scholarly journals Fine tuning of the active site modulates specificity in the interaction of O-acetylserine sulfhydrylase isozymes with serine acetyltransferase

2013 ◽  
Vol 1834 (1) ◽  
pp. 169-181 ◽  
Author(s):  
Francesca Spyrakis ◽  
Paolo Felici ◽  
Alexander S. Bayden ◽  
Enea Salsi ◽  
Riccardo Miggiano ◽  
...  
Keyword(s):  
Active Site ◽  
Fine Tuning ◽  
Serine Acetyltransferase

Entamoeba histolytica: identification of thioredoxin-targeted proteins and analysis of serine acetyltransferase-1 as a prototype example

2013 ◽  
Vol 451 (2) ◽  
pp. 277-288 ◽  
Author(s):  
Sarah Schlosser ◽  
David Leitsch ◽  
Michael Duchêne
Keyword(s):  
Entamoeba Histolytica ◽  
Active Site ◽  
Gel Electrophoresis ◽  
Biosynthetic Pathway ◽  
Interacting Proteins ◽  
Protozoan Parasites ◽  
Serine Acetyltransferase ◽  
Two Dimensional Gel Electrophoresis ◽  
Mixed Disulfide ◽  
Far Western Blot

Entamoeba histolytica, the causative agent of amoebiasis, possesses the dithiol-containing redox proteins Trx (thioredoxin) and TrxR (Trx reductase). Both proteins were found to be covalently modified and inactivated by metronidazole, a 5-nitroimidazole drug that is commonly used to treat infections with microaerophilic protozoan parasites in humans. Currently, very little is known about enzymes and other proteins participating in the Trx-dependent redox network of the parasite that could be indirectly affected by metronidazole treatment. On the basis of the disulfide/dithiol-exchange mechanism we constructed an active-site mutant of Trx, capable of binding interacting proteins as a stable mixed disulfide intermediate to screen the target proteome of Trx in E. histolytica. By applying Trx affinity chromatography, two-dimensional gel electrophoresis and MS, peroxiredoxin and 15 further potentially redox-regulated proteins were identified. Among them, EhSat1 (E. histolytica serine acetyltransferase-1), an enzyme involved in the L-cysteine biosynthetic pathway, was selected for detailed analysis. Binding of Trx to EhSat1 was verified by Far-Western blot analysis. Trx was able to restore the activity of the oxidatively damaged EhSat1 suggesting that the TrxR/Trx system protects sensitive proteins against oxidative stress in E. histolytica. Furthermore, the activity of peroxiredoxin, which is dependent on a functioning TrxR/Trx system, was strongly reduced in metronidazole-treated parasites.

Fine Tuning of the Copper Active Site in Polysaccharide Monooxygenases

2020 ◽  
Vol 124 (10) ◽  
pp. 1859-1865
Author(s):  
Son Tung Ngo ◽  
Han N. Phan ◽  
Chinh N. Le ◽  
Nhung C. T. Ngo ◽  
Khanh Bao Vu ◽  
...  
Keyword(s):  
Active Site ◽  
Fine Tuning ◽  
Polysaccharide Monooxygenases

scholarly journals (−)-Homosalinosporamide A and Its Mode of Proteasome Inhibition: An X-ray Crystallographic Study

Marine Drugs ◽  
2018 ◽  
Vol 16 (7) ◽  
pp. 240 ◽  
Author(s):  
Michael Groll ◽  
Henry Nguyen ◽  
Sreekumar Vellalath ◽  
Daniel Romo
Keyword(s):  
Natural Product ◽  
Active Site ◽  
Late Stage ◽  
Enzyme Assay ◽  
Proteasome Inhibition ◽  
Fine Tuning ◽  
Synthetic Approach ◽  
X Ray ◽  
Salinosporamide A ◽  
Crystallographic Study

Upon acylation of the proteasome by the β-lactone inhibitor salinosporamide A (SalA), tetrahydrofuran formation occurs by intramolecular alkylation of the incipient alkoxide onto the choroethyl sidechain and irreversibly blocks the active site. Our previously described synthetic approach to SalA, utilizing a bioinspired, late-stage, aldol-β-lactonization strategy to construct the bicyclic β-lactone core, enabled synthesis of (–)-homosalinosporamide A (homoSalA). This homolog was targeted to determine whether an intramolecular tetrahydropyran is formed in a similar manner to SalA. Herein, we report the X-ray structure of the yeast 20S proteasome:homoSalA-complex which reveals that tetrahydropyran ring formation does not occur despite comparable potency at the chymotrypsin-like active site in a luminogenic enzyme assay. Thus, the natural product derivative homoSalA blocks the proteasome by a covalent reversible mode of action, opening the door for further fine-tuning of proteasome inhibition.

scholarly journals Atomic design and fine-tuning of sub-nanometric Pt catalysts to tame hydrogen generation

2020 ◽  
Author(s):  
Jie Yang ◽  
Wenzhao Fu ◽  
Chaoqiu Chen ◽  
Wenyao Chen ◽  
Wugen Huang ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Mechanism ◽  
Hydrogen Generation ◽  
Atomic Layer ◽  
Fine Tuning ◽  
Superior Performance ◽  
Single Atom ◽  
Intrinsic Activity ◽  
Atomic Structures ◽  
Metal Metal

Abstract Rational synthesis of sub-nanocatalysts with controllable electronic and atomic structures remains a challenge to break the limits of traditional catalysts for superior performance. Here we report the atomic-level precise synthesis of Pt/graphene sub-nanocatalysts (from single atom, dimer, and to cluster) by atomic layer deposition, achieved by a novel high temperature pulsed ozone strategy to controllably pre-create abundant in-plane epoxy groups on graphene as anchoring sites. The specific in-plane epoxy structure endows the deposited Pt species with outstanding uniformity, controllability and stability. Their size-depended electronic and geometric effects have been observed for ammonia borane hydrolysis, revealing a volcano-type dependence of intrinsic activity on their sizes. Their active site structures have been identified based on extensive characterizations, dynamic compensation effect, kinetic isotope experiments and density function theory simulation. The Pt dimers show the highest catalytic activity and good durability than Pt single atoms and nanoparticles, ascribed to the unique C-Pt-Pt-O (C5Pt2O, metal-metal bond dimer) active site structure. Our work provides new insights into the precise tailoring and catalytic mechanism in sub-nanometer level.

scholarly journals Combination of SAXS and Protein Painting Discloses the Three-Dimensional Organization of the Bacterial Cysteine Synthase Complex, a Potential Target for Enhancers of Antibiotic Action

2019 ◽  
Vol 20 (20) ◽  
pp. 5219
Author(s):  
Brenda Rosa ◽  
Marialaura Marchetti ◽  
Gianluca Paredi ◽  
Heinz Amenitsch ◽  
Nina Franko ◽  
...  
Keyword(s):  
Complex Formation ◽  
Active Site ◽  
Solution Structure ◽  
Three Dimensional ◽  
Direct Interaction ◽  
Site Directed Mutagenesis ◽  
Fine Tuning ◽  
Bacterial Cells ◽  
Saxs Data ◽  
Cysteine Synthase

The formation of multienzymatic complexes allows for the fine tuning of many aspects of enzymatic functions, such as efficiency, localization, stability, and moonlighting. Here, we investigated, in solution, the structure of bacterial cysteine synthase (CS) complex. CS is formed by serine acetyltransferase (CysE) and O-acetylserine sulfhydrylase isozyme A (CysK), the enzymes that catalyze the last two steps of cysteine biosynthesis in bacteria. CysK and CysE have been proposed as potential targets for antibiotics, since cysteine and related metabolites are intimately linked to protection of bacterial cells against redox damage and to antibiotic resistance. We applied a combined approach of small-angle X-ray scattering (SAXS) spectroscopy and protein painting to obtain a model for the solution structure of CS. Protein painting allowed the identification of protein–protein interaction hotspots that were then used as constrains to model the CS quaternary assembly inside the SAXS envelope. We demonstrate that the active site entrance of CysK is involved in complex formation, as suggested by site-directed mutagenesis and functional studies. Furthermore, complex formation involves a conformational change in one CysK subunit that is likely transmitted through the dimer interface to the other subunit, with a regulatory effect. Finally, SAXS data indicate that only one active site of CysK is involved in direct interaction with CysE and unambiguously unveil the quaternary arrangement of CS.

Probing conformational changes in lipoxygenases upon membrane binding: Fine-tuning by the active site inhibitor ETYA

2014 ◽  
Vol 1841 (1) ◽  
pp. 1-10 ◽  
Author(s):  
Almerinda Di Venere ◽  
Eleonora Nicolai ◽  
Igor Ivanov ◽  
Enrico Dainese ◽  
Susan Adel ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Membrane Binding ◽  
Fine Tuning

scholarly journals Interaction of serine acetyltransferase withO-acetylserine sulfhydrylase active site: Evidence from fluorescence spectroscopy

2005 ◽  
Vol 14 (8) ◽  
pp. 2115-2124 ◽  
Author(s):  
Barbara Campanini ◽  
Francesca Speroni ◽  
Enea Salsi ◽  
Paul F. Cook ◽  
Steven L. Roderick ◽  
...  
Keyword(s):  
Fluorescence Spectroscopy ◽  
Active Site ◽  
Serine Acetyltransferase
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