scholarly journals Characterization of FcXTH2, a Novel Xyloglucan Endotransglycosylase/Hydrolase Enzyme of Chilean Strawberry with Hydrolase Activity

2020 ◽  
Vol 21 (9) ◽  
pp. 3380 ◽  
Author(s):  
Luis Morales-Quintana ◽  
Dina Beltrán ◽  
Ángela Mendez-Yañez ◽  
Felipe Valenzuela-Riffo ◽  
Raúl Herrera ◽  
...  
Keyword(s):  
Cell Wall ◽  
Structural Model ◽  
Biochemical Characterization ◽  
Comparative Modeling ◽  
Xyloglucan Endotransglycosylase ◽  
Protein Ligand Interactions ◽  
Modeling Methodology ◽  
Ligand Interactions ◽  
Km Value

Xyloglucan endotransglycosylase/hydrolases (XTHs) are cell wall enzymes with hydrolase (XEH) and/or endotransglycosylase (XET) activities. As they are involved in the modification of the xyloglucans, a type of hemicellulose present in the cell wall, they are believed to be very important in different processes, including growth, development, and fruit ripening. Previous studies suggest that XTHs might play a key role in development and ripening of Fragaria chiloensis fruit, and its characterization is pending. Therefore, in order to provide a biochemical characterization of the FcXTH2 enzyme to explain its possible role in strawberry development, the molecular cloning and the heterologous expression of FcXTH2 were performed. The recombinant FcXTH2 was active and displayed mainly XEH activity. The optimal pH and temperature are 5.5 and 37 °C, respectively. A KM value of 0.029 mg mL−1 was determined. Additionally, its protein structural model was built through comparative modeling methodology. The model showed a typically β-jelly-roll type folding in which the catalytic motif was oriented towards the FcXTH2 central cavity. Using molecular docking, protein-ligand interactions were explored, finding better interaction with xyloglucan than with cellulose. The data provided groundwork for understanding, at a molecular level, the enzymatic mechanism of FcXTH2, an important enzyme acting during the development of the Chilean strawberry.

scholarly journals Molecular Insights into FaEG1, a Strawberry Endoglucanase Enzyme Expressed during Strawberry Fruit Ripening

Plants ◽  
2019 ◽  
Vol 8 (6) ◽  
pp. 140
Author(s):  
Karla Jara ◽  
Ricardo I. Castro ◽  
Patricio Ramos ◽  
Carolina Parra-Palma ◽  
Felipe Valenzuela-Riffo ◽  
...  
Keyword(s):  
Cell Wall ◽  
Phylogenetic Analyses ◽  
Quantitative Polymerase Chain Reaction ◽  
Glycosyl Hydrolase ◽  
Comparative Modeling ◽  
Strawberry Fruit ◽  
Transcript Accumulation ◽  
Protein Ligand Interactions ◽  
Ligand Interactions ◽  
Probable Role

The endo-β-1,4-glucanases (EGs) that belong to the glycosyl hydrolase family 9 (GH9) have roles in cell wall synthesis, remodeling and degradation. Previous studies have suggested that EGs may play a key role in the ripening of different fruits including strawberries. In this study, we used reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assays to determine the transcript accumulation of an endo-β-1,4-glucanase (FaEG1) during fruit development in two different strawberry ‘Camarosa’ and ‘Monterey’ with contrasting softening ratios. Phylogenetic analyses suggest that FaEG1 belongs to the α group of the GH9 family with other proteins previously described with roles in elongation, abscission and ripening. Comparative modeling was used to obtain the FaEG1 structure. The model displays a α-barrel–type structure that is typical of the GH9 enzyme family, and comprises 12 α-helices, 2 310 helices and 6 β-sheets. The catalytic residues were oriented to the solvent in the middle of an open groove. Protein–ligand interactions were explored with cellulose and two xyloglucans as ligands; the results suggest that the FaEG1-cellulose and FaEG1-XXXGXXXG (the most abundant xyloglucan in strawberries) complexes were more stable complexes than XXFGXXFG. The cell wall degradation was observed by scanning electron microscopy (SEM). The data are congruent with the probable role of the FaEG1 protein in the dissembly of the cellulose-hemicellulose fraction during the ripening of strawberry fruit.

Biochemical characterization of a cortexless mutant of a variant of Bacillus cereus

1976 ◽  
Vol 22 (7) ◽  
pp. 1007-1012 ◽  
Author(s):  
Susanne M. Pearce
Keyword(s):  
Cell Wall ◽  
Fatty Acid Composition ◽  
Fatty Acid ◽  
Bacillus Cereus ◽  
Biochemical Characterization ◽  
Diaminopimelic Acid ◽  
Peptidoglycan Synthesis ◽  
Biochemical Lesion ◽  
Do So

Previous studies on this cortexless mutant of Bacillus cereus var. alesti indicated that the forespore membrane was the site of the biochemical lesion. This hypothesis is supported by the results presented here: fatty acid composition of sporulating cells of the mutant is altered, while in vegetative cells it is comparable to the parent; soluble precursors of peptidoglycan synthesis are accumulated in the mutant, at the time of cortex formation; homogenates of the mutant prepared at the time of cortex formation are unable to incorporate tritiated diaminopimelic acid into peptidoglycan, while homogenates of cells forming germ cell wall do so to an extent comparable to that of the parent; lipid-linked intermediates are formed by the mutant as in the parent. Apparently the mutant is unable either to transfer disaccharide penta-peptide units from the carrier lipid to the growing peptidoglycan acceptor, or to transport lipid-linked intermediates across the forespore membrane.

scholarly journals Biochemical characterization of a 27kDa 1,3-β-d-glucanase from Trichoderma asperellum induced by cell wall of Rhizoctonia solani

2012 ◽  
Vol 87 (2) ◽  
pp. 1219-1223 ◽  
Author(s):  
Raquel da Silva Aires ◽  
Andrei Stecca Steindorff ◽  
Marcelo Henrique Soller Ramada ◽  
Saulo José Linhares de Siqueira ◽  
Cirano José Ulhoa
Keyword(s):  
Cell Wall ◽  
Rhizoctonia Solani ◽  
Biochemical Characterization ◽  
Trichoderma Asperellum

scholarly journals Biochemical characterization of wheat straw cell wall with special reference to bioactive profile

2018 ◽  
Vol 21 (1) ◽  
pp. 1303-1310 ◽  
Author(s):  
Tabussam Tufail ◽  
Farhan Saeed ◽  
Muhammad Imran ◽  
Muhammad Umair Arshad ◽  
Faqir Muhammad Anjum ◽  
...  
Keyword(s):  
Cell Wall ◽  
Special Reference ◽  
Wheat Straw ◽  
Biochemical Characterization

scholarly journals Characterization of protein-ligand binding interactions of enoyl-ACP reductase (FabI) by native MS reveals allosteric effects of coenzymes and the inhibitor triclosan

2019 ◽  
Author(s):  
P. Matthew Joyner ◽  
Denise P. Tran ◽  
Muhammad A. Zenaidee ◽  
Joseph A. Loo
Keyword(s):  
Fatty Acid Synthase ◽  
Antibacterial Drug ◽  
Allosteric Effect ◽  
The Past ◽  
Protein Ligand Interactions ◽  
Native Ms ◽  
Ligand Interactions ◽  
Enoyl Acp Reductase ◽  
The Subject

AbstractThe enzyme enoyl-ACP reductase (also called FabI in bacteria) is an essential member of the fatty acid synthase II pathway in plants and bacteria. This enzyme is the target of the antibacterial drug triclosan and has been the subject of extensive studies for the past 20 years. Despite the large number of reports describing the biochemistry of this enzyme, there have been no studies that provided direct observation of the protein and its various ligands. Here we describe the use of native MS to characterize the protein-ligand interactions of FabI with its coenzymes NAD+ and NADH and with the inhibitor triclosan. Measurements of the gas-phase affinities of the enzyme for these ligands yielded values that are in close agreement with solution-phase affinity measurements. Additionally, FabI is a homotetramer and we were able to measure the affinity of each subunit for each coenzyme, which revealed that both coenzymes exhibit a positive homotropic allosteric effect. An allosteric effect was also observed in association with the inhibitor triclosan. These observations provide new insights into this well-studied enzyme and suggest that there may still be gaps in the existing mechanistic models that explain FabI inhibition.

NMR investigation of protein–ligand interactions for G-protein coupled receptors

2019 ◽  
Vol 11 (14) ◽  
pp. 1811-1825 ◽  
Author(s):  
Claire Raingeval ◽  
Isabelle Krimm
Keyword(s):  
Conformational Changes ◽  
Functional Characterization ◽  
Allosteric Modulation ◽  
G Protein Coupled Receptors ◽  
Nmr Studies ◽  
Protein Ligand Interactions ◽  
Ligand Interactions ◽  
Therapeutic Compounds ◽  
G Protein Coupled

In this review, we report NMR studies of ligand–GPCR interactions, including both ligand-observed and protein-observed NMR experiments. Published studies exemplify how NMR can be used as a powerful tool to design novel GPCR ligands and investigate the ligand-induced conformational changes of GPCRs. The strength of NMR also lies in its capability to explore the diverse signaling pathways and probe the allosteric modulation of these highly dynamic receptors. By offering unique opportunities for the identification, structural and functional characterization of GPCR ligands, NMR will likely play a major role for the generation of novel molecules both as new tools for the understanding of the GPCR function and as therapeutic compounds for a large diversity of pathologies.

scholarly journals Biochemical Characterization of a New β-Agarase from Cellulophaga Algicola

2019 ◽  
Vol 20 (9) ◽  
pp. 2143 ◽  
Author(s):  
Han ◽  
Zhang ◽  
Yang
Keyword(s):  
Structural Model ◽  
Biochemical Characterization ◽  
Maximum Activity ◽  
Optimal Temperature ◽  
Salt Tolerant ◽  
Wide Ph Range ◽  
Specific Activities ◽  
Ph Range ◽  
Layer Chromatography

Cellulophaga algicola DSM 14237, isolated from the Eastern Antarctic coastal zone, was found to be able to hydrolyze several types of polysaccharide materials. In this study, a predicted β-agarase (CaAga1) from C. algicola was heterologously expressed in Escherichia coli. The purified recombinant CaAga1 showed specific activities of 29.39, 20.20, 14.12, and 8.99 U/mg toward agarose, pure agar, and crude agars from Gracilaria lemaneiformis and Porphyra haitanensis, respectively. CaAga1 exhibited an optimal temperature and pH of 40 oC and 7, respectively. CaAga1 was stable over a wide pH range from 4 to 11. The recombinant enzyme showed an unusual thermostability, that is, it was stable at temperature below or equal to 40oC and around 70 oC, but was thermolabile at about 50 oC. With the agarose as the substrate, the Km and Vmax values for CaAga1 were 1.19 mg/mL and 36.21 U/mg, respectively. The reducing reagent (dithiothreitol) enhanced the activity of CaAga1 by more than one fold. In addition, CaAga1 was salt-tolerant given that it retained approximately 70% of the maximum activity in the presence of 2 M NaCl. The thin layer chromatography results indicated that CaAga1 is an endo-type β-agarase and efficiently hydrolyzed agarose into neoagarotetraose (NA4) and neoagarohexaose (NA6). A structural model of CaAga1 in complex with neoagarooctaose (NA8) was built by homology modeling and explained the hydrolysis pattern of CaAga1.

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