Discovery of β-N-acetylglucosaminidases from screening metagenomic libraries and their use as thioglycoligase mutants

2021 ◽  
Author(s):  
Gregor Tegl ◽  
Peter Rahfeld ◽  
Katharina Ostmann ◽  
John Hanson ◽  
Stephen G. Withers
Keyword(s):  
Active Site ◽  
Metagenomic Library ◽  
Active Site Residues ◽  
Human Gut ◽  
Protein Targets ◽  
Metagenomic Libraries

Screening of a human gut metagenomic library yielded a set of novel beta-N-acetyl-glucosaminidases. Mutation of active site residues yielded a thioglycoligase that efficiently S-GlcNAcylates protein targets as well as various thiosugar acceptors.

scholarly journals Compilation of Potential Protein Targets for SARS-CoV-2: Preparation of Homology Model and Active Site Determination for Future Rational Antiviral Design

2020 ◽  
Author(s):  
Sourav Pal ◽  
Dr. Arindam Talukdar
Keyword(s):  
Active Site ◽  
Drug Targets ◽  
Active Sites ◽  
Structural Alignment ◽  
Homology Model ◽  
Active Site Residues ◽  
Protein Targets ◽  
Rational Development ◽  
Novel Coronavirus ◽  
Approved Drugs

<p>The recent pandemic due to the novel coronavirus SARS-CoV-2 (COVID-19) is causing significant mortality worldwide. However, there is a lack of specific drugs which can either prevent or treat the patient suffering from COVID-19. To understand the SARS-CoV-2 receptor recognition causing infectivity and pathogenesis, we have compiled a list of 20 probable drug targets on host and virus based on viral life cycle along with their PDB IDs for the rational development of future antivirals. We have prepared nine homology model for vital proteins for which no crystal structure is reported, which includes protein from host, viral membrane proteins and essential non-structural proteins (NSPs) of virus. The generated models were validated followed by Ramachandran plot along with their sequence and structural alignment. The active site residues of all the protein models are calculated by utilizing COACH meta-server and also cross verified with the CASTp webservers. All the active sites of the homology build proteins were evaluated after superimposition of the closely related X-ray crystallized structure bound with the co-crystal ligands. These information present in the manuscript can be used for the discovery effort towards new antivirals as well as repurposing FDA approved drugs against SARS-CoV-2.</p><br>

scholarly journals Compilation of Potential Protein Targets for SARS-CoV-2: Preparation of Homology Model and Active Site Determination for Future Rational Antiviral Design

2020 ◽  
Author(s):  
Sourav Pal ◽  
Dr. Arindam Talukdar
Keyword(s):  
Active Site ◽  
Drug Targets ◽  
Active Sites ◽  
Structural Alignment ◽  
Homology Model ◽  
Active Site Residues ◽  
Protein Targets ◽  
Rational Development ◽  
Novel Coronavirus ◽  
Approved Drugs

<p>The recent pandemic due to the novel coronavirus SARS-CoV-2 (COVID-19) is causing significant mortality worldwide. However, there is a lack of specific drugs which can either prevent or treat the patient suffering from COVID-19. To understand the SARS-CoV-2 receptor recognition causing infectivity and pathogenesis, we have compiled a list of 20 probable drug targets on host and virus based on viral life cycle along with their PDB IDs for the rational development of future antivirals. We have prepared nine homology model for vital proteins for which no crystal structure is reported, which includes protein from host, viral membrane proteins and essential non-structural proteins (NSPs) of virus. The generated models were validated followed by Ramachandran plot along with their sequence and structural alignment. The active site residues of all the protein models are calculated by utilizing COACH meta-server and also cross verified with the CASTp webservers. All the active sites of the homology build proteins were evaluated after superimposition of the closely related X-ray crystallized structure bound with the co-crystal ligands. These information present in the manuscript can be used for the discovery effort towards new antivirals as well as repurposing FDA approved drugs against SARS-CoV-2.</p><br>

Development of Xanthine Based Inhibitors Targeting Phosphodiesterase 9A

2017 ◽  
Vol 14 (10) ◽  
pp. 1122-1137 ◽  
Author(s):  
Nivedita Singh ◽  
Parameswaran Saravanan ◽  
M.S. Thakur ◽  
Sanjukta Patra
Keyword(s):  
Free Energy ◽  
Active Site ◽  
Signal Transduction Pathway ◽  
Docking Study ◽  
Primary Objective ◽  
Cgmp Level ◽  
Active Site Residues ◽  
Aromatic Fragment ◽  
Docking Approach ◽  
Free Energy Of Binding

Background: Phosphodiesterases 9A (PDE9A) is one of the prominent regulating enzymes of the signal transduction pathway having highest catalytic affinity for second messenger, cGMP. When the cGMP level is lowered, an uncontrolled expression of PDE9A may lead to various neurodegenerative diseases. To regulate the catalytic activity of PDE9A, potent inhibitors are needed. Objective: The primary objective of the present study was to develop new xanthine based inhibitors targeting PDE9A. This study was an attempt to bring structural diversification in PDE9A inhibitor development because most of the existing inhibitors are constructed over pyrazolopyrimidinone scaffold. Methods: Manual designing and parallel molecular docking approach were used for the development of xanthine derivatives. In this study, N1, N3, N9 and C8 positions of xanthine scaffold were selected as substitution sites to design 200 new compounds. Reverse docking and pharmaceutical analyses were used for final validation of most promising compounds. Results: By keeping free energy of binding cut-off of -6.0 kcal/mol, 52 compounds were screened. The compounds with substitution at N1, N3 and C8 positions of xanthine showed good occupancy in PDE9A active site pocket with a significant interaction pattern. This was further validated by screening different factors such as free energy of binding, inhibition constant and interacting active site residues in the 5Å region. Substitution at C8 position with phenyl substituent determined the inhibition affinity of compounds towards PDE9A by establishing a strong hydrophobic - hydrophobic interaction. The alkyl chain at N1 position generated selectivity of compounds towards PDE9A. The aromatic fragment at N3 position increased the binding affinity of compounds. Thus, by comparative docking study, it was found that compound 39-42 formed selective interaction towards PDE9A over other members of the PDE superfamily. Conclusion: From the present study, N1, N3 and C8 positions of xanthine were concluded as the best sites for substitution for the generation of potent PDE9A inhibitors.

scholarly journals An enzymatic activation of formaldehyde for nucleotide methylation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Charles Bou-Nader ◽  
Frederick W. Stull ◽  
Ludovic Pecqueur ◽  
Philippe Simon ◽  
Vincent Guérineau ◽  
...  
Keyword(s):  
Amino Acids ◽  
Thymidylate Synthase ◽  
Active Site ◽  
De Novo ◽  
Crystallographic Structure ◽  
De Novo Synthesis ◽  
Active Site Residues ◽  
Enzymatic Activation ◽  
Enzyme Cofactors ◽  
Unique Ability

AbstractFolate enzyme cofactors and their derivatives have the unique ability to provide a single carbon unit at different oxidation levels for the de novo synthesis of amino-acids, purines, or thymidylate, an essential DNA nucleotide. How these cofactors mediate methylene transfer is not fully settled yet, particularly with regard to how the methylene is transferred to the methylene acceptor. Here, we uncovered that the bacterial thymidylate synthase ThyX, which relies on both folate and flavin for activity, can also use a formaldehyde-shunt to directly synthesize thymidylate. Combining biochemical, spectroscopic and anaerobic crystallographic analyses, we showed that formaldehyde reacts with the reduced flavin coenzyme to form a carbinolamine intermediate used by ThyX for dUMP methylation. The crystallographic structure of this intermediate reveals how ThyX activates formaldehyde and uses it, with the assistance of active site residues, to methylate dUMP. Our results reveal that carbinolamine species promote methylene transfer and suggest that the use of a CH2O-shunt may be relevant in several other important folate-dependent reactions.

scholarly journals Important Role for Phylogenetically Invariant PP2Acα Active Site and C-Terminal Residues Revealed by Mutational Analysis in Saccharomyces cerevisiae

Genetics ◽  
2000 ◽  
Vol 156 (1) ◽  
pp. 21-29 ◽  
Author(s):  
David R H Evans ◽  
Brian A Hemmings
Keyword(s):  
Protein Interactions ◽  
Active Site ◽  
Protein Function ◽  
Mutational Analysis ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Active Site Residues ◽  
Catalytic Function

Abstract PP2A is a central regulator of eukaryotic signal transduction. The human catalytic subunit PP2Acα functionally replaces the endogenous yeast enzyme, Pph22p, indicating a conservation of function in vivo. Therefore, yeast cells were employed to explore the role of invariant PP2Ac residues. The PP2Acα Y127N substitution abolished essential PP2Ac function in vivo and impaired catalysis severely in vitro, consistent with the prediction from structural studies that Tyr-127 mediates substrate binding and its side chain interacts with the key active site residues His-118 and Asp-88. The V159E substitution similarly impaired PP2Acα catalysis profoundly and may cause global disruption of the active site. Two conditional mutations in the yeast Pph22p protein, F232S and P240H, were found to cause temperature-sensitive impairment of PP2Ac catalytic function in vitro. Thus, the mitotic and cell lysis defects conferred by these mutations result from a loss of PP2Ac enzyme activity. Substitution of the PP2Acα C-terminal Tyr-307 residue by phenylalanine impaired protein function, whereas the Y307D and T304D substitutions abolished essential function in vivo. Nevertheless, Y307D did not reduce PP2Acα catalytic activity significantly in vitro, consistent with an important role for the C terminus in mediating essential protein-protein interactions. Our results identify key residues important for PP2Ac function and characterize new reagents for the study of PP2A in vivo.

scholarly journals Derived amino acid sequence and identification of active site residues of Escherichia coli beta-hydroxydecanoyl thioester dehydrase.

1988 ◽  
Vol 263 (10) ◽  
pp. 4641-4646 ◽  
Author(s):  
J E Cronan ◽  
W B Li ◽  
R Coleman ◽  
M Narasimhan ◽  
D de Mendoza ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Active Site ◽  
Active Site Residues
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