scholarly journals Structure of a reaction intermediate mimic in t6A biosynthesis bound in the active site of the TsaBD heterodimer from Escherichia coli

2021 ◽  
Author(s):  
Brett J Kopina ◽  
Sophia Missoury ◽  
Bruno Collinet ◽  
Mark G Fulton ◽  
Charles Cirio ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Rational Design ◽  
Trna Modification ◽  
Oxyanion Hole ◽  
Ic50 Value ◽  
Potent Drug ◽  
Unstable Intermediate ◽  
Novel Antibiotics

Abstract The tRNA modification N6-threonylcarbamoyladenosine (t6A) is universally conserved in all organisms. In bacteria, the biosynthesis of t6A requires four proteins (TsaBCDE) that catalyze the formation of t6A via the unstable intermediate l-threonylcarbamoyl-adenylate (TC-AMP). While the formation and stability of this intermediate has been studied in detail, the mechanism of its transfer to A37 in tRNA is poorly understood. To investigate this step, the structure of the TsaBD heterodimer from Escherichia coli has been solved bound to a stable phosphonate isosteric mimic of TC-AMP. The phosphonate inhibits t6A synthesis in vitro with an IC50 value of 1.3 μM in the presence of millimolar ATP and L-threonine. The inhibitor binds to TsaBD by coordination to the active site Zn atom via an oxygen atom from both the phosphonate and the carboxylate moieties. The bound conformation of the inhibitor suggests that the catalysis exploits a putative oxyanion hole created by a conserved active site loop of TsaD and that the metal essentially serves as a binding scaffold for the intermediate. The phosphonate bound crystal structure should be useful for the rational design of potent, drug-like small molecule inhibitors as mechanistic probes or potentially novel antibiotics.

scholarly journals Structure-Function Analysis of Escherichia coli MnmG (GidA), a Highly Conserved tRNA-Modifying Enzyme

2009 ◽  
Vol 191 (24) ◽  
pp. 7614-7619 ◽  
Author(s):  
Rong Shi ◽  
Magda Villarroya ◽  
Rafael Ruiz-Partida ◽  
Yunge Li ◽  
Ariane Proteau ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Conformational Changes ◽  
Function Analysis ◽  
Trna Modification ◽  
Conserved Residues ◽  
Limited Trypsinolysis ◽  
Fad Binding ◽  
Trna Binding

ABSTRACT The MnmE-MnmG complex is involved in tRNA modification. We have determined the crystal structure of Escherichia coli MnmG at 2.4-Å resolution, mutated highly conserved residues with putative roles in flavin adenine dinucleotide (FAD) or tRNA binding and MnmE interaction, and analyzed the effects of these mutations in vivo and in vitro. Limited trypsinolysis of MnmG suggests significant conformational changes upon FAD binding.

scholarly journals Metal-Catalyzed Oxidation of Phenylalanine-Sensitive 3-Deoxy-d-arabino-Heptulosonate-7-Phosphate Synthase from Escherichia coli: Inactivation and Destabilization by Oxidation of Active-Site Cysteines

1999 ◽  
Vol 181 (5) ◽  
pp. 1636-1642 ◽  
Author(s):  
Ohkmae K. Park ◽  
Ronald Bauerle
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Peptide Mapping ◽  
Oxidation Mechanism ◽  
Disulfide Linkage ◽  
Metal Catalyzed Oxidation ◽  
Metal Catalyzed ◽  
Catalyzed Oxidation ◽  
Phosphate Synthase

ABSTRACT The in vitro instability of the phenylalanine-sensitive 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase [DAHPS(Phe)] from Escherichia coli has been found to be due to a metal-catalyzed oxidation mechanism. DAHPS(Phe) is one of three differentially feedback-regulated isoforms of the enzyme which catalyzes the first step of aromatic biosynthesis, the formation of DAHP from phosphoenolpyruvate andd-erythrose-4-phosphate. The activity of the apoenzyme decayed exponentially, with a half-life of about 1 day at room temperature, and the heterotetramer slowly dissociated to the monomeric state. The enzyme was stabilized by the presence of phosphoenolpyruvate or EDTA, indicating that in the absence of substrate, a trace metal(s) was the inactivating agent. Cu2+ and Fe2+, but none of the other divalent metals that activate the enzyme, greatly accelerated the rate of inactivation and subunit dissociation. Both anaerobiosis and the addition of catalase significantly reduced Cu2+-catalyzed inactivation. In the spontaneously inactivated enzyme, there was a net loss of two of the seven thiols per subunit; this value increased with increasing concentrations of added Cu2+. Dithiothreitol completely restored the enzymatic activity and the two lost thiols in the spontaneously inactivated enzyme but was only partially effective in reactivation of the Cu2+-inactivated enzyme. Mutant enzymes with conservative replacements at either of the two active-site cysteines, Cys61 or Cys328, were insensitive to the metal attack. Peptide mapping of the Cu2+-inactivated enzyme revealed a disulfide linkage between these two cysteine residues. All results indicate that DAHPS(Phe) is a metal-catalyzed oxidation system wherein bound substrate protects active-site residues from oxidative attack catalyzed by bound redox metal cofactor. A mechanism of inactivation of DAHPS is proposed that features a metal redox cycle that requires the sequential oxidation of its two active-site cysteines.

scholarly journals Cross-subunit catalysis and a new phenomenon of recessive resurrection in Escherichia coli RNase E

2019 ◽  
Vol 48 (2) ◽  
pp. 847-861 ◽  
Author(s):  
Nida Ali ◽  
Jayaraman Gowrishankar
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Rna Binding ◽  
Initial Step ◽  
Dominant Negative ◽  
Rnase E ◽  
Wild Type ◽  
Catalytic Active Site

Abstract RNase E is a 472-kDa homo-tetrameric essential endoribonuclease involved in RNA processing and turnover in Escherichia coli. In its N-terminal half (NTH) is the catalytic active site, as also a substrate 5′-sensor pocket that renders enzyme activity maximal on 5′-monophosphorylated RNAs. The protein's non-catalytic C-terminal half (CTH) harbours RNA-binding motifs and serves as scaffold for a multiprotein degradosome complex, but is dispensable for viability. Here, we provide evidence that a full-length hetero-tetramer, composed of a mixture of wild-type and (recessive lethal) active-site mutant subunits, exhibits identical activity in vivo as the wild-type homo-tetramer itself (‘recessive resurrection’). When all of the cognate polypeptides lacked the CTH, the active-site mutant subunits were dominant negative. A pair of C-terminally truncated polypeptides, which were individually inactive because of additional mutations in their active site and 5′-sensor pocket respectively, exhibited catalytic function in combination, both in vivo and in vitro (i.e. intragenic or allelic complementation). Our results indicate that adjacent subunits within an oligomer are separately responsible for 5′-sensing and cleavage, and that RNA binding facilitates oligomerization. We propose also that the CTH mediates a rate-determining initial step for enzyme function, which is likely the binding and channelling of substrate for NTH’s endonucleolytic action.

scholarly journals In Vivo Assay for Low-Activity Mutant Forms of Escherichia coli Ribonucleotide Reductase

2003 ◽  
Vol 185 (4) ◽  
pp. 1167-1173 ◽  
Author(s):  
Monica Ekberg ◽  
Pernilla Birgander ◽  
Britt-Marie Sjöberg
Keyword(s):  
Escherichia Coli ◽  
Ribonucleotide Reductase ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
In Vitro Assays ◽  
Tyrosyl Radical ◽  
In Vivo Assay ◽  
Radical Transfer

ABSTRACT Ribonucleotide reductase (RNR) catalyzes the essential production of deoxyribonucleotides in all living cells. In this study we have established a sensitive in vivo assay to study the activity of RNR in aerobic Escherichia coli cells. The method is based on the complementation of a chromosomally encoded nonfunctional RNR with plasmid-encoded RNR. This assay can be used to determine in vivo activity of RNR mutants with activities beyond the detection limits of traditional in vitro assays. E. coli RNR is composed of two homodimeric proteins, R1 and R2. The R2 protein contains a stable tyrosyl radical essential for the catalysis that takes place at the R1 active site. The three-dimensional structures of both proteins, phylogenetic studies, and site-directed mutagenesis experiments show that the radical is transferred from the R2 protein to the active site in the R1 protein via a radical transfer pathway composed of at least nine conserved amino acid residues. Using the new assay we determined the in vivo activity of mutants affecting the radical transfer pathway in RNR and identified some residual radical transfer activity in two mutant R2 constructs (D237N and W48Y) that had previously been classified as negative for enzyme activity. In addition, we show that the R2 mutant Y356W is completely inactive, in sharp contrast to what has previously been observed for the corresponding mutation in the mouse R2 enzyme.

The pyruvate dehydrogenase multi-enzyme complex of Escherichia coli : genetic reconstruction and functional analysis of the lipoyl domains

1986 ◽  
Vol 317 (1540) ◽  
pp. 391-404 ◽  
Keyword(s):  
Escherichia Coli ◽  
Pyruvate Dehydrogenase ◽  
Active Site ◽  
Inner Core ◽  
Polypeptide Chain ◽  
Pyruvate Dehydrogenase Complex ◽  
Site Directed Mutagenesis ◽  
Enzyme Complex ◽  
Site Selective

The dihydrolipoamide acetyltransferase (E2p) component of the pyruvate dehydrogenase complex of Escherichia coli contains three highly homologous lipoyl domains ( ca . 100 residues) that are tandemly repeated to form the N-terminal half of the polypeptide chain. These lipoyl domains are linked to a much larger ( ca . 300 residues) subunit-binding domain that aggregates to form the octahedral inner core of the complex and also contains the acetyltransferase active site. Selective in vitro deletions in the E2p gene ( aceF )have allowed the creation of truncated E2p chains in which one or more of the lipoyl domains has been excised. Site-directed mutagenesis has been used to change individual residues. The effects of these deletions and mutations on the assembly, catalytic activity and active-site coupling in the complex are assessed.

scholarly journals Structural basis of peptidoglycan endopeptidase regulation

2019 ◽  
Author(s):  
Jung-Ho Shin ◽  
Alan G. Sulpizio ◽  
Aaron Kelley ◽  
Laura Alvarez ◽  
Shannon G. Murphy ◽  
...  
Keyword(s):  
Cell Wall ◽  
Vibrio Cholerae ◽  
Active Site ◽  
Normal Growth ◽  
Structural Basis ◽  
Bacterial Cells ◽  
Cell Wall Digestion ◽  
Novel Antibiotics

AbstractMost bacteria surround themselves with a cell wall, a strong meshwork consisting primarily of the polymerized aminosugar peptidoglycan (PG). PG is essential for structural maintenance of bacterial cells, and thus for viability. PG is also constantly synthesized and turned over, the latter process is mediated by PG cleavage enzymes, for example the endopeptidases (EPs). EPs themselves are essential for growth, but also promote lethal cell wall degradation after exposure to antibiotics that inhibit PG synthases (e.g., β-lactams). Thus, EPs are attractive targets for novel antibiotics and their adjuvants. However, we have a poor understanding of how these enzymes are regulated in vivo, depriving us of novel pathways for the development of such antibiotics. Here, we have solved crystal structures of the LysM/M23 family peptidase ShyA, the primary EP of the cholera pathogen Vibrio cholerae. Our data suggest that ShyA assumes two drastically different conformations; a more open form that allows for substrate binding, and a closed form, which we predicted to be catalytically inactive. Mutations expected to promote the open conformation caused enhanced activity in vitro and in vivo, and these results were recapitulated in EPs from the divergent pathogens Neisseria gonorrheae and Escherichia coli. Our results suggest that LysM/M23 EPs are regulated via release of the inhibitory Domain1 from the M23 active site, likely through conformational re-arrangement in vivo.SignificanceBacteria digest their cell wall following exposure to antibiotics like penicillin. The endopeptidases (EPs) are among the proteins that catalyze cell wall digestion processes after antibiotic exposure, but we do not understand how these enzymes are regulated during normal growth. Herein, we present the structure of the major EP from the diarrheal pathogen Vibrio cholerae. Surprisingly, we find that EPs from this and other pathogens appear to be produced as a largely inactive precursor that undergoes a conformational shift exposing the active site to engage in cell wall digestion. These results enhance our understanding of how EPs are regulated and could open the door for the development of novel antibiotics that overactivate cell wall digestion processes.

scholarly journals Remdesivir is a delayed translocation inhibitor of SARS CoV-2 replication in vitro

2020 ◽  
Author(s):  
Jack PK Bravo ◽  
Tyler L Dangerfield ◽  
David W Taylor ◽  
Kenneth A Johnson
Keyword(s):  
Rna Polymerase ◽  
Active Site ◽  
Rational Design ◽  
Antiviral Drugs ◽  
Chain Termination ◽  
Nucleoside Analog ◽  
Rna Dependent Rna Polymerase ◽  
Polymerase Complex

Remdesivir is a nucleoside analog approved by the FDA for treatment of COVID-19. Here, we present a 3.9-Å-resolution cryoEM reconstruction of a remdesivir-stalled RNA-dependent RNA polymerase complex, revealing full incorporation of three copies of remdesivir monophosphate (RMP) and a partially incorporated fourth RMP in the active site. The structure reveals that RMP blocks RNA translocation after incorporation of three bases following RMP, resulting in delayed chain termination, which can guide the rational design of improved antiviral drugs.

Anticancer Evaluation and Docking Study of New Bifunctional Phthalazine Derivatives

2018 ◽  
Vol 15 (3) ◽  
pp. 414-422 ◽  
Author(s):  
Marwa G. El-Gazzar ◽  
Hala M. Aly
Keyword(s):  
Anticancer Activity ◽  
Cell Line ◽  
Anticancer Agents ◽  
Active Site ◽  
Cancer Cell Line ◽  
Docking Study ◽  
Quinazoline Derivative ◽  
Spectroscopic Measurements ◽  
Ic50 Value

Aims and Objective: A series of novel phthalazine derivatives was synthesized with versatile, readily accessible electrophilic and nucleophilic reagents. The newly synthesized compounds were confirmed by the results of spectroscopic measurements. Hence, their potential clinical application investigated in particular for cancer treatment. Materials and Methods: The newly synthesized compounds were characterized by spectroscopic measurements and were tested for their in vitro anticancer activity by MTT assay against human liver cancer cell line. Docking study of all the synthesized compounds was performed within the active site of the enzyme VEGFR-2 (Vascular Endothelial Growth Factor Receptor-2). Results: The quinazoline derivative 12 emerged as the most potent compound in this study with an IC50 value of 5.4 µM. Docking study showed that the synthesized compounds were fit in the VEGFR-2 active site almost at the same position of sorafenib and vatalanib with comparable docking scores (-15.20 to -8.92 was kcal/mol). Conclusion: we have synthesized a novel series of phthalazine derivatives and evaluated their potential anticancer activity against HEPG2 cell line. The quinazoline derivative 12 emerged as the most potent compound in this study with an IC50 value of 5.4 µM. The SAR and docking studies pointed out that rigidification of the structure resulted in better activity and better binding within the active site of VEGFR-2 as in compounds 3, 5, 6 and 12. These results introduced new phthalazine derivatives having promising activity which could lead to the development of more potent anticancer agents.

SYNTHESIS AND INVESTIGATION OF ANTI-ACETYLCHOLINESTERASE ACTIVITY IN SILICO AND IN VITRO OF SOME CHALCONES

2012 ◽  
pp. 98-106
Author(s):  
Thai Son Tran ◽  
Khac Minh Thai ◽  
Thanh Dao Tran
Keyword(s):  
Molecular Docking ◽  
Active Site ◽  
Condensation Reaction ◽  
Acetylcholinesterase Inhibitors ◽  
Acetylcholinesterase Activity ◽  
The Elderly ◽  
Docking Study ◽  
Molecular Docking Study ◽  
Ic50 Value

Background: Alzheimer is a major cause of dementia in the elderly and acetylcholinesterase inhibitors are used to treat the symptoms of this disease. Recently, chalcones have been reported as potential acetylcholinesterase inhibitors. Materials and methods: In this study, Claisen-Schmidt condensation reaction was applied to synthesize chalcones. Anti-acetylcholinesterase activity of these chalcones was determined by Ellman method. Molecular docking studies on acetylcholinesterase were performed to explain the interaction between these chalcone analogues and acetylcholinesterase active site at molecular level. Results: A total of twenty chalcones were synthesized and determined for in vitro anti-acetylcholinesterase activity. The results indicated that six compounds having IC50 value below 100 µM, three compounds having IC50 value in the range of 100 µM and 300 µM, the rest having IC50 value above 300 µM. Chalcone S17 (4’-amino-2-chlorochalcone) shows the strongest anti-acetylcholinesterase activity in the investigated group with IC50 value of 36.10 µM. In combination with the results of the in vitro anti-acetylcholinesterase activity, molecular docking study is used to explain the interaction between chalcone molecules and their active site, and the structure-activity relationship is abstracted. Conclusions: Our study indicated that the 2’-hydroxychalcones with halogen functional groups on B ring are strong acetylcholinesterase inhibitors. Chalcone S17 (4’-amino-2-chlorochalcone) could be considered as a potential lead compound for the development of new acetylcholinesterase inhibitors. Keywords: acetylcholinesterase, AChE, Alzheimer, chalcon, docking. Key words: A cetylcholinesterase, AChE, Alzheimer, chalcon, docking

scholarly journals Protease Activity, Secretion, Cell Entry, Cytotoxicity, and Cellular Targets of Secreted Autotransporter Toxin of Uropathogenic Escherichia coli

2006 ◽  
Vol 74 (11) ◽  
pp. 6124-6134 ◽  
Author(s):  
Nathalie M. Maroncle ◽  
Kelsey E. Sivick ◽  
Rebecca Brady ◽  
Faye-Ellen Stokes ◽  
Harry L. T. Mobley
Keyword(s):  
Escherichia Coli ◽  
Serine Protease ◽  
Active Site ◽  
Protease Activity ◽  
Host Cells ◽  
Single Mutation ◽  
Wild Type ◽  
Passenger Domain ◽  
Hek 293

ABSTRACT The secreted autotransporter toxin (Sat), found predominantly in uropathogenic Escherichia coli, is a member of the SPATE (serine protease autotransporters of Enterobacteriaceae) family and, as such, has serine protease activity and causes cytopathic effects on various cell types. To assess the contribution of the serine protease active site to the mechanism of action of Sat, mutations were made in the first (S256I), in the second (S258A), or in both (S256I/S258A) serine residues within the active site motif. Mutations in the first or both serines reduced protease activity to background levels (P < 0.001); a single mutation in the second serine reduced activity by 60% compared to wild type (P < 0.001). After reversion of the S256I mutation to wild type (I256S), we confirmed S256 as the catalytically active serine. None of these mutations affected secretion of the mature passenger domain or release into the supernatant. The S256I mutation, however, abrogated the cytotoxicity of Sat on human bladder (UM-UC-3) and kidney (HEK 293) epithelial cells, characterized by rounding and elongation, respectively, and a high level of cell detachment. Moreover, S256 is essential for Sat to mediate cytoskeletal contraction and actin loss in host cells as well as to degrade specific membrane/cytoskeletal (fodrin and leukocyte function-associated molecule 1) and nuclear [microtubule-associated proteins, LIM domain-only protein 7, Rap GTPase-activating protein, poly(ADP-ribose) polymerase] proteins in vitro. Lastly, Sat was internalized by host cells and localized to the cytoskeletal fraction where membrane/cytoskeletal target proteins reside.

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