scholarly journals Cryoelectron microscopy structure and mechanism of the membrane-associated electron-bifurcating flavoprotein Fix/EtfABCX

2020 ◽  
Vol 118 (2) ◽  
pp. e2016978118
Author(s):  
Xiang Feng ◽  
Gerrit J. Schut ◽  
Gina L. Lipscomb ◽  
Huilin Li ◽  
Michael W. W. Adams
Keyword(s):  
Electron Donor ◽  
Structural Changes ◽  
Structural Similarity ◽  
Spectroscopic Studies ◽  
Thermophilic Bacterium ◽  
High Potential ◽  
Dimer Interface ◽  
Detailed Mechanism ◽  
Ubiquinone Oxidoreductase ◽  
Electron Transferring Flavoprotein

The electron-transferring flavoprotein-menaquinone oxidoreductase ABCX (EtfABCX), also known as FixABCX for its role in nitrogen-fixing organisms, is a member of a family of electron-transferring flavoproteins that catalyze electron bifurcation. EtfABCX enables endergonic reduction of ferredoxin (E°′ ∼−450 mV) using NADH (E°′ −320 mV) as the electron donor by coupling this reaction to the exergonic reduction of menaquinone (E°′ −80 mV). Here we report the 2.9 Å structure of EtfABCX, a membrane-associated flavin-based electron bifurcation (FBEB) complex, from a thermophilic bacterium. EtfABCX forms a superdimer with two membrane-associated EtfCs at the dimer interface that contain two bound menaquinones. The structure reveals that, in contrast to previous predictions, the low-potential electrons bifurcated from EtfAB are most likely directly transferred to ferredoxin, while high-potential electrons reduce the quinone via two [4Fe-4S] clusters in EtfX. Surprisingly, EtfX shares remarkable structural similarity with mammalian [4Fe-4S] cluster-containing ETF ubiquinone oxidoreductase (ETF-QO), suggesting an unexpected evolutionary link between bifurcating and nonbifurcating systems. Based on this structure and spectroscopic studies of a closely related EtfABCX, we propose a detailed mechanism of the catalytic cycle and the accompanying structural changes in this membrane-associated FBEB system.

scholarly journals Engineering a Pseudo-26-kDa Schistosoma Glutathione Transferase from bovis/haematobium for Structure, Kinetics, and Ligandin Studies

Biomolecules ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 1844
Author(s):  
Neo Padi ◽  
Blessing Oluebube Akumadu ◽  
Olga Faerch ◽  
Chinyere Aloke ◽  
Vanessa Meyer ◽  
...  
Keyword(s):  
Glutathione Transferase ◽  
Specific Activity ◽  
Enzyme Stability ◽  
Competitive Inhibitor ◽  
Spectroscopic Studies ◽  
Complete Sequence ◽  
Detoxification Enzymes ◽  
Ligand Docking ◽  
Sequence Information ◽  
Dimer Interface

Glutathione transferases (GSTs) are the main detoxification enzymes in schistosomes. These parasitic enzymes tend to be upregulated during drug treatment, with Schistosoma haematobium being one of the species that mainly affect humans. There is a lack of complete sequence information on the closely related bovis and haematobium 26-kDa GST isoforms in any database. Consequently, we engineered a pseudo-26-kDa S. bovis/haematobium GST (Sbh26GST) to understand structure–function relations and ligandin activity towards selected potential ligands. Sbh26GST was overexpressed in Escherichia coli as an MBP-fusion protein, purified to homogeneity and catalyzed 1-chloro-2,4-dinitrobenzene-glutathione (CDNB-GSH) conjugation activity, with a specific activity of 13 μmol/min/mg. This activity decreased by ~95% in the presence of bromosulfophthalein (BSP), which showed an IC50 of 27 µM. Additionally, enzyme kinetics revealed that BSP acts as a non-competitive inhibitor relative to GSH. Spectroscopic studies affirmed that Sbh26GST adopts the canonical GST structure, which is predominantly α-helical. Further extrinsic 8-anilino-1-naphthalenesulfonate (ANS) spectroscopy illustrated that BSP, praziquantel (PZQ), and artemisinin (ART) might preferentially bind at the dimer interface or in proximity to the hydrophobic substrate-binding site of the enzyme. The Sbh26GST-BSP interaction is both enthalpically and entropically driven, with a stoichiometry of one BSP molecule per Sbh26GST dimer. Enzyme stability appeared enhanced in the presence of BSP and GSH. Induced fit ligand docking affirmed the spectroscopic, thermodynamic, and molecular modelling results. In conclusion, BSP is a potent inhibitor of Sbh26GST and could potentially be rationalized as a treatment for schistosomiasis.

Spectroscopic Studies of Structural Changes in Two β-Sheet-Forming Peptides Show an Ensemble of Structures that Unfold Noncooperatively†

Biochemistry ◽  
2003 ◽  
Vol 42 (15) ◽  
pp. 4321-4332 ◽  
Author(s):  
Serguei V. Kuznetsov ◽  
Jovencio Hilario ◽  
Timothy A. Keiderling ◽  
Anjum Ansari
Keyword(s):  
Structural Changes ◽  
Spectroscopic Studies ◽  
Sheet Forming ◽  
Β Sheet

Raman Spectroscopic Studies of Structural Changes of Insulin in Controlled Fluid Flows

2010 ◽  
Author(s):  
Jonathan Dusting ◽  
Grant Webster ◽  
Stavroula Balabani ◽  
Ewan W. Blanch ◽  
P. M. Champion ◽  
...  
Keyword(s):  
Structural Changes ◽  
Fluid Flows ◽  
Spectroscopic Studies ◽  
Raman Spectroscopic

scholarly journals Conformational Changes in the Capsid of a Calicivirus upon Interaction with Its Functional Receptor

2010 ◽  
Vol 84 (11) ◽  
pp. 5550-5564 ◽  
Author(s):  
Robert J. Ossiboff ◽  
Yi Zhou ◽  
Patrick J. Lightfoot ◽  
B. V. Venkataram Prasad ◽  
John S. L. Parker
Keyword(s):  
Conformational Changes ◽  
Viral Entry ◽  
Structural Changes ◽  
Feline Calicivirus ◽  
Soluble Receptor ◽  
Viral Capsids ◽  
Dimer Interface ◽  
Metastable Structures ◽  
Growth Properties ◽  
Functional Receptor

ABSTRACT Nonenveloped viral capsids are metastable structures that undergo conformational changes during virus entry that lead to interactions of the capsid or capsid fragments with the cell membrane. For members of the Caliciviridae, neither the nature of these structural changes in the capsid nor the factor(s) responsible for inducing these changes is known. Feline junctional adhesion molecule A (fJAM-A) mediates the attachment and infectious viral entry of feline calicivirus (FCV). Here, we show that the infectivity of some FCV isolates is neutralized following incubation with the soluble receptor at 37°C. We used this property to select mutants resistant to preincubation with the soluble receptor. We isolated and sequenced 24 soluble receptor-resistant (srr) mutants and characterized the growth properties and receptor-binding activities of eight mutants. The location of the mutations within the capsid structure of FCV was mapped using a new 3.6-Å structure of native FCV. The srr mutations mapped to the surface of the P2 domain were buried at the protruding domain dimer interface or were present in inaccessible regions of the capsid protein. Coupled with data showing that both the parental FCV and the srr mutants underwent increases in hydrophobicity upon incubation with the soluble receptor at 37°C, these findings indicate that FCV likely undergoes conformational change upon interaction with its receptor. Changes in FCV capsid conformation following its interaction with fJAM-A may be important for subsequent interactions of the capsid with cellular membranes, membrane penetration, and genome delivery.

scholarly journals Identification of Multiple Soluble Fe(III) Reductases in Gram-Positive Thermophilic BacteriumThermoanaerobacter indiensisBSB-33

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Subrata Pal
Keyword(s):  
Reductase Activity ◽  
Three Dimensional ◽  
Thioredoxin Reductase ◽  
Desulfovibrio Desulfuricans ◽  
Structural Similarity ◽  
Thermophilic Bacterium ◽  
Gram Positive ◽  
Reduction Activity ◽  
Structural Identity ◽  
Disulfide Reductase

Thermoanaerobacter indiensisBSB-33 has been earlier shown to reduce Fe(III) and Cr(VI) anaerobically at 60°C optimally. Further, the Gram-positive thermophilic bacterium contains Cr(VI) reduction activity in both the membrane and cytoplasm. The soluble fraction prepared fromT. indiensiscells grown at 60°C was found to contain the majority of Fe(III) reduction activity of the microorganism and produced four distinct bands in nondenaturing Fe(III) reductase activity gel. Proteins from each of these bands were partially purified by chromatography and identified by mass spectrometry (MS) with the help ofT. indiensisproteome sequences. Two paralogous dihydrolipoamide dehydrogenases (LPDs), thioredoxin reductase (Trx), NADP(H)-nitrite reductase (Ntr), and thioredoxin disulfide reductase (Tdr) were determined to be responsible for Fe(III) reductase activity. Amino acid sequence and three-dimensional (3D) structural similarity analyses of theT. indiensisFe(III) reductases were carried out with Cr(VI) reducing proteins from other bacteria. The two LPDs and Tdr showed very significant sequence and structural identity, respectively, with Cr(VI) reducing dihydrolipoamide dehydrogenase fromThermus scotoductusand thioredoxin disulfide reductase fromDesulfovibrio desulfuricans. It appears that in addition to their iron reducing activityT. indiensisLPDs and Tdr are possibly involved in Cr(VI) reduction as well.

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