scholarly journals The molecular structure of Schistosoma mansoni PNP isoform 2 provides insights into the nucleotide selectivity of PNPs

2018 ◽  
Author(s):  
Juliana Roberta Torini ◽  
Larissa Romanello ◽  
Fernanda Aparecida Heleno Batista ◽  
Vitor Hugo Balasco Serrão ◽  
Muhammad Faheem ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Schistosoma Mansoni ◽  
Binding Site ◽  
Kinetic Parameters ◽  
Isothermal Titration Calorimetry ◽  
Active Site ◽  
Binding Mode ◽  
Titration Calorimetry ◽  
Purine Salvage ◽  
Key Enzyme

AbstractPurine nucleoside phosphorylases (PNPs) play an important role in the blood fluke parasite Schistosoma mansoni as a key enzyme of the purine salvage pathway. Here we present the structural and kinetic characterization of a new PNP isoform from S. mansoni, named as SmPNP2. Screening of different ligands using a thermofluorescence approach indicated cytidine and cytosine as potential ligands. The binding of cytosine was confirmed by isothermal titration calorimetry, with a KD of 27 μM, and kinetic parameters for cytidine catalysis were obtained by ITC resulting in a KM of 76.3 μM. SmPNP2 also displays catalytic activity against inosine and adenosine, making it the first described PNP with robust catalytic activity towards both pyrimidines and purines. Crystallographic structures of SmPNP2 with different ligands were obtained and comparison of these structures with the previously described S. mansoni PNP (SmPNP1) provided clues for the unique capability of SmPNP2 to bind pyrimidines. When compared with the structure of SmPNP1, substitutions in the vicinity of SmPNP2 active site alter the architecture of the nucleoside base binding site allowing an alternative binding mode for nucleosides, with a 180° rotation from the canonical binding mode. The remarkable plasticity of this binding site deepens the understanding of the correlation between structure and nucleotide selectivity, offering new ways to analyses PNP activity.Author SummarySchistosoma mansoni is a human parasite dependent on purine salvage for purine bases supply. Purine nucleoside phosphorylase (PNP) is a key enzyme in this pathway. It carries two PNP isoforms, one previously characterized (SmPNP1) and one unknown (SmPNP2). Here we present the crystallographic structure of SmPNP2 and its complex with cytosine, cytidine, ribose-l-phosphate, adenine, hypoxanthine, and tubercidin. Cytidine and cytosine were identified as ligands of SmPNP2 using a thermofluorescence approach. Binding of cytosine was proven by Isothermal Titration Calorimetry (ITC) and cytidine, inosine, and adenosine kinetic parameters were also obtained. Purine bases showed different binding in the active site, rotated 180° from the canonical binding mode. It’s the first report showing a Low Molecular Mass PNP capable of catalyzing both types of nucleotide bases. The SmPNP2 odd behavior sheds a new light on the Schistosoma mansoni’s life cycle metabolic adaptation.

scholarly journals Modular Glucuronoxylan-Specific Xylanase with a Family CBM35 Carbohydrate-Binding Module

2012 ◽  
Vol 78 (11) ◽  
pp. 3923-3931 ◽  
Author(s):  
Susana Valeria Valenzuela ◽  
Pilar Diaz ◽  
F. I. Javier Pastor
Keyword(s):  
Catalytic Activity ◽  
Kinetic Parameters ◽  
Glucuronic Acid ◽  
Specific Activity ◽  
Dimensional Structure ◽  
Titration Calorimetry ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Content Type ◽  
Catalytic Module

ABSTRACTXyn30D from the xylanolytic strainPaenibacillus barcinonensishas been identified and characterized. The enzyme shows a modular structure comprising a catalytic module family 30 (GH30) and a carbohydrate-binding module family 35 (CBM35). Like GH30 xylanases, recombinant Xyn30D efficiently hydrolyzed glucuronoxylans and methyl-glucuronic acid branched xylooligosaccharides but showed no catalytic activity on arabinose-substituted xylans. Kinetic parameters of Xyn30D were determined on beechwood xylan, showing aKmof 14.72 mg/ml and akcatvalue of 1,510 min−1. The multidomain structure of Xyn30D clearly distinguishes it from the GH30 xylanases characterized to date, which are single-domain enzymes. The modules of the enzyme were individually expressed in a recombinant host and characterized. The isolated GH30 catalytic module showed specific activity, mode of action on xylan, and kinetic parameters that were similar to those of the full-length enzyme. Computer modeling of the three-dimensional structure of Xyn30D showed that the catalytic module is comprised of a common (β/α)8barrel linked to a side-associated β-structure. Several derivatives of the catalytic module with decreasing deletions of this associated structure were constructed. None of them showed catalytic activity, indicating the importance of the side β-structure in the catalysis of Xyn30D. Binding properties of the isolated carbohydrate-binding module were analyzed by affinity gel electrophoresis, which showed that the CBM35 of the enzyme binds to soluble glucuronoxylans and arabinoxylans. Analysis by isothermal titration calorimetry showed that CBM35 binds to glucuronic acid and requires calcium ions for binding. Occurrence of a CBM35 in a glucuronoxylan-specific xylanase is a differential trait of the enzyme characterized.

scholarly journals Crystal structure of Schistosoma mansoni HGPRT isoform 1

2014 ◽  
Vol 70 (a1) ◽  
pp. C833-C833
Author(s):  
Larissa Romanello ◽  
Juliana de Souza ◽  
Louise Bird ◽  
Joanne Nettleship ◽  
Raymond Owens ◽  
...  
Keyword(s):  
Schistosoma Mansoni ◽  
De Novo ◽  
Structural Information ◽  
Guanosine Monophosphate ◽  
New Drugs ◽  
X Rays ◽  
Salvage Pathway ◽  
Purine Salvage ◽  
Space Groups ◽  
Key Enzyme

Schistosoma mansoni is the parasite responsible for schistosomiasis, a disease that affects about 207 million people worldwide [1], and does not have the purine "de novo" pathway, depending entirely on the purine salvage pathway to supply its demands on purines [2]. The purine salvage pathway has been reported as a potential target for developing new drugs against schistosomiasis. Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is a key enzyme in this pathway and the only validated enzyme target of the pathway [3]. HGPRT catalyzes the PRPP dependent conversion of hypoxanthine/guanine to inosine monophosphate or guanine to guanosine monophosphate. HGPRT1 gene was amplified, cloned, expressed and purified at the Oxford Protein Production Facility (OPPF-UK). Robotic crystallization trials were performed and SmHGPRT crystallized in several conditions of the Morpheus crystallization kit: A4, A8, A9, and C9. The crystals appear about a day and have about 30 μM in greatest dimension. About a hundred crystals were screened with x-rays on the macromolecular crystallography beamlines I02 and I24 at Diamond Light Source. 21 datasets was collected from 2.97 to 4.11Å resolution. A solution was obtained for HGPRT1 belongs space group P212121 in a dataset to 3.4Å resolution, with four monomer in the ASU. The structure was solved by the program Phaser using HGPRT human as a search model. The refinement is being carried out by program Phenix. The density map is acceptable for the resolution but a great manual work of interpretation is necessary for the refinement of this structure. The most important is the demonstration that it was possible to crystallize and collect data of SmHGPRT. A major effort will be undertaken to improve the size and diffraction power of HGPRT crystals as well as in the resolution of the structure of HGPRT in other space groups. This structure will increase the structural information available about the Schistosoma mansoni purine salvage pathway.

scholarly journals Characterization of bromosulphophthalein binding to human glutathione S-transferase A1-1: thermodynamics and inhibition kinetics

2004 ◽  
Vol 382 (2) ◽  
pp. 703-709 ◽  
Author(s):  
Doris KOLOBE ◽  
Yasien SAYED ◽  
Heini W. DIRR
Keyword(s):  
Binding Site ◽  
Active Site ◽  
Competitive Inhibition ◽  
Hydrophobic Interactions ◽  
Titration Calorimetry ◽  
Inhibition Kinetics ◽  
High Affinity ◽  
Anionic Ligand ◽  
Catalytic Function ◽  
High Affinity Site

In addition to their catalytic functions, GSTs (glutathione S-transferases) bind a wide variety of structurally diverse non-substrate ligands. This ligandin function is known to result in the inhibition of catalytic function. The interaction between hGSTA1-1 (human class Alpha GST with two type 1 subunits) and a non-substrate anionic ligand, BSP (bromosulphophthalein), was studied by isothermal titration calorimetry and inhibition kinetics. The binding isotherm is biphasic, best described by a set of two independent sites: a high-affinity site and a low-affinity site(s). The binding stoichiometries for these sites are 1 and 3 molecules of BSP respectively. BSP binds to the high-affinity site 80 times more tightly (Kd=0.12 μM) than it does to the low-affinity site(s) (Kd=9.1 μM). Binding at these sites is enthalpically and entropically favourable, with no linkage to protonation events. Temperature- and salt-dependent studies indicate the significance of hydrophobic interactions in the binding of BSP, and that the low-affinity site(s) displays low specificity towards the anion. Binding of BSP results in the release of ordered water molecules at these hydrophobic sites, which more than offsets unfavourable entropic changes during binding. BSP inhibition studies show that the binding of BSP to its high-affinity site does not inhibit hGSTA1-1. This site, located near Trp-20, may be related to the buffer-binding site observed in GSTP1-1. The low-affinity-binding site(s) for BSP is most probably located at or near the active site of hGSTA1-1. Binding to this site(s) results in non-competitive inhibition with respect to CDNB (1-chloro-2,4-dinitrobenzene) (KiBSP=16.8±1.9 μM). Given the properties of the H site and the relatively small size of the electrophilic substrate CDNB, it is plausible that the active site of the enzyme can simultaneously accommodate both BSP and CDNB. This would explain the non-competitive behaviour of certain inhibitors that bind the active site (e.g. BSP).

Active site-targeted carbon dots for the inhibition of human insulin fibrillation

2017 ◽  
Vol 5 (10) ◽  
pp. 2010-2018 ◽  
Author(s):  
Q. Q. Yang ◽  
J. C. Jin ◽  
Z. Q. Xu ◽  
J. Q. Zhang ◽  
B. B. Wang ◽  
...  
Keyword(s):  
Isothermal Titration Calorimetry ◽  
Human Insulin ◽  
Active Site ◽  
Carbon Dots ◽  
Titration Calorimetry ◽  
Inhibitory Mechanism

This work aimed to study the inhibitory mechanism of carbon dots for HI fibrillation using isothermal titration calorimetry.

scholarly journals Modeling of Multivalent Ligand-Receptor Binding Measured by kinITC

Computation ◽  
2019 ◽  
Vol 7 (3) ◽  
pp. 46
Author(s):  
Franziska Erlekam ◽  
Sinaida Igde ◽  
Susanna Röblitz ◽  
Laura Hartmann ◽  
Marcus Weber
Keyword(s):  
Protein Folding ◽  
Kinetic Parameters ◽  
Invariant Subspace ◽  
Isothermal Titration Calorimetry ◽  
Receptor Binding ◽  
Kinetic Data ◽  
Titration Calorimetry ◽  
Subspace Projection ◽  
Binding Process ◽  
Sequential Binding

In addition to the conventional Isothermal Titration Calorimetry (ITC), kinetic ITC (kinITC) not only gains thermodynamic information, but also kinetic data from a biochemical binding process. Moreover, kinITC gives insights into reactions consisting of two separate kinetic steps, such as protein folding or sequential binding processes. The ITC method alone cannot deliver kinetic parameters, especially not for multivalent bindings. This paper describes how to solve the problem using kinITC and an invariant subspace projection. The algorithm is tested for multivalent systems with different valencies.

scholarly journals Reconciling isothermal titration calorimetry analyses of interactions between complexin and truncated SNARE complexes

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Eric A Prinslow ◽  
Chad A Brautigam ◽  
Josep Rizo
Keyword(s):  
Isothermal Titration Calorimetry ◽  
Neurotransmitter Release ◽  
Experimental Error ◽  
Binding Mode ◽  
Snare Complex ◽  
Titration Calorimetry ◽  
Juxtamembrane Region ◽  
C Terminus ◽  
Snare Motif ◽  
Α Helix

Neurotransmitter release depends on the SNARE complex formed by syntaxin-1, synaptobrevin and SNAP-25, as well as on complexins, which bind to the SNARE complex and play active and inhibitory roles. A crystal structure of a Complexin-I fragment bearing a so-called 'superclamp' mutation bound to a truncated SNARE complex lacking the C-terminus of the synaptobrevin SNARE motif (SNAREΔ60) suggested that an 'accessory' α-helix of Complexin-I inhibits release by inserting into the C-terminus of the SNARE complex. Previously, isothermal titration calorimetry (ITC) experiments performed in different laboratories yielded apparently discrepant results in support or against the existence of such binding mode in solution (Trimbuch et al., 2014; Krishnakumar et al., 2015). Here, ITC experiments performed to solve these discrepancies now show that the region containing the Complexin-I accessory helix and preceding N-terminal sequences does interact with SNAREΔ60, but the interaction requires the polybasic juxtamembrane region of syntaxin-1 and is not affected by the superclamp mutation within the experimental error of these experiments.

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