Molecular determinants of ligand specificity in family 11 carbohydrate binding modules - An NMR, X-ray crystallography and computational chemistry approach

12 pags, 6 figs, 1 tabThe direct conversion of plant cell wall polysaccharides into soluble sugars is one of the most important reactions on earth, and is performed by certain microorganisms such as Clostridium thermocellum (Ct). These organisms produce extracellular multi-subunit complexes (i.e. cellulosomes) comprising a consortium of enzymes, which contain noncatalytic carbohydrate-binding modules (CBM) that increase the activity of the catalytic module. In the present study, we describe a combined approach by X-ray crystallography, NMR and computational chemistry that aimed to gain further insight into the binding mode of different carbohydrates (cellobiose, cellotetraose and cellohexaose) to the binding pocket of the family 11 CBM. The crystal structure of C. thermocellum CBM11 has been resolved to 1.98 Å in the apo form. Since the structure with a bound substrate could not be obtained, computational studies with cellobiose, cellotetraose and cellohexaose were carried out to determine the molecular recognition of glucose polymers by CtCBM11. These studies revealed a specificity area at the CtCBM11 binding cleft, which is lined with several aspartate residues. In addition, a cluster of aromatic residues was found to be important for guiding and packing of the polysaccharide. The binding cleft of CtCBM11 interacts more strongly with the central glucose units of cellotetraose and cellohexaose, mainly through interactions with the sugar units at positions 2 and 6. This model of binding is supported by saturation transfer difference NMR experiments and linebroadening NMR studies. © 2008 The Authors.The authors would like to thank the research network REQUIMTE (Project Reqmol), as well as the Portuguese Science and Technology Foundation (FCT-MCTES), for financial support through projectPTDC⁄QUI⁄68286⁄2006 and scholarships SFRH⁄BPD⁄27237⁄2006 and SFRH⁄BD⁄31359⁄200

Unraveling cGAS catalytic mechanism upon DNA activation through molecular dynamics simulations

International audienceCyclic GMP-AMP Synthase (cGAS) is activated upon DNA binding and catalyzes the synthesis of 2′,3′-cGAMP from GTP and ATP. This cyclic dinucleotide is a messenger that triggers the autoimmune system of eukaryotic cells. In this study, we propose a Molecular Dynamics (MD) investigation of cGAS activation. We notably provide insights into the motion of the activation loop, both from a mechanical point of view and considering its role in the catalysis of cGAMP production. We finally shed light on the reaction resulting in cGAMP synthesis. Two possible catalytic routes (referred to as GTP-ATP and ATP–GTP) are proposed based on the active site occupancy, paving the way toward further exploration of the reaction mechanism

Feedback inhibition of DszC, a crucial enzyme for crude oil biodessulfurization

The Ideonella sakaiensis bacterium has a tremendous industrial interest as it can remove sulfur from crude oil through its four-enzyme (DszA-D) 4S metabolic pathway. DszC is one of the rate-limiting enzymes of the pathway and the one that most suffers from feedback inhibition. We have combined molecular docking and molecular dynamics simulations to identify binding sites through which two products of the 4S pathway, 2-hydroxybiphenyl, and 2\u27-hydroxybiphenyl-2-sulfinate, induce DszC feedback inhibition. We have identified four potential binding sites: two adjacent binding sites close to the 280-295 lid loop proposed to contribute to DszC oligomerization and proper binding of the flavin mononucleotide cofactor, and two other close to the active site of DszC and the substrate binding site. By considering i) the occupancy of the binding sites and ii) the similar inhibitor poses, we propose that the mechanism of feedback inhibition of DszC occurs through disturbance of the DszC oligomerization and consequent binding of the flavin mononucleotide due to the weakening of the interactions between the 280-295 lid loop and both the 131-142 loop and the C-terminal tail. Nevertheless, inhibitor binding close to the active site or the substrate binding sites also compromises critical interactions within the active site of DszC. The disclosed molecular details provide valuable insight for future rational enzyme engineering protocols to develop DszC mutants more resistant against the observed feedback inhibition mechanism

Alkyl vs Aryl Modifications: A Comparative Study on Modular Modifications of Triphenylphosphonium Mitochondrial Vectors

Triphenylphosphonium (TPP+) moieties are commonly conjugated to drug molecules to confer mitochondrial selectivity due to their positive charge and high lipophilicity. Although optimisation of lipophilicity can be achieved by modifying the length of the alkyl linkers between the TPP+ moiety and the drug molecule, it is not always possible. While methylation of the TPP+ moiety is a viable alternative to increase lipophilicity and mitochondrial accumulation, there are no studies comparing these two separate modular approaches. Thus, we have systematically designed, synthesised and tested a range of TPP+ molecules with varying alkyl chain lengths and degree of aryl methylation to compare the two modular methodologies for modulating lipophilicity. The ability of aryl/alkyl modified TPP+ to deliver cargo to the mitochondria was also evaluated by confocal imaging with a TPP+-conjugated fluorescein-based fluorophore. Furthermore, we have employed molecular dynamics simulations to understand the translocation of these molecules through biological membrane model systems. These results provides further insights into the thermodynamics of this process and the effect of alkyl and aryl modular modifications<br /

Isomerization of Δ⁵‑Androstene-3,17-dione into Δ⁴‑Androstene-3,17-dione Catalyzed by Human Glutathione Transferase A3-3: A Computational Study Identifies a Dual Role for Glutathione

Glutathione transferases (GSTs) are important enzymes in the metabolism of electrophilic xenobiotic and endobiotic toxic compounds. In addition, human GST A3-3 also catalyzes the double bond isomerization of Δ5-androstene-3,17-dione (Δ⁵-AD) and Δ⁵-pregnene-3,20-dione (Δ⁵-PD), which are the immediate precursors of testosterone and progesterone. In fact, GST A3-3 is the most efficient human enzyme known to exist in the catalysis of these reactions. In this work, we have used density functional theory (DFT) calculations to propose a refined mechanism for the isomerization of Δ⁵-AD catalyzed by GST A3-3. In this mechanism the glutathione (GSH) thiol and Tyr9 catalyze the proton transfer from the Δ⁵-AD C4 atom to the Δ⁵-AD C6 atom, with a rate limiting activation energy of 15.8 kcal·mol^–1. GSH has a dual function, because it is also responsible for stabilizing the negative charge that is formed in the O3 atom of the enolate intermediate. The catalytic role of Tyr9 depends on significant conformational rearrangements of its side chain. Neither of these contributions to catalysis has been observed before. Residues Phe10, Leu111, Ala 208, and Ala 216 complete the list of the important catalytic residues. The mechanism detailed here is based on the GST A3-3:GSH:Δ⁴-AD crystal structure and is consistent with all available experimental data

Mechanism of Glutathione Transferase P1-1-Catalyzed Activation of the Prodrug Canfosfamide (TLK286, TELCYTA)

Canfosfamide (TLK286, TELCYTA) is a prodrug that upon activation by glutathione transferase P1-1 (GST P1-1) yields an anticancer alkylating agent and a glutathione derivative. The rationale underlying the use of TLK286 in chemotherapy is that tumor cells overexpressing GST P1-1 will be locally exposed to the released alkylating agent with limited collateral toxicity to the surrounding normal tissues. TLK286 has demonstrated clinical effects in phase II and III clinical trials for the treatment of malignancies, such as ovarian cancer, nonsmall cell lung cancer, and breast cancer, as a single agent and in combination with other chemotherapeutic agents. In spite of these promising results, the detailed mechanism of GST P1-1 activation of the prodrug has not been elucidated. Here, we propose a mechanism for the TLK286 activation by GST P1-1 on the basis of density functional theory (DFT) and on potential of mean force (PMF) calculations. A catalytic water molecule is instrumental to the activation by forming a network of intermolecular interactions between the active-site Tyr7 hydroxyl and the sulfone and COO^– groups of TLK286. The results obtained are consistent with the available experimental kinetic data and provide an atomistic understanding of the TLK286 activation mechanism