scholarly journals Biochemical and Structural Characterisation of a Novel D-Lyxose Isomerase From the Hyperthermophilic Archaeon Thermofilum sp.

2021 ◽  
Vol 9 ◽  
Author(s):  
Simone Antonio De Rose ◽  
Tom Kuprat ◽  
Michail N. Isupov ◽  
Andreas Reinhardt ◽  
Peter Schönheit ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Disulfide Bond ◽  
Industrial Applications ◽  
Free Form ◽  
Solvent Tolerance ◽  
Dimer Interface ◽  
Hyperthermophilic Archaeon ◽  
Structural Characterisation ◽  
Ligand Free

A novel D-lyxose isomerase has been identified within the genome of a hyperthermophilic archaeon belonging to the Thermofilum species. The enzyme has been cloned and over-expressed in Escherichia coli and biochemically characterised. This enzyme differs from other enzymes of this class in that it is highly specific for the substrate D-lyxose, showing less than 2% activity towards mannose and other substrates reported for lyxose isomerases. This is the most thermoactive and thermostable lyxose isomerase reported to date, showing activity above 95°C and retaining 60% of its activity after 60 min incubation at 80°C. This lyxose isomerase is stable in the presence of 50% (v/v) of solvents ethanol, methanol, acetonitrile and DMSO. The crystal structure of the enzyme has been resolved to 1.4–1.7 A. resolution in the ligand-free form and in complexes with both of the slowly reacting sugar substrates mannose and fructose. This thermophilic lyxose isomerase is stabilised by a disulfide bond between the two monomers of the dimeric enzyme and increased hydrophobicity at the dimer interface. These overall properties of high substrate specificity, thermostability and solvent tolerance make this lyxose isomerase enzyme a good candidate for potential industrial applications.

Crystal Structure of the Urokinase Receptor in a Ligand-Free Form

2012 ◽  
Vol 416 (5) ◽  
pp. 629-641 ◽  
Author(s):  
Xiang Xu ◽  
Henrik Gårdsvoll ◽  
Cai Yuan ◽  
Lin Lin ◽  
Michael Ploug ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Urokinase Receptor ◽  
Free Form ◽  
Ligand Free

Erratum to “Crystal Structure of the Urokinase Receptor in a Ligand-Free Form” [J. Mol. Biol. 416 (2012) 629–641]

2012 ◽  
Vol 422 (1) ◽  
pp. 158
Author(s):  
Xiang Xu ◽  
Henrik Gårdsvoll ◽  
Cai Yuan ◽  
Lin Lin ◽  
Michael Ploug ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Urokinase Receptor ◽  
Free Form ◽  
Ligand Free

scholarly journals Crystal structure of the ligand‐free form of the Vps10 ectodomain of dimerized Sortilin at acidic pH

FEBS Letters ◽  
2018 ◽  
Vol 592 (15) ◽  
pp. 2647-2657 ◽  
Author(s):  
Toshiki Yabe‐Wada ◽  
Shintaro Matsuba ◽  
Masaki Unno ◽  
Nobuyuki Onai
Keyword(s):  
Crystal Structure ◽  
Free Form ◽  
Acidic Ph ◽  
Ligand Free

scholarly journals Functional Heterologous Expression of Mature Lipase LipA from Pseudomonas aeruginosa PSA01 in Escherichia coli SHuffle and BL21 (DE3): Effect of the Expression Host on Thermal Stability and Solvent Tolerance of the Enzyme Produced

2020 ◽  
Vol 21 (11) ◽  
pp. 3925
Author(s):  
Ingrid Yamile Pulido ◽  
Erlide Prieto ◽  
Gilles Paul Pieffet ◽  
Lina Méndez ◽  
Carlos A. Jiménez-Junca
Keyword(s):  
Escherichia Coli ◽  
Molecular Dynamics ◽  
Pseudomonas Aeruginosa ◽  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Residual Activity ◽  
Solvent Tolerance ◽  
Palm Fruit ◽  
E Coli ◽  
Dynamics Simulations

This study aimed to express heterologously the lipase LipA from Pseudomonas aeruginosa PSA01 obtained from palm fruit residues. In previous approaches, LipA was expressed in Escherichia coli fused with its signal peptide and without its disulfide bond, displaying low activity. We cloned the mature LipA with its truncated chaperone Lif in a dual plasmid and overexpressed the enzyme in two E. coli strains: the traditional BL21 (DE3) and the SHuffle® strain, engineered to produce stable cytoplasmic disulfide bonds. We evaluated the effect of the disulfide bond on LipA stability using molecular dynamics. We expressed LipA successfully under isopropyl β-d-1-thio-galactopyranoside (IPTG) and slow autoinducing conditions. The SHuffle LipA showed higher residual activity at 45 °C and a greater hyperactivation after incubation with ethanol than the enzyme produced by E. coli BL21 (DE3). Conversely, the latter was slightly more stable in methanol 50% and 60% (t½: 49.5 min and 9 min) than the SHuffle LipA (t½: 31.5 min and 7.4 min). The molecular dynamics simulations showed that removing the disulfide bond caused some regions of LipA to become less flexible and some others to become more flexible, significantly affecting the closing lid and partially exposing the active site at all times.

Crystal structures of a β-trefoil lectin from Entamoeba histolytica in monomeric and a novel disulfide bond-mediated dimeric forms

Glycobiology ◽  
2020 ◽  
Vol 30 (7) ◽  
pp. 474-488 ◽  
Author(s):  
Farha Khan ◽  
Devanshu Kurre ◽  
K Suguna
Keyword(s):  
Crystal Structure ◽  
Crystal Structures ◽  
Entamoeba Histolytica ◽  
Disulfide Bond ◽  
Structural Basis ◽  
Free Form ◽  
Biophysical Characterization ◽  
Biophysical Studies ◽  
First Time

Abstract β-Trefoil lectins are galactose/N-acetyl galactosamine specific lectins, which are widely distributed across all kingdoms of life and are known to perform several important functions. However, there is no report available on the characterization of these lectins from protozoans. We have performed structural and biophysical studies on a β-trefoil lectin from Entamoeba histolytica (EntTref), which exists as a mixture of monomers and dimers in solution. Further, we have determined the affinities of EntTref for rhamnose, galactose and different galactose-linked sugars. We obtained the crystal structure of EntTref in a sugar-free form (EntTref_apo) and a rhamnose-bound form (EntTref_rham). A novel Cys residue-mediated dimerization was revealed in the crystal structure of EntTref_apo while the structure of EntTref_rham provided the structural basis for the recognition of rhamnose by a β-trefoil lectin for the first time. To the best of our knowledge, this is the only report of the structural, functional and biophysical characterization of a β-trefoil lectin from a protozoan source and the first report of Cys-mediated dimerization in this class of lectins.

scholarly journals Crystal structure of heat-labile enterotoxin from Escherichia coli with increased thermostability introduced by an engineered disulfide bond in the A subunit

2008 ◽  
Vol 6 (12) ◽  
pp. 2644-2649 ◽  
Author(s):  
F. Van Den Akker ◽  
I. K. Feil ◽  
C. Roach ◽  
A. A. Platas ◽  
E. A. Merritt ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Disulfide Bond ◽  
Heat Labile ◽  
A Subunit

scholarly journals Crystal structure of phosphoribulokinase from Synechococcus sp. strain PCC 6301

2019 ◽  
Vol 75 (4) ◽  
pp. 278-289 ◽  
Author(s):  
Robert H. Wilson ◽  
Manajit Hayer-Hartl ◽  
Andreas Bracher
Keyword(s):  
Crystal Structure ◽  
Disulfide Bond ◽  
Carbon Fixation ◽  
Adaptor Protein ◽  
Structural Basis ◽  
Dimer Interface ◽  
Synechococcus Sp ◽  
Β Sheet ◽  
Loop Residue

Phosphoribulokinase (PRK) catalyses the ATP-dependent phosphorylation of ribulose 5-phosphate to give ribulose 1,5-bisphosphate. Regulation of this reaction in response to light controls carbon fixation during photosynthesis. Here, the crystal structure of PRK from the cyanobacterium Synechococcus sp. strain PCC 6301 is presented. The enzyme is dimeric and has an α/β-fold with an 18-stranded β-sheet at its core. Interestingly, a disulfide bond is found between Cys40 and the P-loop residue Cys18, revealing the structural basis for the redox inactivation of PRK activity. A second disulfide bond appears to rigidify the dimer interface and may thereby contribute to regulation by the adaptor protein CP12 and glyceraldehyde-3-phosphate dehydrogenase.

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