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Ram Sasisekharan - One of the best experts on this subject based on the ideXlab platform.
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heparin heparan sulfate 6 o sulfatase from flavobacterium heparinum integrated structural and biochemical investigation of Enzyme Active Site and substrate specificity
Journal of Biological Chemistry, 2009Co-Authors: James R Myette, Rahul Raman, Zachary Shriver, Venkataramanan Soundararajan, Ram SasisekharanAbstract:Abstract Heparin and heparan sulfate glycosaminoglycans (HSGAGs) comprise a chemically heterogeneous class of sulfated polysaccharides. The development of structure-activity relationships for this class of polysaccharides requires the identification and characterization of degrading Enzymes with defined substrate specificity and enzymatic activity. Toward this end, we report here the molecular cloning and extensive structure-function analysis of a 6-O-sulfatase from the Gram-negative bacterium Flavobacterium heparinum. In addition, we report the recombinant expression of this Enzyme in Escherichia coli in a soluble, Active form and identify it as a specific HSGAG sulfatase. We further define the mechanism of action of the Enzyme through biochemical and structural studies. Through the use of defined substrates, we investigate the kinetic properties of the Enzyme. This analysis was complemented by homology-based molecular modeling studies that sought to rationalize the substrate specificity of the Enzyme and mode of action through an analysis of the Active-Site topology of the Enzyme including identifying key Enzyme-substrate interactions and assigning key amino acids within the Active Site of the Enzyme. Taken together, our structural and biochemical studies indicate that 6-O-sulfatase is a predominantly exolytic Enzyme that specifically acts on N-sulfated or N-acetylated 6-O-sulfated glucosamines present at the non-reducing end of HSGAG oligosaccharide substrates. This requirement for the N-acetyl or N-sulfo groups on the glucosamine substrate can be explained through eliciting favorable interactions with key residues within the Active Site of the Enzyme. These findings provide a framework that enables the use of 6-O-sulfatase as a tool for HSGAG structure-activity studies as well as expand our biochemical and structural understanding of this important class of Enzymes.
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the heparin heparan sulfate 2 o sulfatase from flavobacterium heparinum a structural and biochemical study of the Enzyme Active Site and saccharide substrate specificity
Journal of Biological Chemistry, 2003Co-Authors: Rahul Raman, James R Myette, Zachary Shriver, Kevin Pojasek, Ganesh Venkataraman, Ram SasisekharanAbstract:Abstract In the previous paper (Myette, J. R., Shriver, Z., Claycamp, C., McLean, M. W., Venkataraman, G., and Sasisekharan, R. (2003) J. Biol. Chem. 278, 12157–12166), we described the molecular cloning, recombinant expression, and preliminary biochemical characterization of the heparin/heparan sulfate 2-O-sulfatase fromFlavobacterium heparinum. In this paper, we extend our structure-function investigation of the 2-O-sulfatase. First, we have constructed a homology-based structural model of the Enzyme Active Site, using as a framework the available crystallographic data for three highly related arylsulfatases. In this model, we have identified important structural parameters within the Enzyme Active Site relevant to Enzyme function, especially as they relate to its substrate specificity. By docking various disaccharide substrates, we identified potential structural determinants present within these substrates that would complement this unique Active Site architecture. These determinants included the position and number of sulfates present on the glucosamine, oligosaccharide chain length, the presence of a Δ4,5-unsaturated double bond, and the exolytic versusendolytic potential of the Enzyme. The predictions made from our model provided a structural basis of substrate specificity originally interpreted from the biochemical and kinetic data. Our modeling approach was further complemented experimentally using peptide mapping in tandem with mass spectrometry and Site-directed mutagenesis to physically demonstrate the presence of a covalently modified cysteine (formylglycine) within the Active Site. This combinatorial approach of structure modeling and biochemical studies provides insight into the molecular basis of Enzyme function.
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The Heparin/Heparan Sulfate 2-O-Sulfatase from Flavobacterium heparinum A STRUCTURAL AND BIOCHEMICAL STUDY OF THE Enzyme Active Site AND SACCHARIDE SUBSTRATE SPECIFICITY
Journal of Biological Chemistry, 2003Co-Authors: Rahul Raman, James R Myette, Zachary Shriver, Kevin Pojasek, Ganesh Venkataraman, Ram SasisekharanAbstract:Abstract In the previous paper (Myette, J. R., Shriver, Z., Claycamp, C., McLean, M. W., Venkataraman, G., and Sasisekharan, R. (2003) J. Biol. Chem. 278, 12157–12166), we described the molecular cloning, recombinant expression, and preliminary biochemical characterization of the heparin/heparan sulfate 2-O-sulfatase fromFlavobacterium heparinum. In this paper, we extend our structure-function investigation of the 2-O-sulfatase. First, we have constructed a homology-based structural model of the Enzyme Active Site, using as a framework the available crystallographic data for three highly related arylsulfatases. In this model, we have identified important structural parameters within the Enzyme Active Site relevant to Enzyme function, especially as they relate to its substrate specificity. By docking various disaccharide substrates, we identified potential structural determinants present within these substrates that would complement this unique Active Site architecture. These determinants included the position and number of sulfates present on the glucosamine, oligosaccharide chain length, the presence of a Δ4,5-unsaturated double bond, and the exolytic versusendolytic potential of the Enzyme. The predictions made from our model provided a structural basis of substrate specificity originally interpreted from the biochemical and kinetic data. Our modeling approach was further complemented experimentally using peptide mapping in tandem with mass spectrometry and Site-directed mutagenesis to physically demonstrate the presence of a covalently modified cysteine (formylglycine) within the Active Site. This combinatorial approach of structure modeling and biochemical studies provides insight into the molecular basis of Enzyme function.
Khalid Mohammed Khan - One of the best experts on this subject based on the ideXlab platform.
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2,5-Disubstituted thiadiazoles as potent β-glucuronidase inhibitors; Synthesis, in vitro and in silico studies.
Bioorganic Chemistry, 2019Co-Authors: Muhammad Taha, Mohammed Gollapalli, Noor Barak Almandil, Ashik Mosaddik, Mohamed Ibrahim, Umer Rashid, Khalid Mohammed KhanAbstract:Abstract Twenty-five thiadiazole derivatives 1–25 were synthesized from methyl 4-methoxybenzoate via hydrazide and thio-hydrazide intermediates, and evaluated for their potential against β-glucuronidase Enzyme. Most of the compounds including 1 (IC50 = 26.05 ± 0.60 μM), 2 (IC50 = 42.53 ± 0.80 μM), 4 (IC50 = 38.74 ± 0.70 μM), 5 (IC50 = 9.30 ± 0.29 μM), 6 (IC50 = 6.74 ± 0.26 μM), 7 (IC50 = 18.40 ± 0.66 μM), and 15 (IC50 = 18.10 ± 0.53 μM) exhibited superior activity potential than the standard d -saccharic acid-1,4-lactone (IC50 = 48.4 ± 1.25 μM). Molecular docking studies were conducted to correlate the in vitro results and to identify possible mode of interaction with Enzyme Active Site.
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synthesis molecular docking study and in vitro thymidine phosphorylase inhibitory potential of oxadiazole derivatives
Bioorganic Chemistry, 2018Co-Authors: Hayat Ullah, Imad Uddin, Zainul Wahab, Mohsan Nawaz, Fazal Rahim, Muhammad Taha, Rai Khalid Farooq, Syed Adnan Ali Shah, Abdul Wadood, Khalid Mohammed KhanAbstract:Abstract We have synthesized oxadiazole derivatives (1–16), characterized by 1H NMR, 13C NMR and HREI-MS and screened for thymidine phosphorylase inhibitory potential. All derivatives display varied degree of thymidine phosphorylase inhibition in the range of 1.10 ± 0.05 to 49.60 ± 1.30 μM when compared with the standard inhibitor 7-Deazaxanthine having an IC50 value 38.68 ± 1.12 μM. Structure activity relationships (SAR) has been established for all compounds to explore the role of substitution and nature of functional group attached to the phenyl ring which applies imperious effect on thymidine phosphorylase activity. Molecular docking study was performed to understand the binding interaction of the most Active derivatives with Enzyme Active Site.
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novel 2 5 disubtituted 1 3 4 oxadiazoles with benzimidazole backbone a new class of β glucuronidase inhibitors and in silico studies
Bioorganic & Medicinal Chemistry, 2015Co-Authors: Nik Khairunissa Nik Abdullah Zawawi, Norizan Ahmat, Nor Hadiani Ismail, Fazal Rahim, Muhammad Taha, Norishah Abdullah, Abdul Wadood, Khalid Mohammed KhanAbstract:A library of novel 2,5-disubtituted-1,3,4-oxadiazoles with benzimidazole backbone ( 3a – 3r ) was synthesized and evaluated for their potential as β-glucuronidase inhibitors. Several compounds such as 3a – 3d , 3e – 3j , 3l – 3o , 3q and 3r showed excellent inhibitory potentials much better than the standard (IC 50 = 48.4 ± 1.25 μM: d -saccharic acid 1,4-lactone). All the synthesized compounds were characterized satisfactorily by using different spectroscopic methods. We further evaluated the interaction of the Active compounds and the Enzyme Active Site with the help of docking studies.
Marcetta Y. Darensbourg - One of the best experts on this subject based on the ideXlab platform.
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Correlation Between Computed Gas-Phase and Experimentally Determined Solution-Phase Infrared Spectra: Models of the Iron-Iron Hydrogenase Enzyme Active Site
Journal of Computational Chemistry, 2020Co-Authors: Marcetta Y. Darensbourg, Michael B. HallAbstract:Gas-phase density functional theory calculations (B3LYP, double zeta plus polarization basis sets) are used to predict the solution-phase infrared spectra for a series of CO- and CN-containing iron complexes. It is shown that simple linear scaling of the computed CO and CN stretching frequencies yields accurate predictions of the the experimentally determined ν(CO) and ν(CN) values for a variety of complexes of different charges and in solvents of varying polarity. As examples of the technique, the resulting correlation is used to assign structures to spectroscopically observed but structurally ambiguous species in two different systems. For the (μ-SCH2CH2CH2S)[Fe(CO)3]2 complex in tetrahydrofuran solution, our calculations show that the initial electrochemical reduction process leads to a simple one-electron reduced product with a structure very similar to the (μ-SCH2CH2CH2S)[Fe(CO)3]2 parent complex. For the iron–iron hydrogenase Enzyme Active Site, our computations show that the absence or presence of a water molecule near the distal iron center (the iron center further from the [4Fe4S] cluster and protein backbone) has very little effect on the predicted infrared spectra. © 2006 Wiley Periodicals, Inc. J Comput Chem 27: 1454–1462, 2006
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sulfonated diiron complexes as water soluble models of the fe fe hydrogenase Enzyme Active Site
Inorganic Chemistry, 2011Co-Authors: Michael L Singleton, Joseph H. Reibenspies, Danielle J Crouthers, Robert P Duttweiler, Marcetta Y. DarensbourgAbstract:A series of diiron complexes developed as fundamental models of the two-iron subSite in the [FeFe]-hydrogenase Enzyme Active Site show water-solubility by virtue of a sulfonate group incorporated into the −SCH2NRCH2S- dithiolate unit that bridges two FeI(CO)2L moieties. The sulfanilic acid group imparts even greater water solubility in the presence of β-cyclodextrin, β-CyD, for which NMR studies suggest aryl-sulfonate inclusion into the cyclodextrin cavity as earlier demonstrated in the X-ray crystal structure of 1Na·2 β-CyD clathrate, where 1Na = Na+(μ-SCH2N(C6H4SO3–)CH2S-)[Fe(CO)3]2, (Singleton et al., J. Am. Chem. Soc.2010, 132, 8870). Electrochemical analysis of the complexes for potential as electrocatalysts for proton reduction to H2 finds the presence of β-CyD to diminish response, possibly reflecting inhibition of structural rearrangements required of the diiron unit for a facile catalytic cycle. Advantages of the aryl sulfonate approach include entry into a variety of water-soluble derivatives fr...
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Resin-bound models of the [FeFe]-hydrogenase Enzyme Active Site and studies of their reactivity
Dalton Transactions, 2009Co-Authors: Kayla N. Green, Jennifer L. Hess, Christine M. Thomas, Marcetta Y. DarensbourgAbstract:The immobilization of synthetic analogues of the [FeFe]-hydrogenase, [FeFe]H2ase, Enzyme Active Site on polyethyleneglycol-rich polystyrene beads is described. Using the reactivity of the amine termini of the PEG chains with carboxylates incorporated into (μ-SRS)[Fe(CO)3]2 or (μ-SR)2[Fe(CO)3]2 derivative, ν(CO)IR signatures can be used to interrogate the structure and properties of the diiron carbonyl complexes once incorporated into the PEG environment of the polymer beads. Alternatively, the SRS dithiolate was first attached to the resin and the diiron unit assembled via an in situ process on the bead.
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synthetic support of de novo design sterically bulky fefe hydrogenase models
Angewandte Chemie, 2008Co-Authors: Michael L Singleton, Joseph H. Reibenspies, Nattamai Bhuvanesh, Marcetta Y. DarensbourgAbstract:A twisted mimic: Upon oxidation of [(μ-SCH2C(CH3)2CH2S-){FeI(CO)2PMe3}2], rearrangement yields the mixed-valent FeIFeII cation in a square-pyramid/inverted square-pyramid geometry with a semibridging CO ligand, closely mimicking the [FeFe] hydrogenase Enzyme Active Site. According to de novo design principles, the steric effect of bridgehead bulk in the S–S bridging ligand stabilizes this structure in the absence of the protein matrix.
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de novo design of synthetic di iron i complexes as structural models of the reduced form of iron iron hydrogenase
Inorganic Chemistry, 2006Co-Authors: Marcetta Y. Darensbourg, Michael B. HallAbstract:Simple synthetic di-iron dithiolate complexes provide good models of the composition of the Active Site of the iron−iron hydrogenase Enzymes. However, the formally FeIFeI complexes synthesized to date fail to reproduce the precise orientation of the diatomic ligands about the iron centers that is observed in the molecular structure of the reduced form of the Enzyme Active Site. This structural difference is often used to explain the fact that the synthetic di-iron complexes are generally poor catalysts when compared to the Enzyme. Herein, density functional theory computations are used for the rational design of synthetic complexes as structural models of the reduced form of the Enzyme Active Site. These computations suggest several possible synthetic targets. The synthesis of complexes containing five-atom S-to-S linkers of the form S(CH2)2X(CH2)2S (X = CH2, NH, or O) or pendant functionalities attached to the three-carbon framework is one method. Another approach is the synthesis of asymmetrically subst...
Francisco J. Luque - One of the best experts on this subject based on the ideXlab platform.
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Role of the tautomerism of 2-azaadenine and 2-azahypoxanthine in substrate recognition by xanthine oxidase
Journal of Computer-Aided Molecular Design, 1997Co-Authors: Begoña Hernández, Modesto Orozco, Francisco J. LuqueAbstract:The tautomerism of 2-azaadenine and 2-hypoxanthine has been examined in the gas phase and in aqueous solution. The tautomerism in the gas phase has been studied by means of semiempirical and ab initio quantum-mechanical computations, as well as density-functional calculations. The influence of the aqueous solvent on the relative stability between tautomers has been estimated from self-consistent reaction field calculations performed with different high-level continuum models. The results provide a detailed picture of the tautomeric preference for these purine bases. The importance of tautomerism in the substrate recognition by xanthine oxidase is discussed. Finally, the rate of oxidation of 2-azaadenine and 2- hypoxanthine by xanthine oxidase is discussed in terms of the recognition model at the Enzyme Active Site.
Muhammad Taha - One of the best experts on this subject based on the ideXlab platform.
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2,5-Disubstituted thiadiazoles as potent β-glucuronidase inhibitors; Synthesis, in vitro and in silico studies.
Bioorganic Chemistry, 2019Co-Authors: Muhammad Taha, Mohammed Gollapalli, Noor Barak Almandil, Ashik Mosaddik, Mohamed Ibrahim, Umer Rashid, Khalid Mohammed KhanAbstract:Abstract Twenty-five thiadiazole derivatives 1–25 were synthesized from methyl 4-methoxybenzoate via hydrazide and thio-hydrazide intermediates, and evaluated for their potential against β-glucuronidase Enzyme. Most of the compounds including 1 (IC50 = 26.05 ± 0.60 μM), 2 (IC50 = 42.53 ± 0.80 μM), 4 (IC50 = 38.74 ± 0.70 μM), 5 (IC50 = 9.30 ± 0.29 μM), 6 (IC50 = 6.74 ± 0.26 μM), 7 (IC50 = 18.40 ± 0.66 μM), and 15 (IC50 = 18.10 ± 0.53 μM) exhibited superior activity potential than the standard d -saccharic acid-1,4-lactone (IC50 = 48.4 ± 1.25 μM). Molecular docking studies were conducted to correlate the in vitro results and to identify possible mode of interaction with Enzyme Active Site.
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synthesis molecular docking study and in vitro thymidine phosphorylase inhibitory potential of oxadiazole derivatives
Bioorganic Chemistry, 2018Co-Authors: Hayat Ullah, Imad Uddin, Zainul Wahab, Mohsan Nawaz, Fazal Rahim, Muhammad Taha, Rai Khalid Farooq, Syed Adnan Ali Shah, Abdul Wadood, Khalid Mohammed KhanAbstract:Abstract We have synthesized oxadiazole derivatives (1–16), characterized by 1H NMR, 13C NMR and HREI-MS and screened for thymidine phosphorylase inhibitory potential. All derivatives display varied degree of thymidine phosphorylase inhibition in the range of 1.10 ± 0.05 to 49.60 ± 1.30 μM when compared with the standard inhibitor 7-Deazaxanthine having an IC50 value 38.68 ± 1.12 μM. Structure activity relationships (SAR) has been established for all compounds to explore the role of substitution and nature of functional group attached to the phenyl ring which applies imperious effect on thymidine phosphorylase activity. Molecular docking study was performed to understand the binding interaction of the most Active derivatives with Enzyme Active Site.
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synthesis and biological evaluation of novel n arylidenequinoline 3 carbohydrazides as potent β glucuronidase inhibitors
Bioorganic & Medicinal Chemistry, 2016Co-Authors: Muhammad Taha, Herizal Ali Nuzar, Sadia Sultan, Nor Hadiani Ismail, Syahrul Imran, Fazal Rahim, Hayat UllahAbstract:Abstract Thirty N -arylidenequinoline-3-carbohydrazides ( 1 – 30 ) have been synthesized and evaluated against β-glucuronidase inhibitory potential. Twenty four analogs showed outstanding β-glucuronidase activity having IC 50 values ranging between 2.11 ± 0.05 and 46.14 ± 0.95 than standard d -saccharic acid 1,4 lactone (IC 50 = 48.4 ± 1.25 μM). Six analogs showed good β-glucuronidase activity having IC 50 values ranging between 49.38 ± 0.90 and 80.10 ± 1.80. Structure activity relationship and the interaction of the Active compounds and Enzyme Active Site with the help of docking studies were established. Our study identifies novel series of potent β-glucuronidase inhibitors for further investigation.
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novel 2 5 disubtituted 1 3 4 oxadiazoles with benzimidazole backbone a new class of β glucuronidase inhibitors and in silico studies
Bioorganic & Medicinal Chemistry, 2015Co-Authors: Nik Khairunissa Nik Abdullah Zawawi, Norizan Ahmat, Nor Hadiani Ismail, Fazal Rahim, Muhammad Taha, Norishah Abdullah, Abdul Wadood, Khalid Mohammed KhanAbstract:A library of novel 2,5-disubtituted-1,3,4-oxadiazoles with benzimidazole backbone ( 3a – 3r ) was synthesized and evaluated for their potential as β-glucuronidase inhibitors. Several compounds such as 3a – 3d , 3e – 3j , 3l – 3o , 3q and 3r showed excellent inhibitory potentials much better than the standard (IC 50 = 48.4 ± 1.25 μM: d -saccharic acid 1,4-lactone). All the synthesized compounds were characterized satisfactorily by using different spectroscopic methods. We further evaluated the interaction of the Active compounds and the Enzyme Active Site with the help of docking studies.