scholarly journals Updates on enzymatic and structural properties of human glutamine: fructose-6-phosphate amidotransferase 2 (hGFAT2)

2020 ◽  
Author(s):  
Isadora A. Oliveira ◽  
Diego Allonso ◽  
Tácio V. A. Fernandes ◽  
Daniela M. S. Lucena ◽  
Gustavo T. Ventura ◽  
...  
Keyword(s):  
Biosynthetic Pathway ◽  
Three Dimensional ◽  
Dimensional Model ◽  
Expression And Purification ◽  
Three Dimensional Model ◽  
Hexosamine Biosynthetic Pathway ◽  
E Coli ◽  
Cellular Processes ◽  
Catalytically Active ◽  
Dynamics Simulations

AbstractGlycoconjugates play a central role in several cellular processes and alteration in their composition is associated to human pathologies. The hexosamine biosynthetic pathway is a route through which cells obtain substrates for cellular glycosylation, and is controlled by the glutamine: fructose-6-phosphate amidotransferase (GFAT). Human isoform 2 GFAT (hGFAT2) has been implicated in diabetes and cancer, however, there is no information about structural and enzymatic properties of this enzyme. Here, we report a successful expression and purification of a catalytically active recombinant hGFAT2 (rhGFAT2) in E. coli cells fused or not to a HisTag at the C-terminal end. Our enzyme kinetics data suggest that hGFAT2 does not follow the ordered bi-bi mechanism, and performs the glucosamine-6-phosphate synthesis much slowly than previously reported for other GFATs. In addition, hGFAT2 is able to isomerase fructose-6-phosphate into glucose-6-phosphate even in presence of equimolar amounts of glutamine, in an unproductive glutamine hydrolysis. Structural analysis of the generated three-dimensional model rhGFAT2, corroborated by circular dichroism data, indicated the presence of a partially structured loop in glutaminase domain, whose sequence is present in eukaryotic enzymes but absent in the E. coli homolog. Molecular dynamics simulations show such loop as the most flexible portion of the protein, which interacts with the protein mainly through the interdomain region, and plays a key role on conformational states of hGFAT2. Altogether, our study provides the first comprehensive set of data on the structure, kinetics and mechanics of hGFAT2, which will certainly contribute for further studies focusing on drug development targeting hGFAT2.

scholarly journals Enzymatic and structural properties of human glutamine:fructose-6-phosphate amidotransferase 2 (hGFAT2)

2020 ◽  
pp. jbc.RA120.015189
Author(s):  
Isadora A. Oliveira ◽  
Diego Allonso ◽  
Tácio V. A. Fernandes ◽  
Daniela M.S. Lucena ◽  
Gustavo T. Ventura ◽  
...  
Keyword(s):  
Biosynthetic Pathway ◽  
Three Dimensional ◽  
Dimensional Model ◽  
Expression And Purification ◽  
Three Dimensional Model ◽  
Hexosamine Biosynthetic Pathway ◽  
E Coli ◽  
Cellular Processes ◽  
Catalytically Active ◽  
Dynamics Simulations

Glycoconjugates play a central role in several cellular processes and alteration in their composition is associated with numerous human pathologies. Substrates for cellular glycosylation are synthesized in the hexosamine biosynthetic pathway, which is controlled by the glutamine:fructose-6-phosphate amidotransfera-se (GFAT). Human isoform 2 GFAT (hGFAT2) has been implicated in diabetes and cancer; however, there is no information about structural and enzymatic properties of this enzyme. Here, we report a successful expression and purification of a catalytically active recombinant hGFAT2 (rhGFAT2) in E. coli cells fused or not to a HisTag at the C-terminal end. Our enzyme kinetics data suggest that hGFAT2 does not follow the expected ordered bi-bi mechanism, and performs the glucosamine-6-phosphate synthesis much more slowly than previously reported for other GFATs. In addition, hGFAT2 is able to isomerize fructose-6-phosphate into glucose-6-phosphate even in the presence of equimolar amounts of glutamine, which results in unproductive glutamine hydrolysis. Structural analysis of a three-dimensional model of rhGFAT2, corroborated by circular dichroism data, indicated the presence of a partially structured loop in the glutaminase domain, whose sequence is present in eukaryotic enzymes but absent in the E. coli homolog. Molecular dynamics simulations suggest that this loop is the most flexible portion of the protein, and plays a key role on conformational states of hGFAT2. Thus, our study provides the first comprehensive set of data on the structure, kinetics and mechanics of hGFAT2, which will certainly contribute to further studies on the (patho)physiology of hGFAT2.

scholarly journals Mutagenesis in the α3α4 GyrA Helix and in the Toprim Domain of GyrB Refines the Contribution of Mycobacterium tuberculosis DNA Gyrase to Intrinsic Resistance to Quinolones

2008 ◽  
Vol 52 (8) ◽  
pp. 2909-2914 ◽  
Author(s):  
Stéphanie Matrat ◽  
Alexandra Aubry ◽  
Claudine Mayer ◽  
Vincent Jarlier ◽  
Emmanuelle Cambau
Keyword(s):  
Escherichia Coli ◽  
Mycobacterium Tuberculosis ◽  
Primary Structure ◽  
Three Dimensional ◽  
Dna Gyrase ◽  
Quinolone Resistance ◽  
Dimensional Model ◽  
Three Dimensional Model ◽  
Intrinsic Resistance ◽  
E Coli

ABSTRACT The replacement of M74 in GyrA, A83 in GyrA, and R447 in GyrB of Mycobacterium tuberculosis gyrase by their Escherichia coli homologs resulted in active enzymes as quinolone susceptible as the E. coli gyrase. This demonstrates that the primary structure of gyrase determines intrinsic quinolone resistance and was supported by a three-dimensional model of N-terminal GyrA.

scholarly journals Three-Dimensional Model for the Human Cl−/HCO3− Exchanger, AE1, by Homology to the E. coli ClC Protein

2013 ◽  
Vol 425 (14) ◽  
pp. 2591-2608 ◽  
Author(s):  
Pamela Bonar ◽  
Hans-Peter Schneider ◽  
Holger M. Becker ◽  
Joachim W. Deitmer ◽  
Joseph R. Casey
Keyword(s):  
Three Dimensional ◽  
Dimensional Model ◽  
Three Dimensional Model ◽  
E Coli

Molecular Dynamics Simulations of Shocks and Detonations in a Model 3D Energetic Crystal with Defects

1992 ◽  
Vol 296 ◽  
Author(s):  
Lee Phillips
Keyword(s):  
Reaction Rate ◽  
Hot Spot ◽  
Detonation Front ◽  
Three Dimensional ◽  
Perfect Crystal ◽  
Chemical Activity ◽  
Chemical Bonds ◽  
Dimensional Model ◽  
Three Dimensional Model ◽  
Dynamics Simulations

AbstractWe examine shock induced detonation in a three-dimensional model of a nitromethane crystal. The crystal may contain a defect in the form of a small void. Three regimes are identified: the shock can be weak enough that no chemical bonds are broken; the shock can be so strong that a detonation front is established in the perfect crystal; or the shock can be of intermediate strength, where chemical activity requires the existence of the defect. In all regimes, the defect increases the reaction rate and causes a hot spot to appear.

Effect of the Molecular Weight and Density of Ambient Gas on Shock Wave in Laser-Assisted Nanostructuring

2008 ◽  
Author(s):  
Liying Guo ◽  
Xinwei Wang
Keyword(s):  
Shock Wave ◽  
Molecular Weight ◽  
Near Field ◽  
Three Dimensional ◽  
Dimensional Model ◽  
Three Dimensional Model ◽  
Shock Wave Formation ◽  
Ambient Gas ◽  
Dynamics Simulations ◽  
Lower Ratio

This paper presents the results from molecular dynamics simulations that are performed to explore the properties of the shock wave during laser-assisted near field surface nanostructuring. A quasi-three dimensional model is constructed to study systems consisting of over 2 million atoms. This work includes studies on the velocity as well as pressure evolution of shock wave front with respect to different solid/gas molecular mass ratios and different ambient gas densities. The limitation on shock wave formation under the same laser fluence is also investigated. The results show that lower ratio of the solid/gas molecular weight weakens the strength of the shock wave during the nanostructuring process. Additionally, the formation and attenuation of the shock wave under different ambient gas conditions is studied in substantial detail.

scholarly journals Three-Dimensional Model of Lanosterol 14α-Demethylase from Cryptococcus neoformans: Active-Site Characterization and Insights into Azole Binding

2009 ◽  
Vol 53 (8) ◽  
pp. 3487-3495 ◽  
Author(s):  
Chunquan Sheng ◽  
Zhenyuan Miao ◽  
Haitao Ji ◽  
Jianzhong Yao ◽  
Wenya Wang ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Active Site ◽  
Antifungal Agents ◽  
Fungal Infections ◽  
Three Dimensional ◽  
Rational Drug Design ◽  
Dimensional Model ◽  
Three Dimensional Model ◽  
Functional Regions ◽  
Dynamics Simulations

ABSTRACT Cryptococcus neoformans is one of the most important causes of life-threatening fungal infections in immunocompromised patients. Lanosterol 14α-demethylase (CYP51) is the target of azole antifungal agents. This study describes, for the first time, the 3-dimensional model of CYP51 from Cryptococcus neoformans (CnCYP51). The model was further refined by energy minimization and molecular-dynamics simulations. The active site of CnCYP51 was well characterized by multiple-copy simultaneous-search calculations, and four functional regions important for rational drug design were identified. The mode of binding of the natural substrate and azole antifungal agents with CnCYP51 was identified by flexible molecular docking. A G484S substitution mechanism for azole resistance in CnCYP51, which might be important for the conformation of the heme environment, is suggested.

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