SPy1599 is involved in the intracellular degradation of 6‐phosphoglycosides, which are likely to originate from import through one of the organism's many phosphoenolpyruvate phosphotransfer systems (PEP‐PTSs).

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“…The structure will allow us to determine the structural differences that lead to the differences in oligosaccharide hydrolysis between Os4BGlu12 and other rice enzymes such as Os3BGlu6 (Seshadri et al, 2009) and Os3BGlu7 (Chuenchor et al, 2008).…”

Section: Resultsmentioning

confidence: 99%

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

Sansenya¹,

Cairns²,

Opassiri³

2010

Acta Cryst Sect F

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Rice (Oryza sativa L.) Os4BGlu12, a glycoside hydrolase family 1 -glucosidase (EC 3.2.1.21), was expressed as a fusion protein with an N-terminal thioredoxin/ His 6 tag in Escherichia coli strain Origami B (DE3) and purified with subsequent removal of the N-terminal tag. Native Os4BGlu12 and its complex with 2,4-dinitrophenyl-2-deoxy-2-fluoro--d-glucopyranoside (DNP2FG) were crystallized using 19% polyethylene glycol (3350 or 2000, respectively) in 0.1 M TrisHCl pH 8.5, 0.16 M NaCl at 288 K. Diffraction data sets for the apo and inhibitor-bound forms were collected to 2.50 and 2.45 Å resolution, respectively. The space group and the unit-cell parameters of the crystal indicated the presence of two molecules per asymmetric unit, with a solvent content of 50%. The structure of Os4BGlu12 was successfully solved in space group P4 3 2 1 2 by molecular replacement using the white clover cyanogenic -glucosidase structure (PDB code 1cbg) as a search model.

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Structural and Enzymatic Characterization of Os3BGlu6, a Rice β-Glucosidase Hydrolyzing Hydrophobic Glycosides and (1→3)- and (1→2)-Linked Disaccharides

Cited by 73 publications

References 46 publications

Functional Characterization of CsBGlu12, a β-Glucosidase from Crocus sativus, Provides Insights into Its Role in Abiotic Stress through Accumulation of Antioxidant Flavonols

Functional Characterization of CsBGlu12, a β-Glucosidase from Crocus sativus, Provides Insights into Its Role in Abiotic Stress through Accumulation of Antioxidant Flavonols

Structure and activity of theStreptococcus pyogenesfamily GH1 6-phospho-β-glucosidase SPy1599

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

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