scholarly journals Role of Ser11 in the stabilization of the structure of Ochrobactrum anthropi glutathione transferase

2007 ◽  
Vol 403 (2) ◽  
pp. 267-274 ◽  
Author(s):  
Luca Federici ◽  
Michele Masulli ◽  
Daniele Bonivento ◽  
Adele Di Matteo ◽  
Stefano Gianni ◽  
...  
Keyword(s):  
Glutathione Transferase ◽  
Catalytic Efficiency ◽  
Binding Pocket ◽  
Structural Basis ◽  
Ochrobactrum Anthropi ◽  
Structural Water ◽  
Terminal Domain ◽  
Hydrogen Bond Interactions ◽  
Degrading Bacterium

GSTs (glutathione transferases) are a multifunctional group of enzymes, widely distributed and involved in cellular detoxification processes. In the xenobiotic-degrading bacterium Ochrobactrum anthropi, GST is overexpressed in the presence of toxic concentrations of aromatic compounds such as 4-chlorophenol and atrazine. We have determined the crystal structure of the GST from O. anthropi (OaGST) in complex with GSH. Like other bacterial GSTs, OaGST belongs to the Beta class and shows a similar binding pocket for GSH. However, in contrast with the structure of Proteus mirabilis GST, GSH is not covalently bound to Cys10, but is present in the thiolate form. In our investigation of the structural basis for GSH stabilization, we have identified a conserved network of hydrogen-bond interactions, mediated by the presence of a structural water molecule that links Ser11 to Glu198. Partial disruption of this network, by mutagenesis of Ser11 to alanine, increases the Km for GSH 15-fold and decreases the catalytic efficiency 4-fold, even though Ser11 is not involved in GSH binding. Thermal- and chemical-induced unfolding studies point to a global effect of the mutation on the stability of the protein and to a central role of these residues in zippering the terminal helix of the C-terminal domain to the starting helix of the N-terminal domain.

scholarly journals Analysis of the structural requirements of sugar binding to the liver, brain and insulin-responsive glucose transporters expressed in oocytes

1993 ◽  
Vol 294 (3) ◽  
pp. 753-760 ◽  
Author(s):  
C A Colville ◽  
M J Seatter ◽  
G W Gould
Keyword(s):  
Hydrogen Bond ◽  
Binding Sites ◽  
Glucose Transporters ◽  
Binding Pocket ◽  
Structural Basis ◽  
Sugar Binding ◽  
Glucose Analogues ◽  
Sugar Recognition

We have expressed the liver (GLUT 2), brain (GLUT 3) and insulin-responsive (GLUT 4) glucose transporters in oocytes from Xenopus laevis by microinjection of in vitro-transcribed mRNA. Using a range of halogeno- and deoxy-glucose analogues, and other hexoses, we have studied the structural basis of sugar binding to these different isoforms. We show that a hydrogen bond to the C-3 position is involved in sugar binding for all three isoforms, but that the direction of this hydrogen bond is different in GLUT 2 from either GLUT 1, 3 or 4. Hydrogen-bonding at the C-4 position is also involved in sugar recognition by all three isoforms, but we propose that in GLUT 3 this hydrogen bond plays a less significant role than in GLUT 2 and 4. In all transporters we propose that the C-4 position is directed out of the sugar-binding pocket. The role of the C-6 position is also discussed. In addition, we have analysed the ability of fructopyranose and fructofuranose analogues to inhibit the transport mediated by GLUT2. We show that fructofuranose analogues, but not fructopyranose analogues, are efficient inhibitors of transport mediated by GLUT 2, and therefore suggest that GLUT 2 accommodates D-glucose as a pyranose ring, but D-fructose as a furanose ring. Models for the binding sites of GLUT 2, 3 and 4 are presented.

scholarly journals Structural basis for VIPP1 oligomerization and maintenance of thylakoid membrane integrity

2020 ◽  
Author(s):  
Tilak Kumar Gupta ◽  
Sven Klumpe ◽  
Karin Gries ◽  
Steffen Heinz ◽  
Wojciech Wietrzynski ◽  
...  
Keyword(s):  
Membrane Integrity ◽  
Point Mutations ◽  
Binding Pocket ◽  
Thylakoid Membranes ◽  
Lipid Binding ◽  
Structural Basis ◽  
Amphipathic Helices ◽  
Cryo Electron Microscopy

AbstractVesicle-inducing protein in plastids (VIPP1) is essential for the biogenesis and maintenance of thylakoid membranes, which transform light into life. However, it is unknown how VIPP1 performs its vital membrane-shaping function. Here, we use cryo-electron microscopy to determine structures of cyanobacterial VIPP1 rings, revealing how VIPP1 monomers flex and interweave to form basket-like assemblies of different symmetries. Three VIPP1 monomers together coordinate a non-canonical nucleotide binding pocket that is required for VIPP1 oligomerization. Inside the ring’s lumen, amphipathic helices from each monomer align to form large hydrophobic columns, enabling VIPP1 to bind and curve membranes. In vivo point mutations in these hydrophobic surfaces cause extreme thylakoid swelling under high light, indicating an essential role of VIPP1 lipid binding in resisting stress-induced damage. Our study provides a structural basis for understanding how the oligomerization of VIPP1 drives the biogenesis of thylakoid membranes and protects these life-giving membranes from environmental stress.

Understanding the highly efficient catalysis of prokaryotic peptide deformylases by shedding light on the determinants specifying the low activity of the human counterpart

2014 ◽  
Vol 70 (2) ◽  
pp. 242-252 ◽  
Author(s):  
Sonia Fieulaine ◽  
Michel Desmadril ◽  
Thierry Meinnel ◽  
Carmela Giglione
Keyword(s):  
Sequence Similarity ◽  
Three Dimensional ◽  
Binding Pocket ◽  
Dimensional Structure ◽  
Structural Basis ◽  
Human Counterpart ◽  
Low Activity

Peptide deformylases (PDFs), which are essential and ubiquitous enzymes involved in the removal of theN-formyl group from nascent chains, are classified into four subtypes based on the structural and sequence similarity of specific conserved domains. All PDFs share a similar three-dimensional structure, are functionally interchangeablein vivoand display similar propertiesin vitro, indicating that their molecular mechanism has been conserved during evolution. The human mitochondrial PDF is the only exception as despite its conserved fold it reveals a unique substrate-binding pocket together with an unusual kinetic behaviour. Unlike human PDF, the closely related mitochondrial PDF1As from plants have catalytic efficiencies and enzymatic parameters that are similar to those of other classes of PDFs. Here, the aim was to identify the structural basis underlying the properties of human PDF compared with all other PDFs by focusing on plant mitochondrial PDF1A. The construction of a chimaera composed of plant PDF1A with the nonrandom substitutions found in a conserved motif of its human homologue converted it into an enzyme with properties similar to the human enzyme, indicating the crucial role of these positions. The crystal structure of this human-like plant PDF revealed that substitution of two residues leads to a reduction in the volume of the ligand-binding site together with the introduction of negative charges, unravelling the origin of the weak affinity of human PDF for its substrate. In addition, the substitution of the two residues of human PDF modifies the transition state of the reaction through alteration of the network of interactions between the catalytic residues and the substrate, leading to an overall reduced reaction rate.

Critical Role of a Loop at C-Terminal Domain on the Conformational Stability and Catalytic Efficiency of Chondroitinase ABC I

2015 ◽  
Vol 57 (8) ◽  
pp. 727-734 ◽  
Author(s):  
S. Akram Shirdel ◽  
Khosrow Khalifeh ◽  
Abolfazl Golestani ◽  
Bijan Ranjbar ◽  
Khosro Khajeh
Keyword(s):  
Conformational Stability ◽  
Critical Role ◽  
Catalytic Efficiency ◽  
Chondroitinase Abc ◽  
Terminal Domain

scholarly journals Perspective on the Structural Basis for Human Aldo-Keto Reductase 1B10 Inhibition

Metabolites ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 865
Author(s):  
Francesc Xavier Ruiz ◽  
Xavier Parés ◽  
Jaume Farrés
Keyword(s):  
Enzyme Inhibitors ◽  
Binding Pocket ◽  
Anion Binding ◽  
Structural Basis ◽  
Inhibitor Binding ◽  
Reversible Inhibitors ◽  
Alternative Conformation ◽  
Variable Loops ◽  
Cancer Types

Human aldo-keto reductase 1B10 (AKR1B10) is overexpressed in many cancer types and is involved in chemoresistance. This makes AKR1B10 to be an interesting drug target and thus many enzyme inhibitors have been investigated. High-resolution crystallographic structures of AKR1B10 with various reversible inhibitors were deeply analyzed and compared to those of analogous complexes with aldose reductase (AR). In both enzymes, the active site included an anion-binding pocket and, in some cases, inhibitor binding caused the opening of a transient specificity pocket. Different structural conformers were revealed upon inhibitor binding, emphasizing the importance of the highly variable loops, which participate in the transient opening of additional binding subpockets. Two key differences between AKR1B10 and AR were observed regarding the role of external loops in inhibitor binding. The first corresponded to the alternative conformation of Trp112 (Trp111 in AR). The second difference dealt with loop A mobility, which defined a larger and more loosely packed subpocket in AKR1B10. From this analysis, the general features that a selective AKR1B10 inhibitor should comply with are the following: an anchoring moiety to the anion-binding pocket, keeping Trp112 in its native conformation (AKR1B10-like), and not opening the specificity pocket in AR.

scholarly journals Role of Human Glutathione Transferases in Biotransformation of the Nitric Oxide Prodrug JS-K

2021 ◽  
Author(s):  
Birgitta Sjödin ◽  
Bengt Mannervik
Keyword(s):  
Nitric Oxide ◽  
Glutathione Transferase ◽  
Specific Activity ◽  
Physiological Role ◽  
Catalytic Efficiency ◽  
Signal Molecule ◽  
Killing Effect ◽  
Normal Tissues ◽  
Tumor Killing

Abstract Nitric oxide (NO) plays a prominent physiological role as a low-molecular-mass signal molecule involved in diverse biological functions. Great attention has been directed to pharmacologically modulating the release of NO for various therapeutic applications. We have focused on O2-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K) as an example of diazeniumdiolate prodrugs with potential for cancer chemotherapy. JS-K is reportedly activated by glutathione conjugation by glutathione transferase (GST), but the scope of activities among the numerous members of the GSTome is unknown. We demonstrate that all human GSTs tested except GST T1-1 are active with JS-K as a substrate, but their specific activities are notably spanning a 100-fold range. The most effective enzyme was the mu class member GST M2-2 with a specific activity of 273 ± 5 µmol min-1 mg-1 and the kinetic parameters Km 48 ± 4 µM, kcat 501 ± 29 s-1, kcat/Km 10 x106 M-1 s-1. The abundance of the GSTs as an ensemble and their high catalytic efficiency indicate that release of NO occurs rapidly in normal tissues such that other mechanisms play a major role in the tumor-killing effect of JS-K.

scholarly journals Probing the Role of Sigma π Interaction and Energetics in the Catalytic Efficiency of Endo-1,4-β-Xylanase

2012 ◽  
Vol 78 (24) ◽  
pp. 8817-8821 ◽  
Author(s):  
Raushan Kumar Singh ◽  
Manish Kumar Tiwari ◽  
In-Won Kim ◽  
Zhilei Chen ◽  
Jung-Kul Lee
Keyword(s):  
Amino Acid ◽  
Turnover Rate ◽  
Catalytic Efficiency ◽  
Binding Pocket ◽  
Wild Type ◽  
High Turnover ◽  
Substrate Binding Pocket ◽  
Content Type ◽  
Important Residue

ABSTRACTChaetomium globosumendo-1,4-β-xylanase (XylCg) is distinguished from other xylanases by its high turnover rate (1,860 s−1), the highest ever reported for fungal xylanases. One conserved amino acid, W48, in the substrate binding pocket of wild-type XylCg was identified as an important residue affecting XylCg's catalytic efficiency.

Structural basis for the regulation of chemotaxis by MapZ in the presence of c-di-GMP

2017 ◽  
Vol 73 (8) ◽  
pp. 683-691 ◽  
Author(s):  
Yingxiao Zhu ◽  
Zenglin Yuan ◽  
Lichuan Gu
Keyword(s):  
Binding Site ◽  
Crystal Structures ◽  
Second Messenger ◽  
Binding Pocket ◽  
Single Domain ◽  
Structural Basis ◽  
Cyclic Diguanylate ◽  
Bacterial Physiology ◽  
Terminal Domain ◽  
Mechanism Of Inhibition

The bacterial second messenger cyclic diguanylate monophosphate (c-di-GMP) mediates multiple aspects of bacterial physiology through binding to various effectors. In some cases, these effectors are single-domain proteins which only contain a PilZ domain. It remains largely unknown how single-domain PilZ proteins function and regulate their downstream targets. Recently, a single-domain PilZ protein, MapZ (PA4608), was identified to inhibit the activity of the methyltransferase CheR1. Here, crystal structures of the C-terminal domain of CheR1 containing SAH and of CheR1 in complex with c-di-GMP-bound MapZ are reported. It was observed that the binding site of MapZ in CheR1 partially overlaps with the SAH/SAM-binding pocket. Consequently, binding of MapZ blocks SAH/SAM binding. This provides direct structural evidence on the mechanism of inhibition of CheR1 by MapZ in the presence of c-di-GMP.

scholarly journals Structural basis for DNA 5´-end resection by RecJ

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Kaiying Cheng ◽  
Hong Xu ◽  
Xuanyi Chen ◽  
Liangyan Wang ◽  
Bing Tian ◽  
...  
Keyword(s):  
Active Site ◽  
Deinococcus Radiodurans ◽  
Divalent Cations ◽  
Binding Pocket ◽  
Structural Basis ◽  
Phosphate Binding ◽  
Terminal Domain ◽  
Dna Strand ◽  
Recombination Pathway ◽  
End Resection

The resection of DNA strand with a 5´ end at double-strand breaks is an essential step in recombinational DNA repair. RecJ, a member of DHH family proteins, is the only 5´ nuclease involved in the RecF recombination pathway. Here, we report the crystal structures of Deinococcus radiodurans RecJ in complex with deoxythymidine monophosphate (dTMP), ssDNA, the C-terminal region of single-stranded DNA-binding protein (SSB-Ct) and a mechanistic insight into the RecF pathway. A terminal 5´-phosphate-binding pocket above the active site determines the 5´-3´ polarity of the deoxy-exonuclease of RecJ; a helical gateway at the entrance to the active site admits ssDNA only; and the continuous stacking interactions between protein and nine nucleotides ensure the processive end resection. The active site of RecJ in the N-terminal domain contains two divalent cations that coordinate the nucleophilic water. The ssDNA makes a 180° turn at the scissile phosphate. The C-terminal domain of RecJ binds the SSB-Ct, which explains how RecJ and SSB work together to efficiently process broken DNA ends for homologous recombination.

scholarly journals Role of human glutathione transferases in biotransformation of the nitric oxide prodrug JS-K

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Birgitta Sjödin ◽  
Bengt Mannervik
Keyword(s):  
Nitric Oxide ◽  
Glutathione Transferase ◽  
Specific Activity ◽  
Physiological Role ◽  
Catalytic Efficiency ◽  
Signal Molecule ◽  
Killing Effect ◽  
Normal Tissues ◽  
Tumor Killing

AbstractNitric oxide (NO) plays a prominent physiological role as a low-molecular-mass signal molecule involved in diverse biological functions. Great attention has been directed to pharmacologically modulating the release of NO for various therapeutic applications. We have focused on O2-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K) as an example of diazeniumdiolate prodrugs with potential for cancer chemotherapy. JS-K is reportedly activated by glutathione conjugation by glutathione transferase (GST), but the scope of activities among the numerous members of the GSTome is unknown. We demonstrate that all human GSTs tested except GST T1-1 are active with JS-K as a substrate, but their specific activities are notably spanning a > 100-fold range. The most effective enzyme was the mu class member GST M2-2 with a specific activity of 273 ± 5 µmol min−1 mg−1 and the kinetic parameters Km 63 µM, kcat 353 s−1, kcat/Km 6 × 106 M−1 s−1. The abundance of the GSTs as an ensemble and their high catalytic efficiency indicate that release of NO occurs rapidly in normal tissues such that this influence must be considered in clarification of the tumor-killing effect of JS-K.

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