scholarly journals Snapshots during the catalytic cycle of a histidine acid phytase reveal an induced-fit structural mechanism

2020 ◽  
Vol 295 (51) ◽  
pp. 17724-17737
Author(s):  
Isabella M. Acquistapace ◽  
Monika A. Zi¸etek ◽  
Arthur W. H. Li ◽  
Melissa Salmon ◽  
Imke Kühn ◽  
...  
Keyword(s):  
Active Site ◽  
Bifidobacterium Longum ◽  
Catalytic Cycle ◽  
Inositol Polyphosphate ◽  
Positional Specificity ◽  
Structural Mechanism ◽  
Mechanism Of Hydrolysis ◽  
Histidine Phosphatases ◽  
First Time

Highly engineered phytases, which sequentially hydrolyze the hexakisphosphate ester of inositol known as phytic acid, are routinely added to the feeds of monogastric animals to improve phosphate bioavailability. New phytases are sought as starting points to further optimize the rate and extent of dephosphorylation of phytate in the animal digestive tract. Multiple inositol polyphosphate phosphatases (MINPPs) are clade 2 histidine phosphatases (HP2P) able to carry out the stepwise hydrolysis of phytate. MINPPs are not restricted by a strong positional specificity making them attractive targets for development as feed enzymes. Here, we describe the characterization of a MINPP from the Gram-positive bacterium Bifidobacterium longum (BlMINPP). BlMINPP has a typical HP2P-fold but, unusually, possesses a large α-domain polypeptide insertion relative to other MINPPs. This insertion, termed the U-loop, spans the active site and contributes to substrate specificity pockets underpopulated in other HP2Ps. Mutagenesis of U-loop residues reveals its contribution to enzyme kinetics and thermostability. Moreover, four crystal structures of the protein along the catalytic cycle capture, for the first time in an HP2P, a large ligand-driven α-domain motion essential to allow substrate access to the active site. This motion recruits residues both downstream of a molecular hinge and on the U-loop to participate in specificity subsites, and mutagenesis identified a mobile lysine residue as a key determinant of positional specificity of the enzyme. Taken together, these data provide important new insights to the factors determining stability, substrate recognition, and the structural mechanism of hydrolysis in this industrially important group of enzymes.

scholarly journals Characterization of the NiRAN domain from RNA-dependent RNA polymerase provides insights into a potential therapeutic target against SARS-CoV-2

2021 ◽  
Author(s):  
Abhisek Dwivedy ◽  
Richard Mariadasse ◽  
Mohammed Ahmad ◽  
Sayan Chakraborty ◽  
Deepsikha Kar ◽  
...  
Keyword(s):  
Active Site ◽  
Drug Target ◽  
Kinase Inhibitors ◽  
3D Structure ◽  
Drug Repurposing ◽  
Lead Compounds ◽  
Nucleotidyl Transferase ◽  
Domain Organisation ◽  
First Time

AbstractApart from the canonical fingers, palm and thumb domains, the RNA dependent RNA polymerases (RdRp) from the viral order Nidovirales possess two additional domains. Of these, the function of the Nidovirus RdRp associated nucleotidyl transferase domain (NiRAN) remains unanswered. The elucidation of the 3D structure of RdRp from the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), provided the first ever insights into the domain organisation and possible functional characteristics of the NiRAN domain. Using in silico tools, we predict that the NiRAN domain assumes a kinase or phosphotransferase like fold and binds nucleoside triphosphates at its proposed active site. Additionally, using molecular docking we have predicted the binding of three widely used kinase inhibitors and five well characterized anti-microbial compounds at the NiRAN domain active site along with their drug-likeliness as well as DFT properties. For the first time ever, using basic biochemical tools, this study shows the presence of a kinase like activity exhibited by the SARS-CoV-2 RdRp. Interestingly, the proposed kinase inhibitors and a few of the predicted nucleotidyl transferase inhibitors significantly inhibited the aforementioned enzymatic activity. In line with the current global COVID-19 pandemic urgency and the emergence of newer strains with significantly higher infectivity, this study provides a new anti-SARS-CoV-2 drug target and potential lead compounds for drug repurposing against SARS-CoV-2.

scholarly journals Structural Insights into the Catalytic Mechanism of Granzyme B upon Substrate and Inhibitor Binding

2021 ◽  
Author(s):  
Neha Tripathi ◽  
Richard Danger ◽  
Mélanie Chesneau ◽  
Sophie Brouard ◽  
Adèle Laurent
Keyword(s):  
Active Site ◽  
Serine Proteases ◽  
Catalytic Mechanism ◽  
Granzyme B ◽  
Salt Bridge ◽  
Catalytic Triad ◽  
Catalytic Cycle ◽  
Major Step ◽  
Dynamics Simulations ◽  
First Time

Human granzyme B (hGzmB), which is present in various immune cells, has attracted much attention due to its role in various pathophysiological conditions. The hGzmB activity is triggered at a catalytic triad (His59, Asp103, Ser198), cleaving its specific substrates. To date, the drug design strategy against hGzmB mainly targets the catalytic triad, which causes the non-specificity problem of inhibitors due to the highly conserved active site in serine proteases. In the present work, microsecond classical molecular dynamics simulations are devoted to exploring the structural dynamics of the hGzmB catalytic cycle in the presence of Ac-IEPD-AMC, a known substrate (active hGzmB), and Ac-IEPD-CHO, a known inhibitor (inactive hGzmB). By comparing active and inactive forms of hGzmB in the six different stages of the hGzmB catalytic cycle, we revealed, for the very first time, an additional network of interactions involving Arg216, a residue located outside the conventional binding site. Upon activation, the His59∙∙∙Asp103 hydrogen bond is broken due to the formation of the Asp103∙∙∙Arg216 salt bridge, expanding the active site to facilitate the substrate-binding. On the contrary, the binding of inhibitor Ac-IEPD-CHO to hGzmB prevents the Arg216-mediated interactions within the catalytic triad, thus preventing hGzmB activity. In silico Arg216Ala mutation confirms the role of Arg216 in enzyme activity, as the substrate Ac-IEPD-AMC failed to bind to the mutated hGzmB. Importantly, as Arg216 is not conserved amongst the various granzymes, the current findings can be a major step to guide the design of hGzmB specific therapeutics.

Characterization of an Abnormal Antithrombin (Milano 2) with Defective Thrombin Binding

1986 ◽  
Vol 56 (03) ◽  
pp. 349-352 ◽  
Author(s):  
A Tripodi ◽  
A Krachmalnicoff ◽  
P M Mannucci
Keyword(s):  
Venous Thromboembolism ◽  
Active Site ◽  
Inhibitory Activity ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Molecular Defect ◽  
Affinity Adsorption ◽  
Italian Family ◽  
Crossed Immunoelectrophoresis

SummaryFour members of an Italian family (two with histories of venous thromboembolism) had a qualitative defect of antithrombin III reflected by normal antigen concentrations and halfnormal antithrombin activity with or without heparin. Anti-factor Xa activities were consistently borderline low (about 70% of normal). For the propositus’ plasma and serum the patterns of antithrombin III in crossed-immunoelectrophoresis with or without heparin were indistinguishable from those of normal plasma or serum. A normal affinity of antithrombin III for heparin was documented by heparin-sepharose chromatography. Affinity adsorption of the propositus’ plasma to human α-thrombin immobilized on sepharose beads revealed defective binding of the anti thrombin III to thrombin-sepharose. Hence the molecular defect of this variant appears to be at the active site responsible for binding and neutralizing thrombin, thus accounting for the low thrombin inhibitory activity.

TBE in Sweden

2020 ◽  
Keyword(s):  
Genetic Characterization ◽  
Immunoglobulin M ◽  
Enzyme Linked Immunosorbent Assay ◽  
Second Phase ◽  
Serum Samples ◽  
Tick Borne Encephalitis ◽  
Specific Immunoglobulin ◽  
Tick Borne Encephalitis Virus ◽  
First Time

Tick-borne encephalitis virus (TBEV) was isolated for the first time in Sweden in 1958 (from ticks and from 1 tick-borne encephalitis [TBE] patient).1 In 2003, Haglund and colleagues reported the isolation and antigenic and genetic characterization of 14 TBEV strains from Swedish patients (samples collected 1991–1994).2 The first serum sample, from which TBEV was isolated, was obtained 2–10 days after onset of disease and found to be negative for anti-TBEV immunoglobulin M (IgM) by enzyme-linked immunosorbent assay (ELISA), whereas TBEV-specific IgM (and TBEV-specific immunoglobulin G/cerebrospinal fluid [IgG/CSF] activity) was demonstrated in later serum samples taken during the second phase of the disease.

Chemical Constituents and Antibacterial Activity of Cissus Aralioides.

2020 ◽  
Vol 17 ◽  
Author(s):  
Balogun Olaoye Solomon ◽  
Ajayi Olukayode Solomon ◽  
Owolabi Temitayo Abidemi ◽  
Oladimeji Abdulkarbir Oladele ◽  
Liu Zhiqiang
Keyword(s):  
Plant Extract ◽  
Chemical Constituents ◽  
Ethyl Acetate Fraction ◽  
Antibacterial Activities ◽  
Sub Saharan Africa ◽  
Chromatographic Purification ◽  
Whole Plant ◽  
Sub Saharan ◽  
First Time

: Cissus aralioides is a medicinal plant used in sub-Saharan Africa for treatment of infectious diseases; however the chemical constituents of the plant have not been investigated. Thus, in this study, attempt was made at identifying predominant phytochemical constituents of the plant through chromatographic purification and silylation of the plant extract, and subsequent characterization using spectroscopic and GC-MS techniques. The minimum inhibitory concentration (MICs) for the antibacterial activities of the plant extract, chromatographic fractions and isolated compounds were also examined. Chromatographic purification of the ethyl acetate fraction from the whole plant afforded three compounds: β-sitosterol (1), stigmasterol (2) and friedelin (3). The phytosterols (1 and 2) were obtained together as a mixture. The GC-MS analysis of silylated extract indicated alcohols, fatty acids and sugars as predominant classes, with composition of 24.62, 36.90 and 26.52% respectively. Results of MICs indicated that friedelin and other chromatographic fractions had values (0.0626-1.0 mg/mL) comparable with the standard antibiotics used. Characterization of natural products from C. aralioides is being reported for the first time in this study.

Green Synthesis and Spectroscopic Studies of Ag-rGO Nanocomposites for Highly Selective Mercury (II) Sensing

2018 ◽  
Vol 9 (1) ◽  
pp. 101-108 ◽  
Author(s):  
Shubhangi J. Mane-Gavade ◽  
Sandip R. Sabale ◽  
Xiao-Ying Yu ◽  
Gurunath H. Nikam ◽  
Bhaskar V. Tamhankar
Keyword(s):  
Green Synthesis ◽  
Color Change ◽  
Limit Of Detection ◽  
Aqueous Media ◽  
Suitable Condition ◽  
Cost Effective ◽  
Spectroscopic Studies ◽  
Calibration Plot ◽  
First Time

Introduction: Herein we report the green synthesis and characterization of silverreduced graphene oxide nanocomposites (Ag-rGO) using Acacia nilotica gum for the first time. Experimental: We demonstrate the Hg2+ ions sensing ability of the Ag-rGO nanocomposites form aqueous medium. The developed colorimetric sensor method is simple, fast and selective for the detection of Hg2+ ions in aqueous media in presence of other associated ions. A significant color change was noticed with naked eye upon Hg2+ addition. The color change was not observed for cations including Sr2+, Ni2+, Cd2+, Pb2+, Mg2+, Ca2+, Fe2+, Ba2+ and Mn2+indicating that only Hg2+ shows a strong interaction with Ag-rGO nanocomposites. Under the most suitable condition, the calibration plot (A0-A) against concentration of Hg2+ was linear in the range of 0.1-1.0 ppm with a correlation coefficient (R2) value 0.9998. Results & Conclusion The concentration of Hg2+ was quantitatively determined with the Limit of Detection (LOD) of 0.85 ppm. Also, this method shows excellent selectivity towards Hg2+ over nine other cations tested. Moreover, the method offers a new cost effective, rapid and simple approach for the detection of Hg2+ in water samples.

scholarly journals Waste-Based One-Part Alkali Activated Materials

Materials ◽  
2021 ◽  
Vol 14 (11) ◽  
pp. 2911
Author(s):  
Margarida Gonçalves ◽  
Inês Silveirinha Vilarinho ◽  
Marinélia Capela ◽  
Ana Caetano ◽  
Rui Miguel Novais ◽  
...  
Keyword(s):  
Compressive Strength ◽  
Microstructural Analysis ◽  
Construction Materials ◽  
Sodium Metasilicate ◽  
High Compressive Strength ◽  
Hardened State ◽  
First Time ◽  
Furnace Slag ◽  
Alkali Activated

Ordinary Portland Cement is the most widely used binder in the construction sector; however, a very high carbon footprint is associated with its production process. Consequently, more sustainable alternative construction materials are being investigated, namely, one-part alkali activated materials (AAMs). In this work, waste-based one-part AAMs binders were developed using only a blast furnace slag, as the solid precursor, and sodium metasilicate, as the solid activator. For the first time, mortars in which the commercial sand was replaced by two exhausted sands from biomass boilers (CA and CT) were developed. Firstly, the characterization of the slag and sands (aggregates) was performed. After, the AAMs fresh and hardened state properties were evaluated, being the characterization complemented by FTIR and microstructural analysis. The binder and the mortars prepared with commercial sand presented high compressive strength values after 28 days of curing-56 MPa and 79 MPa, respectively. The mortars developed with exhausted sands exhibit outstanding compressive strength values, 86 and 70 MPa for CT and CA, respectively, and the other material’s properties were not affected. Consequently, this work proved that high compressive strength waste-based one-part AAMs mortars can be produced and that it is feasible to use another waste as aggregate in the mortar’s formulations: the exhausted sands from biomass boilers.

scholarly journals Structural Characterization of Two CO Molecules Bound to the Nitrogenase Active Site

2020 ◽  
Author(s):  
Trixia M. Buscagan ◽  
Kathryn A. Perez ◽  
Ailiena O. Maggiolo ◽  
Douglas C. Rees ◽  
Thomas Spatzal
Keyword(s):  
Structural Characterization ◽  
Active Site
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